ASMS 2014 Poster Collection

Transcription

ASMS
Poster collection
Clinical, Forensic
and Pharmaceutical
Applications
• Page 4
Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method
scouting system coupled to LC/MS/MS
• Page 54
Application of a sensitive liquid chromatographytandem mass spectrometric method to pharmacokinetic study of telbivudine in humans
• Page 11
Determination of ∆9 -tetrahydrocannabinol and
two of its metabolites in whole blood, plasma
and urine by UHPLC-MS/MS using QuEChERS
sample preparation
• Page 60
Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass
spectrometry
• Page 17
Determination of opiates, amphetamines and
cocaine in whole blood, plasma and urine by
UHPLC-MS/MS using a QuEChERS sample preparation
• Page 23
Simultaneous analysis for forensic drugs in
human blood and urine using ultra-high speed
LC-MS/MS
• Page 29
Simultaneous screening and quantitation of
amphetamines in urine by on-line SPE-LC/MS
method
• Page 36
Single step separation of plasma from whole
blood without the need for centrifugation applied to the quantitative analysis of warfarin
• Page 42
Development and validation of direct analysis
method for screening and quantitation of
amphetamines in urine by LC/MS/MS
• Page 48
Next generation plasma collection technology
for the clinical analysis of temozolomide by
HILIC/MS/MS
• Page 66
Highly sensitive quantitative analysis of felodipine and hydrochlorothiazide from plasma using
LC/MS/MS
• Page 73
Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation
using LC/MS/MS
• Page 80
Development of 2D-LC/MS/MS method for quantitative analysis of 1,25-Dihydroxylvitamin D3 in
human serum
• Page 86
Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with
mass spectrometer
• Page 93
A rapid and reproducible Immuno-MS platform
from sample collection to quantitation of IgG
• Page 99
Simultaneous determinations of 20 kinds of
common drugs and pesticides in human blood
by GPC-GC-MS/MS
• Page 103
Low level quantitation of loratadine from plasma
using LC/MS/MS
PO-CON1452E
Rapid development of analytical
method for antiepileptic drugs in
plasma using UHPLC method scouting
system coupled to LC/MS/MS
ASMS 2014
ThP 672
Miho Kawashima1, Satohiro Masuda2, Ikuko Yano2,
Kazuo Matsubara2, Kiyomi Arakawa3, Qiang Li3,
Yoshihiro Hayakawa3
1 Shimadzu Corporation, Tokyo, JAPAN,
2 Kyoto University Hospital, Kyoto, JAPAN,
3 Shimadzu Corporation, Kyoto, JAPAN
Rapid development of analytical method for antiepileptic drugs
in plasma using UHPLC method scouting system coupled to LC/MS/MS
Introduction
Method development for therapeutic drug monitoring
(TDM) is indispensable for managing drug dosage based on
the drug concentration in blood in order to conduct a
rational and efficient drug therapy. Liquid chromatography
coupled with tandem quadrupole mass spectrometry is
increasingly used in TDM because it can perform selective
and sensitive analysis by simple sample pretreatment. The
UHPLC method scouting system coupled to tandem
O
O
N
+
H
N
O-
O
O
NH 2
Carbamazepine
N
Gabapentin
O
O
H
N
Lamotrigine
S
H 3C
S
N
OH
O
CH 3
Primidone
O
S
H 2N
O
O
O
O
O
O
N
O
S
H 2C
O
Tiagabine
Phenytoin
CH 3
CH 3
H 3C
O
O
Phenobarbial
Nitrazepam
CH 3
O
NH
O
N
H
O
O
Levetiracetam
Felbamate
HN
H
N
O
H 3C
N
NH 2
NH
H 3C
H
N
Cl
CH 3
O
+
O-
NH 2
O
Ethomuximide
O
N
O
O
N
N
NH 2
O
O
Diazepam
O
O
O
CH 3
Cl
Clonazepam
NH 2
Cl
H 2N
CH 3
N
Cl
NH 2
NH
O
N
OH
H 3C
N
Carbamazepine- 10,11-epoxide
H 2N
O
O
N
N
N
quadrupole mass spectrometer used in this study can
dramatically shorten the total time for optimization of
analytical conditions because this system can make
enormous combinatorial analysis methods and run batch
program automatically. In this study, we developed a
high-speed and sensitive method for measurement of
seventeen antiepileptics in plasma by UHPLC coupled with
tandem quadrupole mass spectrometer.
OH
CH 3
O
NH 2
Topiramate
Vigabatrin
Zonisamide
Figure 1 Antiepileptic drugs used in this assay
Experimental
Instruments
UHPLC based method scouting system (Nexera X2 Method
Scouting System, Shimadzu Corporation, Japan) is
configured by Nexera X2 UHPLC modules. For the detection,
tandem quadrupole mass spectrometer (LCMS-8050,
Shimadzu Corporation, Japan) was used. The system can be
operated at a maximum pressure of 130 MPa, and it enables
to automatically select up to 96 unique combinations of
eight different mobile phases and six different columns. A
dedicated software was newly developed to control the
system (Method Scouting Solution, Shimadzu Corporation,
Japan), which provides a graphical aid to configure the
different type of columns and mobile phases. The software
is integrated into the LC/MS/MS workstation (LabSolutions,
Shimadzu Corporation, Japan) so that selected conditions
are seamlessly translated into method files and registered to
a batch queue, ready for analysis instantly.
2
Figure 2 Nexera Method Scoutuing System and LCMS-8050 triple quadrupole mass spectrometer
Calibration standards and QC samples
The main standard mixture was prepared in methanol
from individual stock solutions. The calibration standards
were prepared by diluting the standard mixture with
methanol.
QC sample was prepared by adding 4 volume of
acetonitrile to 1 volume of control plasma, thereby
precipitating proteins, and subsequently adding the
standard mixture to the supernatant to contain plasma
concentration equivalents stated in Table 4. The QC
samples were further diluted 100 times (10 μL sample
added to 990μL methanol) before injection.
Next step of preparation procedure was divided into three
groups by the intensity of each compound. For
ethomuximide, phenobarbial and phenytoin, the
supernatant was used for the LC/MS/MS analysis without
further dilution. For zonisamide, 10 μL supernatant was
further diluted with 990 μL methanol. For others, 100 μL
supernatant was further diluted with 900 μL methanol.
The diluted solutions were used for the LC/MS/MS
analysis.
Result
MRM condition optimization
The MS condition optimization was performed by flow
injection analysis (FIA) of ESI positive and negative ionization
mode, and the compound dependent parameters such as
CID and pre-bias voltage were adjusted using automatic
MRM optimization function. The transition that gave highest
intensity was used for quantification. The MRM transitions
used in this assay are listed in Table 1.
3
Table 1 Compounds, Ionization polarity and MRM transition
Compound
Retaintion (min)
Polarity
Precursor m/z
Product m/z
Carbamazepine
3.84
+
237.1
194.2
Carbamazepine-10,11-epoxide
3.24
+
253.1
180.15
Clonazepam
3.93
+
316.1
269.55
Diazepam
4.79
+
284.9
154.15
Ethomuximide
2.50
+
239.3
117.20
Felbamate
2.86
+
172.2
154.25
Gabapentin
2.27
+
256.2
211.05
Lamotrigine
2.96
+
171.2
126.15
Levetiracetam
2.32
+
281.9
236.20
Nitrazepam
3.90
+
219.2
162.15
Phenobarbial
3.06
+
376.2
111.15
Phenytoin
3.64
+
130.2
71.15
Primidone
2.83
+
213.1
132.10
Tiagabine
4.28
-
140.0
42.00
Topiramate
3.14
-
231.0
42.05
Vigabatrin
0.82
-
337.9
78.00
Zonisamide
2.58
-
143.1
143.10
UHPLC condition optimization
36 analytical conditions, comprising combinations of 9
mobile phase and 4 columns, were automatically
investigated using Method Scouting System. Schematic
representation of scouting system was shown in Figure 3.
From the result of scouting, the combination of 10 mM
ammonium acetate water and methanol for mobile phase
and Inertsil-ODS4 for separation column were selected.
Using this combination of mobile phase and column, the
gradient condition was further optimized. The final analytical
condition was shown in Table 2.
Kinetex XB-C18 (Phenomenex)
2.1 x 50 mm
Kinetex PFP (Phenomenex)
2.1 x 50 mm
Pump A
InertsilODS-4 (GL Science)
2.1 x 50 mm
Discovery HS F5-5 (SPELCO)
2.1 x 50 mm
1
2
3
4
Auto Sampler
LPGE Unit
LCMS-8050
Column Oven
Pump B
(A)
(B)
1
2
3
4
1 – 10mM Ammonium Acetate
2 – 10mM Ammonium Formate
3 – 0.1%FA - 10mM Ammonium Acetate
1 – Methanol
2 – Acetonitrile
3 – Methanol/Acetonitrile=1/1
Figure. 3 Schematic representation and features of the Nexera Method Scouting System.
4
Table.2 UHPLC analytical conditions
Column
Mobile phase
: Inertsil ODS-4 (50 mmL. x 2.1mmi.d., 2um)
: A) 10mM Ammonium Acetate
B) Methanol
: B conc. 3% (0.65 min) → 40% (1.00 min) → 85% (5.00 min)
→ 100% (5.01-8.00 min) → 3% (8.01-10.00 min)
: 0.4 mL/min
: 1 μL
: 40 deg. C
Binary gradient
Flow Rate
Injection vol.
Column Temp.
Precision, accuracy and linearity of AEDs
Figure 4 shows MRM chromatograms of the 17 AEDs. It took only 10 minutes per one UHPLC/MS/MS analysis, including
column rinsing.
Felbamate
239.30>117.20(+)
Vigabatrin
130.20>71.15(+)
0.0
1.0
2.0
3.0
4.0
5.0
min
0.0
1.0
2.0
3.0
1.0
2.0
3.0
4.0
5.0
min
0.0
1.0
2.0
3.0
4.0
Levetiracetam
171.20>126.15(+)
0.0
1.0
2.0
3.0
4.0
5.0
min
1.0
2.0
3.0
4.0
5.0
min
0.0
1.0
2.0
1.0
2.0
3.0
4.0
5.0
min
3.0
0.0
1.0
2.0
1.0
2.0
3.0
4.0
5.0
min
min
4.0
5.0
min
3.0
4.0
5.0
min
253.10>180.15(+)
0.0
1.0
2.0
3.0
Primidone
219.20>162.15(+)
0.0
5.0
Topiramate
337.85>78.00(-)
Zonisamide
213.10>132.10(+)
0.0
min
Phenobarbial
231.00>42.05(-)
Ethomuximide
140.00>42.00(-)
0.0
5.0
Lamotrigine
256.20>211.05(+)
Gabapentin
172.20>154.25(+)
0.0
4.0
4.0
5.0
min
Carbamazepine
237.10>194.20(+)
0.0
1.0
2.0
3.0
4.0
5.0
min
3.0
4.0
5.0
min
3.0
4.0
5.0
min
2.0
3.0
4.0
5.0
min
2.0
3.0
4.0
5.0
min
Nitrazepam
281.90>236.20(+)
0.0
1.0
2.0
Clonazepam
316.10>269.55(+)
0.0
1.0
2.0
Tiagabine
376.20>111.15(+)
0.0
1.0
Diazepam
284.90>154.15
0.0
1.0
Phenytoin
251.00>208.20(-)
0.0
1.0
2.0
3.0
4.0
5.0
min
Figure. 4 Chromatogram of 17 AEDs calibration standards
5
Table 3 illustrates linearity of 17 AEDs and Table 4 illustrates
accuracy and precision of the QC samples at three
concentration levels. Determination coefficient (r2) of all
calibration curves was larger than 0.995, and the precision
and accuracy were within +/- 15%. Excellent linearity,
accuracy and precision for all 17 AEDs were obtained at
only 1 μL injection volume.
Table.3 Linearity of 17 AEDs QC sample
Compound
Linarity (ng/mL)
r2
Carbamazepine
0.25
-
50
0.999
0.25
-
50
0.998
Clonazepam
0.005
-
2.5
0.998
Diazepam
0.01
-
5
0.999
Ethomuximide
25
-
2500
0.998
Felbamate
0.5
-
100
0.998
Gabapentin
2
-
50
0.999
Lamotrigine
0.25
-
50
0.999
Levetiracetam
0.5
-
100
0.999
Nitrazepam
0.005
-
1
0.999
Phenobarbial
5
-
500
0.996
Phenytoin
5
-
500
0.998
Primidone
0.25
-
10
0.996
Tiagabine
0.25
-
50
0.998
Topiramate
0.5
-
100
0.998
Vigabatrin
0.5
-
50
0.998
Zonisamide
0.5
-
20
0.996
6
Table.4 Accuracy and precision of 17 AEDs QC sample
Compound
Plasma concentration
equivalents (µg/mL)
Precision (%)
Accuracy (%)
Low
Middle
High
Low
Middle
High
Low
Middle
High
Carbamazepine
1.8
18
71
2.2
0.9
0.9
106.1
103.9
95.8
1.8
18
71
2.4
1.9
1.3
104.2
105.0
98.2
Clonazepam
0.04
0.9
1.8
3.3
0.7
0.5
106.7
102.1
90.1
Diazepam
0.1
0.7
2.9
3.2
1.7
1.4
105.8
106.6
100.6
Ethomuximide
18
446
714
7.8
1.5
1.4
104.3
99.9
97.0
Felbamate
3.6
89
179
1.7
0.4
0.8
97.1
106.3
91.7
Gabapentin
18
36
143
1.3
0.7
0.7
85.8
98.8
89.5
Lamotrigine
1.8
45
71
10.5
1.2
1.7
107.7
98.4
99.2
Levetiracetam
3.6
89
179
2.1
0.5
1.1
99.5
104.9
90.4
Nitrazepam
0.04
0.4
1.4
3.3
1.4
1.5
105.0
105.2
97.9
Phenobarbial
3.6
71
143
3.5
6.2
1.6
100.9
108.4
95.8
Phenytoin
3.6
89
143
7.8
1.9
1.2
103.2
100.1
96.2
Primidone
1.8
18
45
3.2
0.7
0.7
99.5
112.6
97.1
Tiagabine
1.8
18
71
1.8
1.8
1.0
107.6
105.7
97.5
Topiramate
3.6
36
143
12.5
1.5
1.2
105.4
101.6
96.1
Vigabatrin
8.9
18
89
1.4
1.1
2.1
105.9
101.6
88.8
Zonisamide
36
89
179
3.3
1.3
1.6
111.7
100.4
95.2
Conclusions
• We could select the most suitable combination of mobile phase and column from 36 analytical condition without
time-consuming investigation.
• We have measured plasma sample as it is after 100-10,000 times dilution by methanol without making tedious sample
pretreatment. Excellent linearity, precision and accuracy for all 17 AEDs were obtained at only 1 uL injection volume.
First Edition: June, 2014
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PO-CON1446E
Determination of Δ9-tetrahydrocannabinol
and two of its metabolites in whole blood,
plasma and urine by UHPLC-MS/MS using
QuEChERS sample preparation
ASMS 2014
ThP600
Sylvain DULAURENT1, Mikaël LEVI2, Jean-michel GAULIER1,
Pierre MARQUET1,3 and Stéphane MOREAU2
1
CHU Limoges, Department of Pharmacology and Toxicology,
Unit of clinical and forensic toxicology, Limoges, France ;
2
Shimadzu France SAS, Le Luzard 2, Boulevard Salvador
Allende, 77448 Marne la Vallée Cedex 2
3
Univ Limoges, Limoges, France
Determination of Δ9-tetrahydrocannabinol and two of its
metabolites in whole blood, plasma and urine by UHPLC-MS/MS
using QuEChERS sample preparation
Introduction
In France, as in other countries, cannabis is the most
widely used illicit drug. In forensic as well as in clinical
contexts, ∆9-tetrahydrocannabinol (THC), the main active
compound of cannabis, and two of its metabolites
[11-hydroxy-∆9-tetrahydrocannabinol (11-OH-THC) and
11-nor-∆9-tetrahydrocannabinol-9-carboxylic acid
(THC-COOH)] are regularly investigated in biological fluids
for example in Driving Under the Influence of Drug
context (DUID) (figure 1).
Historically, the concentrations of these compounds were
determined using a time-consuming extraction procedure
and GC-MS. The use of LC-MS/MS for this application is
relatively recent, due to the low response of these
compounds in LC-MS/MS while low limits of quantification
need to be reached. Recently, on-line
Solid-Phase-Extraction coupled with UHPLC-MS/MS was
described, but in our hands it gave rise to significant
carry-over after highly concentrated samples. We propose
here a highly sensitive UHPLC-MS/MS method with
straightforward QuEChERS sample preparation (acronym
for Quick, Easy, Cheap, Effective, Rugged and Safe).
CH 3
H
H
H 3C
H 3C
OH
O
THC
O
OH
OH
H 2C
H
H
H 3C
H 3C
OH
H
H
H 3C
H 3C
O
11-OH-THC
OH
O
THC-COOH
Figure 1: Structures of THC and two of its metabolites
Methods and Materials
Isotopically labeled internal standards (one for each target
compound in order to improve method precision and
accuracy) at 10 ng/mL in acetonitrile, were added to 100
µL of sample (urine, whole blood or plasma) together
with 50 mg of QuEChERS salts (MgSO4 /NaCl/Sodium
citrate dehydrate/Sodium citrate sesquihydrate) and 200
µL of acetonitrile. Then the mixture was shaken and
centrifuged for 10 min at 12,300 g. Finally, 15 µL of the
upper layer were injected in the UHPLC-MS-MS system.
The whole acquisition method lasted 3.4 min.
2
UHPLC conditions (Nexera MP system)
Column
Mobile phase A
B
Flow rate
Time program
Column temperature
:
:
:
:
:
:
Kinetex C18 50x2.1 mm 2.6 µm (Phenomenex)
5mM ammonium acetate in water
CH3CN
0.6 mL/min
B conc. 20% (0-0.25 min) - 90% (1.75-2.40 min) - 20% (2.40-3.40 min)
50 °C
MS conditions (LCMS-8040)
Ionization
Ion source temperatures
Gases
: ESI, negative MRM mode
: Desolvation line: 300°C
Heater Block: 500°C
: Nebulization: 2.5 L/min
Drying: 10 L/min
MRM Transitions:
Compound
Pause time
Loop time
MRM
Dwell time (msec)
THC
313.10>245.25 (Quan)
313.10>191.20 (Qual)
313.10>203.20 (Qual)
60
60
60
THC-D3
316.10>248.30 (Quan)
316.10>194.20 (Qual)
5
5
11-OH-THC
329.20>311.30 (Quan)
329.20>268.25 (Qual)
329.20>173.20 (Qual)
45
45
45
11-OH-THC-D3
332.30>314.40 (Quan)
332.30>271.25 (Qual)
5
5
THC-COOH
343.20>245.30 (Quan)
343.20>325.15 (Qual)
343.20>191.15 (Qual)
343.20>299.20 (Qual)
50
50
50
50
THC-COOH-D3
346.20>302.25 (Quan)
346.20>248.30 (Qual)
5
5
: 3 msec
: 0.4 sec (minimum 20 points per peak for each MRM transition)
3
Results
Chromatographic conditions
A typical chromatogram of the 6 compounds is presented in figure 1.
Figure 1: Chromatogram obtained after an injection of a 15 µL whole blood extract spiked at 50 µg/L
Extraction conditions
As described by Anastassiades et al. J. AOAC Int 86 (2003)
412-31, the combination of acetonitrile and QuEChERS salts
allowed the extraction/partitioning of compounds of interest
from matrix. This extraction/partitioning process is not only
A
obtained with whole blood and plasma-serum where
deproteinization occurred and allowed phase separation,
but also with urine as presented in figure 2.
B
Figure 2: influence of QuEChERS salts on urine extraction/partitioning: A: acetonitrile with urine sample lead to one phase /
B: acetonitrile, QuEChERS salts and urine lead to 2 phases.
4
Validation data
One challenge for the determination of cannabinoids in
blood using LC-MS/MS is the low quantification limits that
need to be reached. The French Society of Analytical
Toxicology proposed 0.5 µg/L for THC et 11-OH-THC and
2.0 µg/L for THC-COOH. With the current application, the
THC-COOH
lower limit of quantification was fixed at 0.5 µg/L for the
three compounds (3.75 pg on column). The corresponding
extract ion chromatograms at this concentration are
presented in figure 3.
11-OH-THC
THC
Figure 3: Chromatogram obtained after an injection of a 15 µL whole blood extract spiked at 0.5 µg/L (lower limit of quantification).
The upper limit of quantification was set at 100 µg/L.
Calibration graphs of the cannabinoids-to-internal standard
peak-area ratios of the quantification transition versus
THC-COOH
expected cannabinoids concentration were constructed
using a quadratic with 1/x weighting regression analysis
(figure 4).
11-OH-THC
THC
Figure 4: Calibration curves of the three cannabinoids
Contrary to what was already observed with on-line
Solid-Phase-Extraction no carry-over effect was noted using
the present method, even when blank samples were
injected after patient urine samples with concentrations
exceeding 2000 µg/L for THC-COOH.
5
Conclusions
• Quick sample preparation based on QuEChERS salts extraction/partitioning, almost as short as on-line Solid Phase
Extraction.
• Low limit of quantification compatible with determination of DUID.
• No carry over effect noticed.
PO-CON1445E
Determination of opiates, amphetamines
and cocaine in whole blood, plasma
and urine by UHPLC-MS/MS using
a QuEChERS sample preparation
ASMS 2014
ThP599
Sylvain DULAURENT1, Mikaël LEVI2, Jean-michel GAULIER1,
Pierre MARQUET1,3 and Stéphane MOREAU2
1
CHU Limoges, Department of Pharmacology and Toxicology,
Unit of clinical and forensic toxicology, Limoges, France ;
2
Allende, 77448 Marne la Vallée Cedex 2
3
Univ Limoges, Limoges, France
Determination of opiates, amphetamines and cocaine
in whole blood, plasma and urine by UHPLC-MS/MS using a
Introduction
The determination of drugs of abuse (opiates,
amphetamines, cocaine) in biological fluids is still an
important issue in toxicology, in cases of driving under the
influence of drugs (DUID) as well as in forensic toxicology.
At the end of the 20th century, the analytical methods able
to determine these three groups of narcotics were mainly
based on a liquid-liquid-extraction with derivatization
followed by GC-MS. Then LC-MS/MS was proposed,
coupled with off-line sample preparation. Recently, on-line
Solid-Phase-Extraction coupled with UHPLC-MS/MS was
described, but in our hands it gave rise to significant
carry-over after highly concentrated samples. We propose
here another approach based on the QuEChERS (acronym
for Quick, Easy, Cheap, Effective, Rugged and Safe) sample
preparation principle, followed by UHPLC-MS/MS.
This method involves 40 compounds of interest (13
opiates, 22 amphetamines, as well as cocaine and 4 of its
metabolites) and 18 isotopically labeled internal standards
(designed with *) (Table1).
Table 1: list of analyzed compounds with their associate internal standard (*)
Cocaine and metabolites
• Anhydroecgonine methylester
• Benzoylecgonine*
• Cocaethylene*
• Cocaine*
• Ecgonine methylester*
Amphetamines or related
compounds
• 2-CB
• 2-CI
• 4-MTA
• Ritalinic acid
• Amphetamine*
• BDB
• Ephedrine*
• MBDB
• m-CPP
• MDA*
• MDEA*
• MDMA*
• MDPV
• Mephedrone
• Metamphetamine*
• Methcathinone
• Methiopropamine
• Methylphenidate
• Norephedrine
• Norfenfluramine
• Norpseudoephedrine
• Pseudoephedrine
Opiates
• 6-monoacetylmorphine*
• Dextromethorphan
• Dihydrocodeine*
• Ethylmorphine
• Hydrocodone
• Hydromorphone
• Methylmorphine*
• Morphine*
• Naloxone*
• Naltrexone*
• Noroxycodone*
• Oxycodone*
• Pholcodine
2
To 100 µL of sample (urine, whole blood or plasma) were
added isotopically labeled internal standards (in order to
improve method precision and accuracy) at 20 µg/L in
acetonitrile (20 µL), and 200 µL of acetonitrile. After a 15 s
shaking, the mixture was placed at -20°C for 10 min. Then
approximately 50 mg of QuEChERS salts
(MgSO4 /NaCl/Sodium citrate dehydrate/Sodium citrate
sesquihydrate) were added and the mixture was shaken
again for 15 s and centrifuged for 10 min at 12300 g. The
upper layer was diluted (1/3; v/v) with a 5 mM ammonium
formate buffer (pH 3). Finally, 5 µL were injected in the
UHPLC-MS/MS system. The whole acquisition method
lasted 5.5 min.
UHPLC conditions (Nexera MP system, figure 1)
Column
Mobile phase A
B
Flow rate
Time program
Column temperature
:
:
:
:
:
Restek Pinnacle DB PFPP 50x2.1 mm 1.9 µm
5mM Formate ammonium with 0.1% formic acid in water
90% CH3OH/ 10% CH3CN (v/v) with 0.1 % formic acid
0.474 mL/min
B conc. 15% (0-0.16 min) - 20% (1.77 min) - 90% (2.20 min) –
100% (4.00 min) – 15% (4.10-5.30 min)
: 50 °C
MS conditions (LCMS-8040, figure 1)
Ionization
Ion source temperatures
Gases
MRM Transitions
Pause time
Loop time
: ESI, Positive MRM mode
: Desolvation line: 300°C
Heater Block: 500°C
: Nebulization: 2.5 L/min
Drying: 10 L/min
: 2 Transitions per compounds were dynamically scanned for 1 min except
pholcodine (2 min)
: 3 msec
: 0.694 sec (minimum 17 points per peak for each MRM transition)
Figure 1: Shimadzu UHPLC-MS/MS Nexera-8040 system
3
Results
Chromatographic conditions
The analytical conditions allowed the chromatographic
separation of two couples of isomers: norephedrine and
norpseudoephedrine; ephedrine and pseudoephedrine
A
(figure 2). A typical chromatogram of the 58 compounds is
presented in figure 3.
B
Figure 2: Chromatograms obtained after an injection of a 5 µL whole blood extract spiked at 200 µg/L.
Order of retention - A: norephedrine and norpseudoephedrine / B: ephedrine and pseudoephedrine
Figure 3: Chromatogram obtained after an injection of a 5 µL whole blood extract spiked at 200 µg/L
4
Extraction conditions
As described by Anastassiades et al. J. AOAC Int 86 (2003)
412-31, the combination of acetonitrile and QuEChERS salts
allowed the extraction/partitioning of compounds of interest
from matrix. This extraction/partitioning process is not only
A
obtained with whole blood and plasma-serum where
deproteinization occurred and allowed phase separation,
but also with urine as presented in figure 4.
B
Figure 4: influence of QuEChERS salts on urine extraction/partitioning: A: acetonitrile with urine sample lead to one phase /
B: acetonitrile, QuEChERS salts and urine lead to 2 phases.
Validation data
Among the 40 analyzed compounds, 38 filled the validation
conditions in term of intra- and inter-assay precision and
accuracy were less than 20% at the lower limit of
quantification and less than 15% at the other
concentrations.
Despite the quick and simple sample preparation, no
significant matrix effect was observed and the lower limit of
quantification was 5 µg/L for all compounds, while the
upper limit of quantification was set at 500 µg/L. The
concentrations obtained with a reference (GC-MS) method
in positive patient samples were compared with those
obtained with this new UHPLC-MS/MS method and showed
satisfactory results.
Contrary to what was already observed with on-line
Solid-Phase-Extraction, no carry-over effect was noted using
the present method, even when blank samples were
injected after patient urine samples with analytes
concentrations over 2000 µg/L.
5
Conclusions
• Separation of two couples of isomers with a run duration less than 6 minutes and using a 5 cm column.
• Quick sample preparation based on QuEChERS salts extraction/partitioning, almost as short as on-line Solid Phase
Extraction.
• Lower limit of quantification compatible with determination of DUID.
• No carry over effect noticed.
PO-CON1442E
Simultaneous analysis for forensic
drugs in human blood and urine
using ultra-high speed LC-MS/MS
ASMS 2014
ThP-592
Toshikazu Minohata1, Keiko Kudo2, Kiyotaka Usui3,
Noriaki Shima4, Munehiro Katagi4, Hitoshi Tsuchihashi5,
Koichi Suzuki5, Noriaki Ikeda2
1
Shimadzu Corporation, Kyoto, Japan
2
Kyushu University, Fukuoka, Japan
3
Tohoku University Graduate School of Medicine, Sendai, Japan
4
Osaka Prefectural Police, Osaka, Japan
5
Osaka Medical Collage, Takatsuki, Japan
Simultaneous analysis for forensic drugs in human
blood and urine using ultra-high speed LC-MS/MS
Introduction
In Forensic Toxicology, LC/MS/MS has become a preferred
method for the routine quantitative and qualitative analysis
of drugs of abuse. LC/MS/MS allows for the simultaneous
analysis of multiple compounds in a single run, thus
enabling a fast and high throughput analysis. In this study,
we report a developed analytical system using ultra-high
speed triple quadrupole mass spectrometry with a new
extraction method for pretreatment in forensic analysis.
The system has a sample preparation utilizing modified
QuEChERS extraction combined with a short
chromatography column that results in a rapid run time
making it suitable for routine use.
Sample Preparation
Whole blood sample preparation was carried out by the
modified QuEChERS extraction method (1) using Q-sep™
QuEChERS Sample Prep Packets purchased from RESTEK
(Bellefonte, PA).
1) Add 0.5 mL of blood and 1 mL of distilled water into
the 15 mL centrifugal tube and agitate the mixture
using a vortex mixer.
2) Add two 4 mm stainless steel beads, 1.5 mL of
acetonitrile and 100 µL of acetonitrile solution
containing 1 ng/µL of Diazepam-d5. Then agitate using
the vortex mixer.
3) Add 0.5 g of the filler of the Q-sep™ QuEChERS
Extraction Salts Packet.
4) Vigorously shake the tube by hand several times, agitate
well using the vortex mixer for approximately 20
seconds. Then centrifuge the tube for 10 minutes at
3000 rpm.
5) Move the supernatant to a different 15 mL centrifugal
tube and add 100 µL of 0.1 % TFA acetonitrile solution.
Then, dry using a nitrogen-gas-spray concentration and
drying unit or a similar unit.
6) Reconstitute with 200 µL of methanol using the vortex
mixer. Then move it to a microtube, and centrifuge for
5 minutes at 10,000 rpm.
7) Transfer 150 µL of the supernatant to a 1.5 mL vial for
HPLC provided with a small-volume insert.
[ ref.] (1) Usui K et al, Legal Medicine 14 (2012), 286-296
Water 1 mL
ACN 1.5 mL
Diazepam-d5 (IS) 100ng
Stainless-Steel Beads (4mm x 2)
Transfer supernatant
Add 100uL of 0.1% TFA
Dry
Reconstitution with 200
uL MeOH
Q-sep QuEChERS
Extraction Salts
(MgSO4,NaOAc)
Sample
0.5 mL
LC/MS/MS analysis
[Shake]
[Centrifuge]
Figure 1 Scheme of the modified QuEChERS procedure
2
LC-MS/MS Analysis
Treated samples were analyzed using a Nexera UHPLC
system coupled to a LCMS-8050 triple quadrupole mass
spectrometer (Shimadzu Corporation, Japan) with
LC/MS/MS Rapid Tox. Screening Database. The Database
contains product ion scan spectra for 106 forensic and
toxicology-related compounds of Abused drugs,
Psychotropic drugs and Hypnotic drugs etc (Table 1) and
provides Synchronized Survey Scan® parameters (product
ion spectral data acquisition parameters based on the
MRM intensity as threshold) optimized for screening
analysis.
Samples were separated on a YMC Triart C18 column. A
flow rate of 0.3 mL/min was used together with a gradient
elution.
Analytical Conditions
HPLC (Nexera UHPLC system)
Column
Mobile Phase A
Mobile Phase B
Gradient Program
Flow Rate
Column Temperature
Injection Volume
: YMC Triart C18 (100x2mm, 1.9μm)
: 10 mM Ammonium formate - water
: Methanol
: 5%B (0 min) - 95%B (10 min - 13min) - 5%B (13.1 min - 20 min)
: 0.3 mL / min
: 40 ºC
: 5 uL
Mass (LCMS-8050 triple quadrupole mass spectrometry)
Ionization
Polarity
Probe Voltage
Nebulizing Gas Flow
Drying Gas Pressure
Heating gas flow
DL Temperature
BH Temperature
MRM parameter
Analytes
Ret. Time
Diazepam-d5
9.338
Alprazolam
8.646
Atropine
Estazolam
Ethyl loflazepate
Etizolam
Haloperidol
5.378
8.408
9.350
8.786
8.253
: heated ESI
: Positive & Negative
: +4.5 kV (ESI-Positive mode); -3.5 kV (ESI-Negative mode)
: 3 L / min
: 10 L / min
: 10 L / min
: 250 ºC
: 400 ºC
:
Collision
Energy
Q1 m/z
Q3 m/z
290.15
154.05
-27
290.15
198.20
-34
309.10
281.10
-24
309.10
205.10
-41
290.15
124.15
-23
290.15
93.20
-30
295.05
267.15
-24
295.05
205.25
-37
361.15
259.10
-30
361.15
287.15
-19
343.05
314.10
-24
343.05
138.15
-36
376.15
165.15
-24
376.15
123.10
-39
Analytes
Ret. Time
Risperidone
7.993
Triazolam
8.573
Amobarbital
(neg)
Barbital
(neg)
Phenobarbital
(neg)
Thiamylal
(neg)
8.093
5.243
6.762
8.883
Collision
Energy
Q1 m/z
Q3 m/z
411.20
191.05
-28
411.20
69.05
-55
343.05
315.00
-27
343.05
308.20
-25
225.15
42.00
25
225.15
182.00
14
183.10
42.10
21
183.10
140.10
15
231.10
42.20
19
231.10
85.10
14
253.00
58.10
23
253.00
101.00
16
3
positive
negative
Figure 2 LCMS-8050 triple quadrupole mass spectrometer
Results and Discussion
Alprazolam
Etizolam
(x103) 309.10>281.10(+)
2.0
0.01
ng/mL
S/N 39.5
Triazolam
(x102) 343.05>315.00(+)
2.5
S/N 145.5
1.0
1.0
0.0
(x104) 343.05>314.10(+)
0.0
(x104) 309.10>281.10(+)
0.1
ng/mL
Risperidone
(x103) 411.20>191.05(+)
(x103) 343.05>314.10(+)
S/N 107.6
S/N 18.8
2.5
0.0
(x103) 343.05>315.00(+)
0.0
(x104) 411.20>191.05(+)
1.0
2.5
0.5
2.5
0.5
0.0
0.0
0.0
8.0
Area Ratio
1.0
8.5
9.0
9.5
r2=0.998
8.0
8.5
Area Ratio (x0.1)
7.5
9.0
9.5
r2=0.998
5.0
0.0
0.00
Conc.
0.01
0.1
1
Area
9,004
8,288
9,519
75,236
75,983
74,023
829,519
831,098
849,597
0.50
0.75 Conc. Ratio
Accuracy
112.1
105.1
119.3
89.6
89.6
80.6
99.9
99.6
104.2
0.0
0.00
%RSD
Conc.
6.57
0.01
6.04
0.1
2.53
1
8.5
8.0
8.5
Area Ratio (x0.1)
r2=0.998
4.0
9.0
9.5
r2=0.998
2.0
2.5
2.5
0.25
8.0
3.0
5.0
0.5
0.0
7.0
7.5
Area Ratio
1.0
0.25
Area
4,865
5,109
4,321
48,038
49,152
54,497
604,640
581,207
579,390
0.50
0.75 Conc. Ratio
Accuracy
114.4
119.9
105.7
84.0
85.1
87.0
103.7
99.2
101.2
0.0
0.00
%RSD
Conc.
8.71
0.01
1.82
0.1
2.22
1
0.25
Area
29,832
32,436
30,461
335,202
309,273
343,172
3,826,373
3,718,854
3,705,165
0.50
0.75 Conc. Ratio
Accuracy
108.4
116.7
110.8
91.3
83.7
85.6
102.8
99.4
101.4
0.0
0.00
%RSD
Conc.
5.14
0.01
4.74
0.1
1.66
1
0.25
Area
3,047
3,064
3,356
27,991
25,542
26,317
288,776
297,332
294,788
0.50
0.75 Conc. Ratio
Accuracy
107.0
109.2
118.5
94.8
85.7
81.5
99.0
101.5
102.9
%RSD
5.63
7.83
1.96
4
Amobarbital (neg)
Barbital (neg)
(x102) 225.15>42.00(-)
Phenobarbital (neg)
Thiamylal (neg)
(x102) 253.00>58.10(-)
(x102) 231.10>42.20(-)
(x10) 183.10>42.10(-)
5.0
S/N 40.2
2.5
1
ng/mL
S/N 15.3
5.0
S/N 38.2
1.0
S/N 167.9
2.5
0.5
0.0
(x102) 183.10>42.10(-)
0.0
(x103) 225.15>42.00(-)
0.0
(x103) 231.10>42.20(-)
0.0
(x103) 253.00>58.10(-)
5.0
10 2.5
ng/mL
5.0
1.0
2.5
0.5
0.0
0.0
7.5
8.0
8.5
0.0
0.0
4.5
9.0
Area Ratio (x0.1)
2.5
5.0
5.5
6.0
Area Ratio (x0.01)
r2=0.999
r2=0.999
2.0
5.0
1.0
2.5
6.0
6.5
7.0
7.5
0.0
Conc.
1
10
100
25.0
Area
1,837
1,862
2,041
21,685
22,169
20,654
227,698
223,480
225,079
50.0
Conc. Ratio
Accuracy
100.2
99.1
105.8
99.6
102.4
92.5
101.3
98.3
100.9
0.0
0.0
%RSD
Conc.
4.53
1
5.30
10
1.62
100
25.0
Area
521
464
509
5,078
5,033
5,424
55,420
55,658
53,484
50.0
Conc. Ratio
Accuracy
108.7
96.6
103.4
95.6
95.4
99.4
101.4
100.8
98.7
8.5
Area Ratio (x0.1)
4.0
2
0.75
3.0
0.50
2.0
0.00
9.0
9.5
r =0.999
1.0
0.25
0.0
8.0
Area Ratio (x0.1)
1.00
r2=0.999
0.0
%RSD
Conc.
7.10
1
2.38
10
1.42
100
25.0
Area
725
693
617
7,909
8,564
7,939
81,987
83,274
82,656
50.0
Conc. Ratio
Accuracy
106
100.2
91
98.8
107.5
96.7
99.2
99.7
100.8
0.0
0.0
%RSD
Conc.
9.82
1
5.82
10
0.85
100
25.0
Area
2,520
2,192
2,288
30,808
29,623
31,379
318,233
317,214
313,399
50.0
Accuracy
107
95.3
97.5
101.4
98.3
100.6
100.7
99.3
100
Conc. Ratio
%RSD
8.99
1.68
0.71
Figure 3 Results of 8 drugs spiked in human whole blood using LCMS-8050
In this experiment, two different matrices consisting of
human whole blood and urine were prepared and 18
drugs were spiked into extract solution. Calibration curves
constructed in the range from 0.01 to 1 ng/mL for 12
drugs (Alprazolam, Aripiprazole, Atropine, Brotizolam,
Estazolam, Ethyl loflazepate, Etizolam, Flunitrazepam,
Haloperidol, Nimetazepam, Risperidone and Triazolam) and
from 1 to 100 ng/mL for 6 drugs (Bromovalerylurea,
Amobarbital, Barbital, Loxoprofen, Phenobarbital and
Thiamylal). All calibration curves displayed linearity with an
R2 > 0.997 and excellent reproducibility was observed for
all compounds (CV < 12%) at low concentration level.
5
Amobarbital (neg)
Barbital (neg)
(x102) 225.15>42.00(-)
Phenobarbital (neg)
(x102) 183.10>42.10(-)
Thiamylal (neg)
(x102) 253.00>58.10(-)
(x102) 231.10>42.20(-)
5.0
2.5
S/N 14.7
1
ng/mL
S/N 9.4
1.0
0.0
(x103) 225.15>42.00(-)
S/N 97.4
2.5
0.0
(x103) 253.00>58.10(-)
0.0
(x103) 231.10>42.20(-)
(x102) 183.10>42.10(-)
2.5
S/N 18.3
1.0
1.0
5.0
0.5
2.5
5.0
10
ng/mL
2.5
0.0
0.0
7.5
8.0
8.5
Area Ratio (x0.1)
5.0
5.5
r2=0.999
6.5
7.0
7.5
8.0
8.5
9.0
9.5
Area Ratio (x0.1)
r2=0.999
1.0
0.50
1.0
6.0
Area Ratio (x0.1)
0.75
2.0
0.0
6.0
Area Ratio (x0.1)
r2=0.999
3.0
0.0
0.0
4.5
9.0
r2=0.999
5.0
0.5
2.5
0.25
0.0
Conc.
1
10
100
25.0
Area
1,468
1,233
1,245
17,241
20,546
18,689
211,917
251,963
234,789
50.0
Conc. Ratio
Accuracy
102.2
86.6
87.6
104.4
114.7
106.9
96.8
103
97.9
0.00
0.0
%RSD
Conc.
12.73
1
5.10
10
3.34
100
25.0
Area
651
695
654
4,989
5,613
5,443
55,392
69,481
66,327
50.0
Conc. Ratio
Accuracy
93.6
96.1
89
105.2
109.6
108.6
92.6
104
101.3
0.0
0.0
%RSD
Conc.
2.77
1
2.07
10
5.98
100
25.0
Area
612
545
609
5,656
6,632
6,384
71,965
88,685
82,091
50.0
Conc. Ratio
Accuracy
103.6
89.4
99.3
97.9
106.1
104.4
95.2
105
99.1
0.0
0.0
%RSD
Conc.
8.16
1
4.24
10
4.95
100
25.0
Area
3,142
3,470
3,153
27,257
34,377
32,933
365,563
431,826
390,719
50.0
Conc. Ratio
Accuracy
95.1
100.5
91.4
94.9
110.8
108.5
98.5
104.1
96.1
%RSD
4.54
8.15
4.15
Figure 4 Results of 4 drugs spiked in human urine using LCMS-8050
Conclusions
• The validated sample preparation protocol can get adequate recoveries in quantitative works for all compounds ranging
from acidic to basic.
• The combination of the modified QuEChERS extraction method and high-speed triple quadrupole LC/MS/MS with a
simple quantitative method enable to acquire reliable data easily.
PO-CON1460E
Simultaneous Screening and Quantitation
of Amphetamines in Urine
by On-line SPE-LC/MS Method
ASMS 2014
ThP587
Helmy Rabaha1, Lim Swee Chin1, Sun Zhe2,
Jie Xing2 & Zhaoqi Zhan2
1
Department of Scientific Services, Ministry of Health,
Brunei Darussalam;
2
Shimadzu (Asia Pacific) Pte Ltd, Singapore, SINGAPORE
of Amphetamines in Urine by On-line SPE-LC/MS Method
Introduction
Amphetamines belong to stimulant drugs and are also
controlled as illicit drugs worldwide. The conventional
analytical procedure of amphetamines in human urine
includes initial immunological screening followed by GCMS
confirmation and quantitation [1]. With new SAMHSA
guidelines effective in Oct 2010 [2], screening,
confirmation and quantitation of illicit drugs including
amphetamines were allowed to employ LC/MS and
LC/MS/MS, which usually does not require a derivatization
step as used in the GCMS method [1]. The objective of this
study was to develop an on-line SPE-LC/MS method for
analysis of five amphetamines in urine without sample
pre-treatment except dilution with water. The compounds
studied include amphetamine (AMPH), methamphetamine
(MAMP) and three newly added MDMA, MDA and MDEA
by the new SAMHSA guideline (group A in Table 1). Four
potential interferences (group B in) and PMPA (R) as a
control reference were also included to enhance the
method reliability in identification of the five targeted
amphetamines from those structurally similar analogues
which potentially present in forensic samples.
Experimental
The test stock solutions of the ten compounds (Table 1)
were prepared in the toxicology laboratory in the
Department of Scientific Services (MOH, Brunei). Five urine
specimens were collected from healthy adult volunteers.
The urine samples used as blank and matrix to prepare
spiked amphetamine samples were not pre-treated off-line
by any means except dilution of 10 times with pure water.
An on-line SPE-LC/MS was set up on the LCMS-2020, a
single quadrupole system, with a switching valve and a
trapping column kit (Shimadzu Co-Sense configuration)
installed in the column oven and controlled by the
LabSolutions workstation. The analytical column used was
Shim-pack VP-ODS 150 x 2mm (5um) and the trapping
column was Synergi Polar-RP 50 x 2mm (2.5um), instead of
a normal SPE cartridge. The injected sample first passed
through the trapping column where the amphetamines
were trapped, concentrated and washed by pure water for
3 minutes followed by switching to the analytical flow line.
The trapped compounds were then eluted out with a
gradient program: 0.01min, valve at position 0 & B=5%; 3
min, valve at position 1; 3.01-10 min, B=5% → 15%;
10.5-12 min, B=65%; 12.1 min, B=5%; 14 min stop, valve
to position 0. The mobile phases A and B were water and
MeOH both with 0.1% formic acid and mobile C was pure
water. The total flow rates of the trapping line and
analytical line are 0.6 and 0.3 mL/min, respectively. The
injection volume was 20uL in all experiments.
2
Table 1: Amphetamines & relevant compounds
No
Name
Abbr. Name
Formula
A1
Amphetamine
AMPH
C9H13N
A2
Methampheta-mine
MAMP
C10H15N
A3
3,4-methylene-dioxyamphetamine
MDA
C10H13NO2
A4
3,4-methylene-dioxymetham phetamine
MDMA
C11H15NO2
A5
3,4-methylene dioxy-N-ethyl amphetamine
MDEA
C12H17NO2
B1
Nor pseudo-ephedrine
Nor pseudo-E
C9H13NO
B2
Ephedrine
Ephe
C10H15NO
B3
Pseudo-Ephedrine
Pseudo-E
C10H15NO
B4
Phentermine
Phent
C10H15N
R
Propyl-amphetamine
PAMP
C12H19N
Pump A
Mixer
SPE Trapping
Column
Structure
Manual
injector
Analytical LCMS-2020
column
5
1
3
Waste
Pump B
Switching
Valve
Auto
sampler
Pump C
Figure 1: Schematic diagram of on-line SPE-LC/MS system
3
Development of on-line SPE-LC/MS method
With ESI positive SIM and scan mode, all of the 10
compounds formed protonated ions [M+H]+ which were
used as quantifier ions. The scan spectra were used for
confirmation to reduce false positive results. Mixed
standards of the ten compounds in Table 1 spiked in urine
was used for method development. An initial difficulty
encountered was that the normal reusable SPE cartridges
(10-30 mmL) for on-line SPE could not trap all of the ten
compounds. With using a 50mmL C18-column to replace
the SPE cartridge, the ten compounds studied were
trapped efficiently. Furthermore, the trapped compounds
were well-separated and eluted out in 8~13 minutes as
sharp peaks (Figure 2) by the fully automated on-line
SPE-LC/MS method established.
(x1,000,000)
(x1,000,000)
2.0 2:136.10(+)
1.0
0.5
0.5
0.0
0.0
MDEA
Phent PAMP
MDMA
AMPH
MDA
1.0
(b) spiked samples
Ephedrine
Pseudo
2:150.10(+)
2:178.10(+)
2:180.10(+)
2:194.10(+)
1.5 2:208.20(+)
2:166.10(+)
2:152.10(+)
(a) Urine blank
MAMP
2.0 2:136.10(+)
Norpseudo
2:150.10(+)
2:178.10(+)
2:180.10(+)
2:194.10(+)
1.5 2:208.20(+)
2:166.10(+)
2:152.10(+)
0.0
2.5
5.0
7.5
10.0
12.5 min
0.0
2.5
5.0
7.5
10.0
12.5 min
Figure 2: SIM chromatograms of urine blank (a) and five amphetamines and related
compounds (125 ppb each) spiked in urine (b) by on-line SPE-LC/MS.
curves with R2> 0.999 were obtained for every compound
(Figure 3 & Table 2).
Calibration curves of the on-line SPE-LC/MS method were
established using mixed standard samples with
concentrations from 2.5 ppb to 500 ppb. Linear calibration
Area (x1,000,000)
Area (x10,000,000)
AMPH
7.5
1.5
5.0
1.0
2.5
0.5
Area (x10,000,000)
MAMP
Area (x10,000,000)
MDA
1.0
0
250
Conc.
Area (x1,000,000)
0.0
Nor pseudo-E
0
250
Conc.
Ephedrine
1.5
0
250
Conc.
0.0
1.0
0.5
0.5
0
250
0
250
Conc.
Conc.
0.0
1.0
0.0
0
250
Conc.
0.0
Area (x10,000,000)
1.0
Pseudo-E
1.5
1.0
0.0
1.0
Area (x10,000,000)
2.5
0.0
MDEA
2.0
Area (x10,000,000)
5.0
MDMA
2.0
0.5
0.0
Area (x10,000,000)
3.0
0
Phent
0.5
250
Conc.
0.0
Conc.
PAMP
2.0
0
250
Area (x10,000,000)
1.0
0
250
Conc.
0.0
0
250
Conc.
Figure 3: Calibration curves of five amphetamines and five related compounds with
concentrations from 2.5 ppb to 500 ppb by on-line SPE-LC/MS method
4
Table 2: Peak detection, retention, calibration curves and method performance evaluation
Name
SIM ion
(+)
RT
(min)
Conc. range
(ppb)
Linearity
(r2)
Rec. %
(62.5ppb)
M.E %
(62.5ppb)
RSD%(n=6)
(62.5ppb)
S/N
(2.5ppb)
LOD/LOQ
(ppb)
Norpseudo-E
152.1
8.0
2.5 - 500
0.9982
97.3
69.3
1.67
11.3
0.71/2.17
Ephe
166.1
8.4
2.5 - 500
0.9960
84.4
111.0
0.54
33.7
0.25/0.76
Pseudo-E
166.1
9.0
2.5 - 500
0.9976
78.9
109.2
0.41
28.5
0.29/0.88
AMPH
136.1
9.6
2.5 - 500
0.9983
85.6
71.1
0.98
17.5
0.48/1.46
MAMP
150.1
10.2
2.5 - 500
0.9968
76.5
96.8
0.94
30.3
0.26/0.80
MDA
180.1
10.4
2.5 - 500
0.9989
71.8
70.3
1.94
18.2
0.45/1.36
MDMA
194.1
10.8
2.5 - 500
0.9973
72.2
116.3
1.08
36.6
0.23/0.70
MDEA
208.1
12.2
2.5 - 500
0.9908
74.8
107.1
2.18
41.9
0.19/0.57
Phent
150.1
12.4
2.5 - 500
0.9960
74.5
69.9
1.82
12.7
0.66/2.01
PAMP (Ref)
178.1
12.7
2.5 - 500
0.9912
69.5
96.8
5.30
37.7
0.22/0.66
Performance evaluation of on-line SPE-LCMS method
The trapping efficiency of the on-line SPE is critical and
must be evaluated first, because it determines the recovery
of the method. In this study, the recovery of the on-line
SPE was determined by injecting a same mixed standard
sample from a manual injector installed before the
analytical column (by-pass on-line SPE) and also from the
Autosampler (See Figure 1). The peaks areas obtained by
the two injections were used to calculate recovery value of
the on-line SPE method. As shown in Table 2, the recovery
obtained with 62.5 ppb mixed standards are at 69.5% ~
97.3%. The recovery with 250 ppb and 500 ppb mixed
samples were also determined and similar results were
obtained.
Matrix effect was determined with 62.5 ppb and 250 ppb
levels of mixed samples in clear solution and in urine. The
results (Table 2) show a variation between 69.3% and
116% with compounds. The matrix effect with different
urine specimens did not show significant differences.
Repeatability was evaluated with spiked mixed samples of
62.5 ppb and 250 ppb. The results of 62.5 ppb is shown in
Table 2, RSD between 0.41% and 5.3%. The sensitivity of
the on-line SPE-LC/MS method was evaluated with spiked
sample of 2.5 ppb level. The SIM chromatograms are
shown in Figure 4. The S/N ratios obtained ranged
11.3~42, which were suitable to determine LOQ (S/N = 10)
and LOD (S/N = 3). Since the urine samples were diluted
for 10 times with water before injection, the LOD and LOQ
of the method for source urine samples were at 1.9~7.1
and 5.7~21.7 ng/mL, respectively. The confirmation cutoff
values of the five targeted amphetamines (Group A) in
urine enforced by the new SMAHSA guidelines are 250
ng/mL [2]. The on-line SPE-LC/MS method established has
sufficient allowance in terms of sensitivity and confirmation
reliability for analysis of actual urine samples.
(x10,000)
2.0
1.0
7.5
PAMP
MDEA
Phent
MDMA
10.0
MAMP
MDA
Norpseudo
3.0
AMPH
4.0
Ephedrine
5.0
2:136.10(+)
2:150.10(+)
2:178.10(+)
2:180.10(+)
2:194.10(+)
2:208.20(+)
2:166.10(+)
2:152.10(+)
Pseudo
6.0
12.5
min
Figure 4: SIM chromatograms of 10 compounds with
2.5 ppb each by on-line SPE-LC/MS method.
5
Durability of on-line SPE trapping column
(x1,000,000)
0.5
1.0
0.5
0.0
0.0
200th injection
spiked mixed std 125ppb in urine
inj vol: 20 µL
Phent
PAMP
MDEA
1.5
2:136.10(+)
2:150.10(+)
2:178.10(+)
2:180.10(+)
2:194.10(+)
2:208.20(+)
2:166.10(+)
2:152.10(+)
AMPH
MAMP
MDA
MDMA
1.0
2.0
Phent
1st injection
spiked mixed std 125ppb in urine
inj vol: 20 µL
Norpseudo
Ephedrine
Pseudo
AMPH
MAMP
MDA
MDMA
1.5
2:136.10(+)
2:150.10(+)
2:178.10(+)
2:180.10(+)
2:194.10(+)
2:208.20(+)
2:166.10(+)
2:152.10(+)
Norpseudo
Ephedrine
Pseudo
(x1,000,000)
2.0
spiked sample. The results show that the variations of peak
area and retention time of the 200th injection compared to
the 1st injection were at 89.5%~117.8% and
89.5%~99.8% respectively.
MDEA PAMP
The durability of the trapping column was tested purposely
by continuous injections of spiked urine samples (125 ppb)
for 200 times in a few days. Figure 5 shows the
chromatograms of the first and 200th injections of a same
0.0
2.5
5.0
7.5
10.0
12.5
min
0.0
2.5
5.0
7.5
10.0
12.5
min
Figure 5: Durability test of on-line SPE-LC/MS method, comparison of 1st and 200th injections.
Confirmation Reliability
Confirmation reliability of LC/MS and LC/MS/MS methods
must be proven to be equivalent to the GCMS method
according to the SMAHSA guidelines [2]. Validation of
confirmation reliability of the on-line SPE-LC/MS method
has not be carried out systematically. The high sensitivity of
MS detection in SIM mode is a key factor to ensure no
false-negative and the scan spectra acquired
simultaneously is used for excluding false-positive. In this
work, the confirmation reliability was evaluated using five
different urine specimens as matrix to prepare spiked
samples of 2.5 ppb (correspond 25 ng/mL in source urine)
and above. The results show that false-positive and false
negative results were not found.
Conclusions
A novel high sensitivity on-line SPE-LC/MS method was
developed for screening, conformation and quantification
of five amphetamines: AMPH, MAMP, MDMA, MDA and
MDEA in urines. The recovery of the on-line SPE by
employing a 50mmL Synergi Polar-RP column was at
72%~86% for the five amphetamines, which are
considerably high if comparing with conventional on-line
SPE cartridges. The method performance was evaluated
thoroughly with urine spiked samples. The results
demonstrate that the on-line SPE-LC/MS method is suitable
for direct analysis of the amphetamines and relevant
compounds in urine samples without off-line sample
pre-treatment.
6
References
1. Kudo K, Ishida T, Hara K, Kashimura S, Tsuji A, Ikeda N, J Chromatogr B, 2007, 855, 115-120.
2. SAMHSA “Manual for urine laboratories, National laboratory certification program”, Oct 2010, US Department of
Health and Human Services.
PO-CON1481E
Single step separation of plasma
from whole blood without the need
for centrifugation applied
to the quantitative analysis of warfarin
ASMS 2014
MP762
Alan J. Barnes1, Carrie-Anne Mellor2,
Adam McMahon2, Neil J. Loftus1
1
Shimadzu, Manchester, UK
2
WMIC, University of Manchester, UK
Single step separation of plasma from whole blood
without the need for centrifugation applied to the quantitative
analysis of warfarin
Introduction
Dried plasma sample collection and storage from whole
blood without the need for centrifugation separation and
refrigeration opens new opportunities in blood sampling
strategies for quantitative LC/MS/MS bioanalysis. Plasma
samples were generated by gravity filtration of a whole
blood sample through a laminated membrane stack
allowing plasma to be collected, dried, transported and
analysed by LC/MS/MS. This novel plasma separation card
(PSC) technology was applied to the quantitative
LC/MS/MS analysis of warfarin, in blood samples. Warfarin
is a coumarin anticoagulant vitamin-K antagonist used for
the treatment of thrombosis and thromboembolism. As a
result of vitamin-K recycling being inhibited, hepatic
synthesis is in-turn inhibited for blood clotting factors as
well as anticoagulant proteins. Whilst the measurement of
warfarin activity in patients is normally measured by
prothrombin time by international normalized ratio (INR) in
some cases the quantitation of plasma warfarin
concentration is needed to confirm patient compliance,
resistance to the anticoagulant drug, or diet related issues.
In this preliminary evaluation, warfarin concentration was
measured by LC/MS/MS to evaluate if PSC technology
could complement INR when sampling patient blood.
Materials and Methods
Sample preparation
Warfarin standard was dissolved in water containing 50%
ethanol + 0.1% formic acid, spiked (60uL) to whole human
blood (1mL) and mixed gently. 50uL of spiked blood was
deposited onto the PSC. After 3 minutes, the primary
filtration overlay was removed followed by 15 minutes air
drying at room temperature. The plasma sample disc was
prepared directly for analysis after drying. LC/MS/MS
sample preparation involved vortexing the sample disk in
40uL methanol, followed by centrifugation 16,000g 5 min.
20uL supernatant was added directly to the LCMS/MS
sample vial already containing 80uL water (2uL analysed).
Control plasma comparison was prepared by centrifuging
remaining blood at 1000g for 10min. 2.5uL supernatant
plasma was taken, 40uL methanol added, and prepared as
PSC samples. LCMS/MS sample injection volume, 2uL.
LC-MS/MS analysis
Warfarin was measured by MRM, positive negative switching mode (15msec).
LC/MS/MS System
Flow rate
Mobile phase
Gradient
Analytical column
Column temperature
Ionisation
Desolvation line
Drying/Nebulising gas
Heating block
: Nexera UHPLC system + LCMS-8040 Shimadzu Corporation
: 0.4mL/min (0-7.75min), 0.5mL/min (7.5-14min), 0.4mL/min (15min)
: A= Water + 0.1% formic acid
B= Methanol + 0.1% formic acid
: 20% B (0-0.5 min), 100% B (8-12 min), 20% B (12.01-15 min)
: Phenomenex Kinetex XB C18 100 x 2.1mm 1.7um 100A
: 50ºC
: Electrospray, positive, negative switching mode
: 250ºC
: 10L/min, 2L/min
: 400ºC
2
Design of plasma separator technology
Spreading Layer
[Lateral spreading layer rapidly spreads blood so it will
enter the filtration layer as a front while adding buffers and
anticoagulants. The lateral spreading rate is 150um/sec].
Control Spot:
[Determines whether enough
blood was placed on the card].
Filtration Layer
[Filtration layer captures blood
cells by a combination of filtration
and adsorption. The average
linear vertical migration rate is
approximately 1um/sec].
Isolation Screen
[Precludes lateral wicking along the
card surface].
Collection Layer
[Loads with a specific aliquot of plasma onto a 6.35mm disc]. Although flow through
the filtration membrane is unlikely to be constant throughout the plasma extraction
process, the average loading rate of the Collection Disc was 13 nL/sec. This
corresponds to a volumetric flow rate into the Collection Disc of 400 pL/mm2/sec.
Plasma separation workflow
1
2
3
4
The collection disc is
removed from the
card and is ready for
extraction for
LC-MS/MS analysis.
A NoviPlex card is
removed from foil
packaging.
Approximately 50uL
of whole blood is
added to the test
area.
After 3 minutes, the
top layer is completely
removed (peeled
back).
The collection disc
contains 2.5uL of
plasma. Card is air
dried for 15 minutes.
Figure 1. Noviplex workflow.
3
Figure 2. Applying a blood sample, either as a finger prick or by accurately measuring the blood volume, to the laminated membrane stack retains red cells and
allows a plasma sample to be collected. The red cells are retained by a combination of adsorption and filtration whilst plasma advances through the membrane stack
by capillary action. After approximately three minutes the plasma Collection Disc was saturated with an aliquot of plasma and was ready for LC/MS/MS analysis.
Results
Comparison between plasma separation cards (PSC) and plasma
(x100,000)
2.00
1.75
1.50
1.25
Plasma separation card
Positive ion
Warfarin m/z 309.20 > 163.05
(x100,000)
3.00
2.75
2.50
2.25
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
Q1 (V) -22
Collision energy -15
Q3 (V) -15
1.00
2.5ug/mL
0.75
Calibration standard
0.50
0.4ug/mL
0.25
0.00
0.0
(x100,000)
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
min
0.0
(x100,000)
Negative ion
Warfarin m/z 307.20 > 161.25
1.50
Plasma
Positive ion
Warfarin m/z 309.20 > 163.05
Q1 (V) -22
Q3 (V) -15
2.5ug/mL
0.4ug/mL
1.0
2.0
3.0
Q1 (V) 14
Collision energy 19
Q3 (V) 30
1.25
1.00
Q1 (V) 14
Collision energy 19
Q3 (V) 30
2.5ug/mL
0.75
2.5ug/mL
5.0
6.0
7.0
min
5.0
6.0
7.0
min
0.50
0.4ug/mL
4.0
Plasma
Negative ion
Warfarin m/z 307.20 > 161.05
0.4ug/mL
0.25
0.00
1.0
2.0
3.0
4.0
5.0
6.0
7.0
min
1.0
2.0
3.0
4.0
Figure 3. Comparison between the warfarin response in both positive and negative ion modes for warfarin calibration standards at 2.5ug/mL and
0.4ug/mL extracted from the plasma separation cards and a conventional plasma sample. There is a broad agreement in ion signal intensity between
the 2 sample preparation techniques.
4
450000
700,000
Positive ion
Warfarin m/z 309.20 > 163.05
600,000
Replicate calibration points at
2.5ug/mL and 0.4ug/mL (n=3)
350000
800,000
Negative ion
Warfarin m/z 309.20 > 163.05
400000
2.5ug/mL and 0.4ug/mL (n=3)
300000
500,000
250000
400,000
200000
300,000
100,000
0
150000
Linear regresson analysis
y = 246527x + 14796
R² = 0.9986
200,000
0
0.5
1
1.5
2
2.5
Linear regression analysis
y = 133197x + 15795
R² = 0.9954
100000
50000
3
0
3.5
Blood concentration ( ug/mL)
0
0.5
1
1.5
2
2.5
3
3.5
Blood concentration ( ug/mL)
Figure 4. In both ion modes, the calibration curve was linear over the therapeutic range studied for warfarin extracted from PSC’s (calibration range
0-3ug/mL, single point calibration standards at each level with the exception of replicate calibration points at 2.5ug/mL and 0.4ug/mL (n=3); r2>0.99 for
PSC analysis [r2>0.99 for a conventional plasma extraction]).
(x10,000)
1.75
1.50
1.25
(x10,000)
Matrix blank comparison
Positive ion
Plasma separation card matrix blank
Plasma matrix blank
1.75
1.50
1.25
Negative ion
Plasma separation card matrix blank
Plasma matrix blank
1.00
1.00
0.75
0.75
0.50
0.50
0.25
0.25
0.00
0.00
0.0
2.5
5.0
min
2.5
5.0
min
Figure 5. Matrix blank comparison. In both ion modes, the MRM chromatograms for PSC and plasma are comparable. Warfarin ion signals were not
detected in the any PSC or plasma matrix blank.
Plasma separation card comparison
The drive to work with smaller sample volumes offers
significant ethical and economical advantages in
pharmaceutical and clinical workflows and dried blood
spot sampling techniques have enabled a step change
approach for many toxicokinetic and pharmacokinetic
studies. However, the impressive growth of this technique
in the quantitative analysis of small molecules has also
discovered several limitations in the case of sample
instability (some enzyme labile compounds, particularly
prodrugs, analyte stability can be problematic), hematocrit
effect and background interferences of DBS. DBS also
shows noticeable effects on many lipids dependent on the
sample collection process. To compare PSC to plasma lipid
profiles the same blood sample extraction procedure
applied for warfarin analysis was measured by a high mass
accuracy system optimized for lipid profiling.
5
Monoacylglycerophosphoethanolamines
Monoacylglycerophosphocholines
Ceramide Diacylglycerophosphocholines phosphocholines
sample
Positive ion
LCMS-IT-TOF
Lipid profiling
7.5
10.0
12.5
15.0
17.5
20.0
22.5
Conventional plasma
sample
Positive ion
LCMS-IT-TOF
Lipid profiling
25.0
27.5
30.0 min
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0 min
Figure 6. Lipid profiles from the same human blood sample extracted using a plasma separation card (left hand profile) compared to a conventional
plasma samples (centrifugation). Both lipid profiles are comparable in terms of distribution and the number of lipids detected (the scaling has been
normalized to the most intense lipid signal).
Conclusions
• In this limited study, plasma separation card (PSC) sampling delivered a quantitative analysis of warfarin spiked into
human blood.
• PSC generated a linear calibration curve in both positive and negative ion modes (r2>0.99; n=5);
• The warfarin plasma results achieved by using the PSC technique were in broad agreement with conventional plasma
sampling data.
• The plasma generated by the filtration process appears broadly similar to plasma derived from conventional
centrifugation.
• Further work is required to consider the robustness and validation in a routine analysis.
References
• Jensen, B.P., Chin, P.K.L., Begg, E.J. (2011) Quantification of total and free concentrations of R- and S-warfarin in
human plasma by ultrafiltration and LC-MS/MS. Anal Bioanal Chem., 401, 2187-2193
• Radwan, M.A., Bawazeer, G.A., Aloudah, N.M., Aluadeib, B.T., Aboul-Enein, H.Y. (2012) Determination of free and total
warfarin concentrations in plasma using UPLC MS/MS and its application to patient samples. Biochemical
Chromatography, 26, 6-11
PO-CON1462E
Development and Validation
of Direct Analysis Method for Screening
and Quantitation of Amphetamines
in Urine by LC/MS/MS
ASMS 2014
MP535
Zhaoqi Zhan1, Zhe Sun1, Jie Xing1, Helmy Rabaha2
and Lim Swee Chin2
1
Shimadzu (Asia Pacific) Pte Ltd, Singapore, SINGAPORE;
2
Department of Scientific Services, Ministry of Health,
Brunei Darussalam
Development and Validation of Direct Analysis Method for
Screening and Quantitation of Amphetamines in Urine by LC/MS/MS
Introduction
Amphetamines are among the most commonly abused
drugs type worldwide. The conventional analytical
procedure of amphetamines in human urine in forensic
laboratory involves initial immunological screening
followed by GCMS confirmation and quantitation [1]. The
new guidelines of SAMHSA under U.S. Department of
Health and Human Services effective in Oct 2010 [2]
allowed use of LC/MS/MS for screening, confirmation and
quantitation of illicit drugs including amphetamines. One
of the advantages by using LC/MS/MS is that derivatization
of amphetamines before analysis is not needed, which was
a standard procedure of GCMS method. Since analysis
speed and throughput could be enhanced significantly,
development and use of LC/MS/MS methods are in
demand and many such efforts have been reported
recently [3]. The objective of this study is to develop a fast
LC/MS/MS method for direct analysis of amphetamines in
urine without sample pre-treatment (except dilution with
water) on LCMS-8040, a triple quadrupole system featured
as ultra fast mass spectrometry (UFMS). The compounds
studied include amphetamines (AMPH), methamphetamine
(MAMP) and three newly added MDMA, MDA and MDEA
by the new SAMHSA guidelines, four potential
interferences as well as PMPA as a control reference (Table
1). Very small injection volumes of 0.1uL to 1uL was
adopted in this study, which enabled the method suitable
for direct injection of untreated urine samples without
causing significant contamination to the ESI interface.
Experimental
The stock standard solutions of amphetamines and related
compounds as listed in Table 1 were prepared in the
Toxicology Laboratory in the Department of Scientific
Services (MOH, Brunei). Five urine specimens were
collected from healthy adult volunteers. The urine samples
used as blank and spiked samples were not pre-treated by
any means except dilution of 10 times with Milli-Q water.
An LCMS-8040 triple quadrupole coupled with a Nexera
UHPLC system (Shimadzu Corporation) was used. The
analytical column used was a Shim-pack XR-ODS III UHPLC
column (1.6 µm) 50mm x 2mm. The mobile phases used
were water (A) and MeOH (B), both with 0.1% formic acid.
A fast gradient elution program was developed for analysis
of the ten compounds: 0-1.6min, B=2%->14%;
1.8-2.3min, B=70%; 2.4min, B=2%; end at 4min. The
total flow rate was 0.6 mL/min. Positive ESI ionization
mode was applied with drying gas flow of 15 L/min,
nebulizing gas flow of 3 L/min, heating block temperature
of 400 ºC and DL temperature of 250 ºC. Various injection
volumes from 0.1 uL to 5 uL were tested to develop a
method with a lower injection volume to reduce
contamination of untreated urine samples to the interface.
Method development of direct injection of amphetamines in urine
MRM optimization of the ten compounds (Table 1) was
performed using an automated MRM optimization
program with LabSolutions workstation. Two MRM
transitions were selected for each compound, one for
quantitation and second one for confirmation (Table 1).
The ten compounds were separated and eluted in
0.75~2.2 minutes as sharp peaks as shown in Figure 1. In
addition to analysis speed and detection sensitivity, this
method development was also focused on evaluation of
small to ultra-small injection volumes to develop a method
suitable for direct injection of urine samples without any
pre-treatment while it should not cause significant
contamination to the interface. The Nexera SIL-30A
auto-sampler enables to inject as low as 0.10 uL of sample
with excellent precision.
Figure 1 shows a few selected results of direct injection of
urine blank (a) and mixed standards spiked in urine with 1
uL (c and d) and 0.1 uL (b) injection. It can be seen that all
compounds (12.5 ppb each in urine) could be detected
with 0.1uL injection except MDA and Norpseudo-E. With
1uL injection, all of them were detected.
2
Table 1: MRMs of amphetamines and related compounds
Cat.
Compound
B1
Abbr.
Nor pseudo-E
Nor pseudo ephedrine
B2
RT (min)
0.75
Ephe
Ephedrine
0.94
B3
Pseudo ephedrine
Pseudo-E
1.01
A1
Amphetamine
AMPH
1.20
A2
Methampheta-mine
MAMP
1.42
A3
3,4-methylenedi oxyamphetamine
A4
1.49
MDA
3,4-methylene dioxymeth amphetamine
1.59
MDMA A5
3,4-methylene dioxy-N-ethyl amphetamine
MDEA 1.94
B4
Phentermine
Phent 1.93
R
Propyl amphetamine
PAMP
2.20
2.5
min
0.0
0.0
(x100,000)
0.5
1.0
0.5
1.0
1.5
-23
166>148
-14
166>91
-31
166>148
-14
166>91
-30
136>91
-20
136>119
-14
150>91
-20
150>119
-14
180>163
-12
180>163
-38
194>163
-13
194>105
-22
208>163
-12
208>105
-24
150>91
-20
150>119
-40
178>91
-22
178>65
-47
PAMP
Phent
MDEA
2.5
min
0.5
2.5
min
0.0
0.0
0.5
1.0
1.5
2.5
min
PAMP
Phent
MDEA
1.0
2.0
2.0
(d) 62.5ppb in urine, 1uL inj
MAMP
PAMP
1.5
1.5
Phent
MDEA
AMPH
Ephedrine
Pseudo
Norpseudo
1.0
MAMP
MDA
MDMA
(c) 12.5ppb, 1uL inj
2.0
0.0
0.0
152>115
(x1,000,000)
MDA
MDMA
2.0
AMPH
1.5
Ephedrine
Pseudo
1.0
Norpseudo
0.5
MDMA
1.0
MAMP
MDA
1.0
AMPH
2.0
Norpseudo
2.0
3.0
-13
(b) 12.5ppb in urine, 0.1uL inj
3.0
Ephedrine
Pseudo
(a) Urine blank, 1 uL inj
0.0
0.0
CE (V)
(x10,000)
(x10,000)
3.0
MRM
152>134
2.0
Figure 1: MRM chromatograms of urine blank (a) and spiked samples of amphetamines and related
compounds in urine by LC/MS/MS method with 1uL and 0.1uL injection volumes.
3
Calibration curves with small and ultra-small injection volumes
Linear calibration curves were established for the ten
compounds spiked in urine with different injection
volumes: 0.1, 0.2, 0.5, 1, 2 and 5 uL. Good linearity of
calibration curves (R2>0.999) were obtained for all
injection volumes including 0.1uL, an ultra-small injection
Area (x100,000)
7.5
Area (x1,000,000)
1.25
AMPH
5.0
Area (x100,000)
5.0
MAMP
1.00
volume. The calibration curves with 0.1 uL injection volume
are shown in Figure 2. The linearity (r2) of all compounds
with 0.1 uL and 1 uL injection volumes are equivalently
good as shown in Table 2.
Area (x100,000)
Area (x100,000)
MDA
7.5
MDMA
5.0
0.75
5.0
2.5
0.50
2.5
2.5
2.5
0.25
0.0
0
250
Conc.
Area (x100,000)
3.0
0.00
0
250
Conc.
Area (x100,000)
Nor pseudo-E
0.0
0
Conc.
Area (x100,000)
Ephedrine
5.0
250
0.0
0.0
0
250
Conc.
Pseudo-E
2.5
1.0
0
250
Conc.
0.0
0
250
Conc.
0.0
1.0
2.5
0
250
Conc.
0.0
Conc.
1.5
5.0
2.5
250
PAMP
Phent
7.5
5.0
0
Area (x1,000,000)
Area (x100,000)
2.0
0.0
MDEA
0.5
0
250
Conc.
0.0
0
250
Conc.
Figure 2: Calibration Curves of amphetamines spiked in urine with 0.1uL injection
Performance validation
Repeatability of peak area was evaluated with a same
loading amount (6.25 pg) but with different injection
volumes. The RSD shown in Table 2 were 1.6% ~ 7.9%
and 1.6 ~ 7.8% for 0.1uL and 1uL injection, respectively. It
is worth to note that the repeatability of every compounds
with of 0.1uL injection is closed to that of 1uL injection as
well as 5uL injection (data not shown).
Matrix effect of the method was determined by
comparison of peak areas of mixed standards in pure water
and in urine matrix. The results of 62.5ppb with 1uL
injection were at 102-115% except norpseudoephedrine
(79%) as shown in Table 2.
Accuracy and sensitivity of the method were evaluated
with spiked samples of low concentrations. The results of
LOD and LOQ of the ten compounds in urine are shown in
Table 3. Since the working samples (blank and spiked)
were diluted for 10 times with water before injection, the
concentrations and LOD/LOQ of the method described
above for source urine samples have to multiply a factor of
10. Therefore, the LOQs of the method for urine specimens
are at 2.1-17.1 ng/mL for AMPH, PAMP, MDMA and
MDEA and 53 ng/mL for MDA. The LOQs for the potential
interferences (Phentermine, Ephedrine, Pseudo-Ephedrine
and Norpseudo-Ephedrine) are at 17-91 ng/mL, 2.4 ng/mL
for the internal reference MAMP. The sensitivity of the
direct injection LC/MS/MS method are significantly higher
than the confirmation cutoff (250 ng/mL) required by the
SAMHSA guidelines.
4
Table 2: Method Performance with different inj. volumes
Name
Calibration curve, R2
RSD% area (n=6)
M.E. %1
(ppb)2
(0.1uL)
(1uL)
(0.1uL)
(1uL)
(1uL)
Norpseudo-E
1-500
0.9992
0.9996
4.5
5.7
79
Ephe
2.5-500
0.9995
0.9998
3.2
2.9
115
Pseudo-E
1-500
0.9994
0.9986
3.7
3.3
113
AMPH
1-500
0.9997
0.9998
3.5
2.4
102
MAMP
1-500
0.9998
0.9999
1.6
2.3
110
MDA
2.5-500
0.9978
0.9995
7.9
7.8
103
MDMA
1-500
0.9993
0.9998
1.8
4.5
115
MDEA
1-500
0.9996
0.9998
3.5
2.9
115
Phent
2.5-500
0.9998
0.9998
4.1
1.6
106
PAMP
1-500
0.9998
0.9932
2.9
2.0
102
1: Measured with mixed stds of 62.5 ppb in clear solution and spiked in urine
2: For 0.1uL injection, the lowest conc. is 2.5 or 12.5 ppb
Table 3: Method performance: sensitivity & accuracy (1uL)
Name
Conc. (ppb)
Accuracy
Sensitivity (ppb)
Prep.
Meas.
(%)
S/N
LOD
LOQ
Norpseudo-E
1.0
1.2
118.7
2.3
1.53
5.09
Ephe
2.5
2.2
88.2
2.7
2.41
8.04
Pseudo-E
1.0
1.0
99.5
5.9
0.50
1.67
AMPH
1.0
1.1
114.1
6.7
0.51
1.71
MAMP
1.0
1.0
103.6
21.8
0.14
0.47
MDA
2.5
2.4
96.3
4.5
1.60
5.34
MDMA
1.0
1.1
106.4
51.9
0.06
0.21
MDEA
1.0
1.1
111.8
28.5
0.12
0.39
Phent
2.5
2.6
105.3
2.9
2.73
9.10
PAMP
1.0
1.0
101.7
42.2
0.07
0.24
Method operational stability
The method operational stability with 1uL injection was
tested with spiked samples of 25 ppb in five urine
specimens, corresponding to 250 ng/mL in the source urine
samples. Continuous injections of accumulated 120 times
was carried out in about 10 hours. The purpose of the
experiment was to evaluate the operational stability against
the ESI source contamination by urine samples without
pre-treatment. Figure 3 shows the first injection and the
120th injection of the same spiked sample (S1) as well as
other spiked samples (S2, S3, S4 and S5) in between.
Decrease in peak areas of the compounds occurred, but the
degree of the decrease in average was about 17% from the
first injection to the last injection. This result indicates that it
is possible to carry out direct analysis of urine samples (10
times dilution with water) by the high sensitivity LC/MS/MS
method with a very small injection volume.
5
(x100,000)
0.0
0.0
2.0
min
0.0
0.0
1.0
2.0
min
0.0
PAMP
Phent
MDEA
2.0
min
1.0
PAMP
S1 (110th inj)
Phent
MDEA
MDA
MDMA
MAMP
5.0
MAMP
Phent
MDEA
MAMP
AMPH
MDA
MDMA
5.0
2.5
1.0
(x100,000)
S5 (41st inj)
Norpseudo
0.0
Norpseudo
Ephedrine
Pseudo
5.0
2.5
1.0
(x100,000)
PAMP
(x100,000)
S4 (31st inj)
MAMP
0.0
0.0
PAMP
min
2.5
0.0
2.0
min
0.0
Phent
MDEA
2.0
MDA
MDMA
1.0
Ephedrine
Pseudo
AMPH
0.0
AMPH
2.5
MDA
MDMA
PAMP
Phent
MDEA
5.0
Norpseudo
Ephedrine
Pseudo
AMPH
0.0
S3 (21st inj)
7.5
Norpseudo
Ephedrine
Pseudo
2.5
AMPH
5.0
Norpseudo
Ephedrine
Pseudo
2.5
S2 (11 inj)
7.5
Phent
MDEA
Norpseudo
Ephedrine
Pseudo
AMPH
5.0
MDA
MDMA
MAMP
7.5
(x100,000)
th
MAMP
S1 (1 inj)
st
MDA
MDMA
PAMP
(x100,000)
1.0
2.0
min
Figure 3: Selected chromatograms of continuous injections of spiked samples (25 ppb) with 1 µL injection.
Five urine specimens S1, S2, S3, S4 and S5 were used to prepare these spiked samples.
Conclusions
In this study, we developed a fast LC/MS/MS method for
direct analysis of five amphetamines and related
compounds in human urine for screening and quantitative
confirmation. Very small injection volumes of 0.1~1.0 uL
were adopted to minimize ESI contamination and enhance
operational stability. The good performance results
observed reveals that screening and confirmation of
amphetamines in human urine by direct injection to
LC/MS/MS is possible and the method could be an
alternative choice in forensic and toxicology analysis.
References
1. Kudo K, Ishida T, Hara K, Kashimura S, Tsuji A, Ikeda N, J Chromatogr B, 2007, 855, 115-120.
2. Mandatory guidelines for Federal Workplace Drug Testing Program, 73 FR 71858-71907, Nov. 25, 2008.
3. Huei-Ru Lina, Ka-Ian Choia, Tzu-Chieh Linc, Anren Hu,, Journal of Chromatogr B, 2013, 929, 133–141.
PO-CON1482E
Next generation plasma collection
technology for the clinical analysis of
temozolomide by HILIC/MS/MS
ASMS 2014
WP641
Alan J. Barnes1, Carrie-Anne Mellor2,
Adam McMahon2, Neil Loftus1
1
Shimadzu, Manchester, UK
2
2WMIC, University of Manchester, UK
for the clinical analysis of temozolomide by HILIC/MS/MS
Introduction
Plasma extraction technology is a novel technique achieved
by applying a blood sample to a laminated membrane
stack which allows plasma to flow through the asymmetric
filter whilst retaining the cellular components of the blood
sample.
Plasma separation card technology was applied to the
quantitative analysis of temozolomide (TMZ); an oral
imidazotetrazine alkylating agent used for the treatment of
Grade IV astrocytoma, an aggressive form of brain tumour.
Under physiological conditions TMZ is rapidly converted to
5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC)
which in-turn degrades by hydrolysis to
5-aminoimidazole-4-carboxamide (AIC). Storage of plasma
has previously shown that both at -70C and 4C
degradation still occurs. In these experiments, whole blood
containing TMZ standard was applied to NoviPlex plasma
separation cards (PSC). The aim was to develop a robust
LC/MS/MS quantitative method for TMZ.
Plasma separation
TMZ spiked human blood calibration standards (50uL) were applied to the PSC as described below in figure 1.
1
2
3
4
The collection disc is
removed from the
card and is ready for
extraction for
LC-MS/MS analysis.
A NoviPlex card is
removed from foil
packaging.
Approximately 50uL
of whole blood is
added to the test
area.
After 3 minutes, the
top layer is completely
removed (peeled
back).
The collection disc
contains 2.5uL of
plasma. Card is air
dried for 15 minutes.
Figure 1. Noviplex plasma separation card workflow
2
Spreading Layer
[Lateral spreading layer rapidly spreads blood so it will
enter the filtration layer as a front while adding buffers and
anticoagulants. The lateral spreading rate is 150um/sec].
Control Spot:
[Determines whether enough
blood was placed on the card].
Filtration Layer
[Filtration layer captures blood
cells by a combination of filtration
and adsorption. The average
linear vertical migration rate is
approximately 1um/sec].
Isolation Screen
[Precludes lateral wicking along the
card surface].
Collection Layer
[Loads with a specific aliquot of plasma onto a 6.35mm disc]. Although flow through
the filtration membrane is unlikely to be constant throughout the plasma extraction
process, the average loading rate of the Collection Disc was 13 nL/sec. This
corresponds to a volumetric flow rate into the Collection Disc of 400 pL/mm2/sec.
Figure 1. Noviplex plasma separation card workflow (Cont'd)
Figure 2. Applying a blood sample, either as a finger prick or by accurately measuring the blood volume, to the laminated membrane stack retains red cells and
allows a plasma sample to be collected. The red cells are retained by a combination of adsorption and filtration whilst plasma advances through the membrane stack
by capillary action. After approximately three minutes the plasma Collection Disc was saturated with an aliquot of plasma and was ready for LC/MS/MS analysis.
Sample preparation
TMZ was extracted from the dried plasma collection discs
by adding 40uL acetonitrile + 0.1% formic acid, followed
by centrifugation 16,000g for 5 min. 30uL supernatant
was added directly to the LC/MS/MS sample vial for
analysis.
As a control, conventional plasma samples were prepared
by centrifuging the human blood calibration standards at
1000g for 10min. TMZ was extracted from 2.5uL of plasma
using the same extraction protocol as applied for PSC.
3
LC/MS/MS analysis
Ionisation
: Electrospray, positive mode
MRM 195.05 >138.05 CE -10
Desolvation line
Drying/Nebulising gas
Heating block
: 300ºC
: 10L/min, 2L/min
: 400ºC
HPLC
: HILIC
Nexera UHPLC system
: 0.5mL/min (0-7min), 1.8mL/min (7.5min-17.5min)
: A= Water + 0.1% formic acid
B= Acetonitrile + 0.1% formic acid
: 95% B – 30%% B (6.5 min),
30% B (7.5 min), 95% B (18 min)
: ZIC HILIC 150 x 4.6mm 5um 200ª
: 40ºC
: 10uL
Flow rate
Mobile phase
Gradient
Analytical column
Column temperature
Injection volume
Reverse Phase
Nexera UHPLC system
0.4mL/min
A= Water + 0.1% formic acid
B= methanol + 0.1% formic acid
5% B – 100%% B (3 min),
100% B (7 min), 5% B (10 min)
Phenomenex Kinetex XB C18 100 x 2.1mm 1.7um 100A
50ºC
2µL
Results
HILIC LC/MS/MS
Temozolomide is known to be unstable under physiological
conditions and is converted to
5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) by
(x10,000)
5.0
4.0
a nonenzymatic, chemical degradation process. Previous
studies have used HILIC to analyze the polar compound
and to avoid degradation in aqueous solutions.
Peak Area
HILIC analysis
TMZ m/z 195.05> 138.05
HILIC analysis
TMZ
700000
600000
Q1 (V) -20
Q3 (V) -12
Single point calibration standards
Calibration curve 0.2-10ug/mL
500000
3.0
400000
2.0
300000
200000
8.0ug/mL calibn std
1.0
0.0
0
0.0
2.5
y = 64578x + 18473
R² = 0.9988
100000
0.5ug/mL calibn std
5.0
min
0
2
4
6
8
10
12
Blood Concentration (ug/mL)
Figure 3. HILIC LC/MS/MS chromatograms for PSC TMZ analysis at 0.5 and 8ug/mL. The PSC calibration curve was linear between 0.2-10ug/mL (r2>0.99).
HILIC was considered in response to previous published data and to minimize potential stability issues. However, to reduce sample cycle times a reverse
phase method was also developed.
4
Reversed Phase LC/MS/MS
(x10,000)
9.0
RP analysis
TMZ m/z 195.05 > 138.05
8.0
800,000
Q1 (V) -20
Q3 (V) -12
7.0
6.0
RP analysis
TMZ calibration curve
Peak Area
0.5ug/mL and 8ug/mL (n=3)
700,000
600,000
500,000
5.0
8.0ug/mL
4.0
400,000
3.0
300,000
0.5ug/mL
2.0
y = 72219x - 355.54
R² = 0.9997
200,000
1.0
100,000
0.0
0
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 min
2
4
6
8
10
12
Blood Concentration (ug/mL)
Figure 4. Reverse phase LC/MS/MS chromatograms for PSC TMZ analysis at 0.5 and 8ug/mL. The PSC calibration curve was linear between
0.2-10ug/mL (r2>0.99; replicate samples were included in the liner regression analysis at 0.5 and 8ug/mL; n=3).
Due to the relatively long cycle time (18 min), a faster
reversed phase method was developed (10 min). Sample
preparation was modified with PSC sample disk placed in
40uL methanol + 0.1% formic acid, followed by
centrifugation 16,000g 5 min. 20uL supernatant was
added directly to the LC/MS sample vial plus 80uL water +
0.1% formic acid. In addition to reversed phase being
faster, the sample injection volume was reduced to just 2uL
as a result of higher sensitivity from narrower peak width
(reversed phase,13 sec; HILIC, 42 sec).
Comparison between PSC and plasma
MRM 195.05>67.05
(x100)
4.0
matrix blank
3.5
500ng/mL comparison
MRM 195.05>67.05
(x1,000)
1.50
500ng/mL calibration standard
1.25
Plasma
matrix blank
3.0
Plasma
1.00
2.5
0.75
2.0
1.5
0.50
1.0
0.25
0.5
0.00
0.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
min
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
min
Figure 5. Human blood TMZ calibration standards were prepared using PSC and conventional plasma. Using the confirmatory ion transition
195.05>67.05 both the PSC and plasma sample are in broad agreement with regard to matrix ion signal distribution.
5
(x10,000)
MRM 195.05>138.05
Matrix peak
matrix blank
1.50
(x10,000)
Matrix peak 500ng/mL comparison
MRM 195.05>138.05
1.50
1.25
1.25
Plasma
Plasma
matrix blank
1.00
1.00
TMZ
0.75
0.75
0.50
0.50
TMZ
Rt
1.7mins
0.25
0.25
0.00
0.00
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
min
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
min
Figure 6. Human blood TMZ calibration standards were prepared using PSC and conventional plasma. Using the quantitation ion transition
195.05>138.05 both the PSC and plasma sample are in broad agreement in signal distribution and intensity including the presence of
a matrix peak at 2.4mins.
Conclusions
This technology has the potential for a simplified clinical sample collection by the finger prick approach, with future work
aimed to evaluate long term sample stability of PSC samples, stored at room temperature. Quantitation of drug
metabolites MTIC and AIC also could help provide a measure of sample stability.
References
• Andrasia, M., Bustosb, R., Gaspara,A., Gomezb, F.A. & Kleknerc, A. (2010) Analysis and stability study of
temozolomide using capillary electrophoresis. Journal of Chromatography B. Vol. 878, p1801-1808
• Denny, B.J., Wheelhouse, R.T., Stevens, M.F.G., Tsang, L.L.H., Slack, J.A., (1994) NMR and molecular modeliing
investigation of the mechanism of activation of the antitumour drug temozolomide and its Interaction with
DNA. Biochemistry, Vol. 33, p9045-9051
PO-CON1475E
Application of a Sensitive Liquid
Chromatography-Tandem Mass Spectrometric
Method to Pharmacokinetic Study of
Telbivudine in Humans
ASMS 2014
WP 629
Bicui Chen1, Bin Wang1, Xiaojin Shi1, Yuling Song2,
Jinting Yao2, Taohong Huang2, Shin-ichi Kawano2,
Yuki Hashi2
1 Pharmacy Department, Huashan Hospital,
Fudan University,
2 Shimadzu Global COE, Shimadzu (China) Co., Ltd.
Application of a Sensitive Liquid Chromatography-Tandem
Mass Spectrometric Method to Pharmacokinetic Study of
Introduction
Telbivudine is a synthetic L-nucleoside analogue, which is
phosphorylated to its active metabolite, 5’-triphosphate, by
cellular kinases. The telbivudine 5’-triphosphate inhibits
HBV DNA polymerase (a reverse transcriptase) by
competing with the natural substrate, dTTP. Incorporation
of 5’-triphosphorylated-telbivudine into viral DNA obligates
DNA chain termination, resulting in inhibition of HBV
replication. The objectives of the current studies were to
develop a selective and sensitive LC-MS/MS method to
determine of telbivudine in human plasma.
Method
Sample Preparation
(1) Add 100 μL of plasma into the polypropylene tube, add 40 μL of internal standard working solution (33 µg/mL, with
thymidine phosphorylase) to all other tubes.
(2) Incubate the tubes for 1 h at 37 ºC in dark.
(3) Add 200 μL of acetonitrile to all tubes, seal and vortex for 1 minutes.
(4) Centrifuge the tubes for 5 minutes at 13000 rpm.
(5) Transfer 200 μL supernatant to a clean glass bottle and inject into the HPLC-MS/MS system.
LC-MS/MS Analysis
The analysis was performed on a Shimadzu Nexera UHPLC
instrument (Kyoto, Japan) equipped with LC-30AD pumps,
CTO-30A column oven, DGU-30A5 on-line egasser, and
SIL-30AC autosampler. The separation was carried out on
GL Sciences InertSustain C18 column (3.0 mmI.D. x 100
mmL.) with the column temperature at 40 ºC. A triple
quadruple mass spectrometer (Shimadzu LCMS-8050,
Kyoto, Japan) was connected to the UHPLC instrument via
an ESI interface.
HPLC (Nexera UHPLC system)
Column
Mobile Phase A
Mobile Phase B
Gradient Program
Flow Rate
Column Temperature
Injection Volume
:
:
:
:
:
:
:
InertSustain (3.0 mmI.D. x 100 mmL., 2 μm, GL Sciences)
water with 0.1% formic acid
acetonitrile
as shown in Table 1
0.4 mL/min
40 ºC
2 µL
Table 1 Time Program
Time (min)
Module
Command
Value
0.00
Pumps
Pump B Conc.
5
4.00
Pumps
Pump B Conc.
80
4.10
Pumps
Pump B Conc.
5
6.00
Controller
Stop
2
MS (LCMS-8050 triple quadrupole mass spectrometer)
Ionization
Polarity
Ionization Voltage
Nebulizing Gas Flow
Heating Gas Flow
Drying Gas Flow
Interface Temperature
Heat Block Temperature
DL Temperature
Mode
:
:
:
:
:
:
:
:
:
:
ESI
Positive
+0.5 kV (ESI-Positive mode)
3.0 L/min
8.0 L/min
12.0 L/min
250 ºC
300 ºC
350 ºC
MRM
Table 2 MRM Parameters
Compound
Precursor
m/z
Product
m/z
Dwell Time
(ms)
Q1 Pre Bias
(V)
CE (V)
Q3 Pre Bias
(V)
Telbivudine
243.10
127.10
100
-26
-10
-13
Telbivudine-D3
246.10
130.10
100
-16
-9
-25
Human plasma samples containing telbivudine ranging
from 1.0 to 10000 ng/mL were prepared and extracted
by protein precipitation and the final extracts were
analyzed by LC-MS/MS. MRM chromatograms of
telbivudine (1 ng/mL) and deuterated internal standard
are presented in Fig. 1 (blank) and Fig. 2 (spiked). The
linear regression for telbivudine was found to be
>0.9999. The calibration curve with human plasma as
the matrix were shown in Fig. 3. Excellent precision and
accuracy were maintained for four orders of magnitude,
demonstrating a linear dynamic range suitable for
real-world applications. LLOQ for telbivudine was 1.0
ng/mL, which met the criteria for bias (%) and precision
within ±15% both within run and between run. The
intra-day and inter-day precision and accuracy of the
assay were investigated by analyzing QC samples.
Intra-day precision (%RSD) at three concentration levels
(3, 30, and 8000 ng/mL) were below 2.5% and inter-day
precision (%RSD) was below 2.5%. The recoveries of
telbivudine were 100.6±2.5 %, 104.5±1.5% and
104.3±1.6% at three concentration levels, respectively.
The stability data showed that the processed samples
were stable at the room temperature for 8 h, and there
was no significant degradation during the three
freeze/thaw cycles at -20 ºC. The reinjection
reproducibility results indicated that the extracted
samples could be stable for 72 h at 10 ºC.
3
(x100)
4.0 1:Telbivudine 243.10>127.10(+) CE: -10.0
4.0
3.0
(x1,000)
2:Telbivudine-D3 246.10>130.10(+) CE: -9.0
3.0
2.0
2.0
1.0
1.0
0.0
0.0
0.0
1.0
2.0
3.0
4.0
5.0
min
1.0
2.0
3.0
4.0
5.0
min
5.0
min
Figure 1 Representative MRM chromatograms of blank human plasma
(left: transition for telbivudine, right: transition for internal standard)
(x100)
1:Telbivudine 243.10>127.10(+) CE: -10.0
7.5
Telbivudine-D3
Telbivudine
(x1,000,000)
1.50 2:Telbivudine-D3 246.10>130.10(+) CE: -9.0
1.25
1.00
5.0
0.75
0.50
2.5
0.25
0.0
0.0
0.00
1.0
2.0
3.0
4.0
5.0
min
1.0
2.0
3.0
4.0
Figure 2 Representative MRM chromatograms of telbivudine (left, 1 ng/mL) and internal standard (right) in human plasma
Area Ratio
2.5
2.0
1.5
1.0
0.5
0.0
0
2500
5000
7500
Conc. Ratio
Figure 3 Calibration curve of telbivudine in human plasma
4
Compound
Calibration
Curve
Linear Range
(ng/mL)
Accuracy
(%)
r
Telbivudine
Y = (2.77×10-4)X + (3.39×10-5)
1~10000
93.1~116.6%
0.9998
Table 3 Accuracy and precision for the analysis of amlodipine in human plasma
(in pre-study validation, n=3 days, six replicates per day)
Added Conc.
(ng/mL)
Intra-day Precision
(%RSD)
Inter-day Precision
(%RSD)
Accuracy
(%)
3
2.18
2.11
107.7~114.4
400
1.52
1.58
91.6~95.9
8000
1.76
1.68
95.4~101.3
Table 4 Recovery for QC samples (n=6)
QC Level
Concentartion
(ng/mL)
Recovery
(%)
LQC
3
100.6
MQC
400
104.5
HQC
8000
104.3
Table 5 Matrix effect for QC samples (n=6)
3.0
QC Level
Added Conc.
(ng/mL)
Matrix Factor
IS-Normalized
Matrix Factor
LQC
3
82.3%
99.0%
MQC
400
81.7%
101.0%
HQC
8000
90.8%
101.5%
(x10,000)
1:Telbivudine 243.10>127.10(+) CE: -10.0
(x1,000,000)
2:Telbivudine-D3 246.10>130.10(+) CE: -9.0
1.00
0.75
2.0
0.50
1.0
0.25
0.0
0.0
0.00
1.0
2.0
3.0
4.0
5.0
min
1.0
2.0
3.0
4.0
5.0
min
Figure 4 Representative MRM chromatograms of real-world sample
5
Conclusion
Results of parameters for method validation such as dynamic range, linearity, LLOQ, intra-day precision, inter-day
precision, recoveries, and matrix effect factors were excellent. The sensitive LC-MS/MS technique provides a powerful
tool for the high-throughput and highly selective analysis of telbivudine in clinical trial study.
PO-CON1449E
Accelerated and robust monitoring
for immunosuppressants using triple
quadrupole mass spectrometry
ASMS 2014
WP628
Natsuyo Asano1, Tairo Ogura1, Kiyomi Arakawa1
1 Shimadzu Corporation. 1, Nishinokyo Kuwabara-cho,
Nakagyo-ku, Kyoto 604–8511, Japan
Accelerated and robust monitoring for immunosuppressants
using triple quadrupole mass spectrometry
Introduction
Immunosuppressants are drugs which lower or suppress
activity of the immune system. They are used to prevent
the rejection after transplantation or treat autoimmune
disease. To avoid immunodeficiency as adverse effect, it is
recommended to monitor blood level of therapeutic drug
with high throughput and high reliability. There are several
analytical technique to monitor drugs, LC/MS is superior in
terms of cross-reactivity at low level and throughput of
HO
analysis. Therefore, it is important to analyze these drugs in
blood by using ultra-fast mass spectrometer to accelerate
monitoring with high quantitativity. We have developed
analytical method for four immunosuppressants
(Tacrolimus, Rapamycin, Everolimus and Cyclosporin A)
with two internal standards (Ascomycin and Cyclosporin D)
using ultra-fast mass spectrometer.
O
HO
O
HO
O
O
O
O
N
OH
O
O
O
O
O
HO
O
HO
O
H
O
O
N
O
OH
O
O
O
N
N
H
O
O
O
O
Rapamycin
Everolimus
MW: 804.02
MW: 914.17
MW: 958.22
N
O
OH
Tacrolimus
OH
O
O
HO
O
O
O
N
O
O
O
O
N
HO
H H
N
N
O
N
O
H
N
N
N
HO
H
O
N
H
N
HN
O
O
H
N
O
O
N
O
HO
H
O
O
O
N
O
N
H
N
O
O
N
O
O
O
OH
N
O
O
O
O
O
O
O
O
O
N
N
H
N
O
O
Cyclosporin A
Ascomycin (IS)
Cyclosporin D (IS)
MW: 1202.61
MW: 792.01
MW: 1216.64
Figure 1 Structure of immunosuppressants and internal standards (IS)
2
Standard samples of each compound were analyzed to optimize conditions of liquid chromatograph and mass
spectrometer. Whole blood extract was prepared based on liquid-liquid extraction described bellow.
2.7 mL of Whole blood and 20.8 mL of Water
↓
Vortex for 15 seconds
↓
Add 36 mL of MTBE/Cyclohexane (1:3)
↓
Vortex for 15 seconds and Centrifuge with 3000 rpm at 20 ºC for 10 minutes
↓
Extract an Organic phase
↓
Evaporate and Dry under a Nitrogen gas stream
↓
Redissolve in 1.8 mL of 80 % Methanol solution with 1 mmol/L Ammonium acetate
↓
Vortex for 1 minute and Centrifuge with 3000 rpm at 4 ºC for 5 minutes
↓
Filtrate and Transfer into 1 mL glass vial
Table 1 Analytical conditions
UHPLC
Liquid Chromatograph
Analysis Column
Mobile Phase A
Mobile Phase B
Gradient Program
Flow Rate
Column Temperature
Injection Volume
:
:
:
:
:
Nexera (Shimadzu, Japan)
YMC-Triart C18 (30 mmL. × 2 mmI.D.,1.9 μm)
1 mmol/L Ammonium acetate - Water
1 mmol/L Ammonium acetate - Methanol
60 % B. (0 min) – 75 % B. (0.10 min) – 95 % B. (0.70 – 0.90 min) –
60 % B. (0.91 – 1.80 min)
: 0.45 mL/min
: 65 ºC
: 1.5 µL
MS
MS Spectrometer
Ionization
Probe Voltage
Nebulizing Gas Flow
Drying Gas Flow
Heating Gas Flow
DL Temperature
HB Temperature
:
:
:
:
:
:
:
:
:
LCMS-8050 (Shimadzu, Japan)
ESI (negative)
-4.5 ~ -3 kV
3.0 L/min
5.0 L/min
15.0 L/min
400 ºC
150 ºC
390 ºC
3
Result
Immunosuppressants, which we have developed a method
for monitoring of, has been often observed as ammonium
or sodium adduct ion by using positive ionization. In
general, protonated molecule (for positive) or
deprotonated molecule (for negative) is more preferable
for reliable quantitation than adduct ions such as
ammonium, sodium, and potassium adduct. In this study,
each compound was detected as deprotonated molecule in
negative mode by using heated ESI source of LCMS-8050
(Table 2).
The separation of all compounds was achieved within 1.8
min, with a YMC-Triart C18 column (30 mmL. × 2
mmI.D.,1.9 μm) and at 65 ºC of column oven temperature.
(x100,000)
5
1.4
1.2
6
1.0
0.8
0.6
4
0.4
3
0.2
2
1
0.0
0.75
1.25
1.00
min
Figure 2 MRM chromatograms of immnosuppresants in human whole blood (50 ng/mL)
Table 2 MRM transitions
Peak No.
Compound
Porality
Precursor ion (m/z)
Product ion (m/z)
1
Ascomysin (IS)
neg
790.40
548.20
2
Tacrolimus
neg
802.70
560.50
3
Rapamycin
neg
912.70
321.20
4
Everolimus
neg
956.80
365.35
5
Cyclosporin A
neg
1200.90
1088.70
6
Cyclosporin D (IS)
neg
1215.10
1102.60
4
a) Tacrolimus
0.5 ng/mL
Ascomycin
40 ng/mL
0.5 – 1000 ng/mL
b) Rapamycin
0.5 ng/mL
Ascomycin
40 ng/mL
0.5 – 500 ng/mL
c) Everolimus
0.5 ng/mL
Ascomycin
40 ng/mL
0.5 – 100 ng/mL
d) Cyclosporin A
0.5 ng/mL
Cyclosporin D
100 ng/mL
0.5 – 1000 ng/mL
Figure 3 MRM chromatograms at LLOQ and ISTD (left), and calibration curves (right) for four immnosuppresants in human whole blood
5
Figure 3 illustrates both a calibration curve and chromatogram at the lowest calibration level for all immunosuppressants
analyzed. Table 3 lists both the reproducibility and accuracy for each immunosuppressant that has been simultaneously
measured in 1.8 minutes.
Table 3 Reproducibility and Accuracy
Compound
Concentration
CV % (n = 6)
Accuracy %
Tacrolimus
Low (0.5 ng/mL)
Low-Mid (2 ng/mL)
High (1000 ng/mL)
18.0
13.0
2.87
99.4
99.5
88.7
Rapamycin
Low (0.5 ng/mL)
Low-Mid (5 ng/mL)
High (500 ng/mL)
6.87
2.88
3.41
95.6
109.3
90.0
Everolimus
Low (0.5 ng/mL)
Low-Mid (5 ng/mL)
High (100 ng/mL)
10.4
5.11
2.26
95.3
104.4
93.3
Cyclosporin A
Low (0.5 ng/mL)
Low-Mid (10 ng/mL)
High (1000 ng/mL)
7.31
2.36
2.67
95.1
99.9
94.9
In high speed measurement condition, we have achieved high sensitivity and wide dynamic range for all analytes.
Additionally, the accuracy of each analyte ranged from 88 to 110 % and area reproducibility at the lowest calibration level
of each analyte was less than 20%. Conclusions
• Monitoring with negative mode ionization permitted more sensitive, robust and reliable quantitation for four
immunosuppressants.
• A total of six compounds were measured in 1.8 minutes. The combination of Nexera and LCMS-8050 provided a faster
run time without sacrificing the quality of results.
• Even with a low injection volume of 1.5 μL, the lower limit of quantitation (LLOQ) for all compounds was 0.5 ng/mL.
• In this study, it is demonstrated that LCMS-8050 is useful for the rugged and rapid quantitation for immunosuppressants
in whole blood.
Acknowledgement
We appreciate suggestions from Prof. Kazuo Matsubara and Assoc. Prof. Ikuko Yano from the department of pharmacy,
Kyoto University Hospital, and Prof. Satohiro Masuda from the department of pharmacy, Kyusyu University Hospital.
PO-CON1468E
Highly sensitive quantitative analysis
of Felodipine and Hydrochlorothiazide
from plasma using LC/MS/MS
ASMS 2014
TP497
Shailendra Rane, Rashi Kochhar, Deepti Bhandarkar,
Shruti Raju, Shailesh Damale, Ajit Datar,
Pratap Rasam, Jitendra Kelkar
Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh
Chambers, Makwana Road, Marol, Andheri (E),
Mumbai-400059, Maharashtra, India.
Highly sensitive quantitative analysis of Felodipine
and Hydrochlorothiazide from plasma using LC/MS/MS
Introduction
Felodipine is a calcium antagonist (calcium channel
blocker), used as a drug to control hypertension[1].
Hydrochlorothiazide is a diuretic drug of the thiazide class
that acts by inhibiting the kidney’s ability to retain water. It
is, therefore, frequently used for the treatment of
hypertension, congestive heart failure, symptomatic
edema, diabetes insipidus, renal tubular acidosis and the
prevention of kidney stones[2].
Efforts have been made here to develop high sensitive
methods of quantitation for these two drugs using
LCMS-8050 system from Shimadzu Corporation, Japan.
Presence of heated Electro Spray Ionization (ESI) probe in
LCMS-8050 ensured good quantitation and repeatability
even in the presence of a complex matrix like plasma. Ultra
high sensitivity of LCMS-8050 enabled development
quantitation method at low ppt level for both Felodipine
and Hydrochlorthiazide.
Felodipine
Felodipine is a calcium antagonist (calcium channel
blocker). Felodipine is a dihydropyridine derivative that is
chemically described as ± ethyl methyl
4-(2,3-dichlorophenyl)1,4-dihydro-2,6-dimethyl-3,5-pyridin
edicarboxylate. Its empirical formula is C18H19Cl2NO4 and its
structure is shown in Figure 1.
Figure 1. Structure of Felodipine
Hydrochlorothiazide
Figure 2. Structure of Hydrochlorothiazide
Hydrochlorothiazide, abbreviated HCTZ (or HCT, HZT), is a
diuretic drug of the thiazide class that acts by inhibiting the
kidney‘s ability to retain water.
Hydrochlorothiazide is
6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine7-sulfonamide.Its empirical formula is C7H8ClN3O4S2 and its
structure is shown in Figure 2.
Method of Analysis
Preparation of matrix matched plasma by protein precipitation method
using cold acetonitrile
To 100 µL of plasma, 500 µL of cold acetonitrile was added
for protein precipitation then put in rotary shaker at 20
rpm for 15 minutes for uniform mixing. It was centrifuged
at 12000 rpm for 15 minutes. Supernatant was collected
and evaporated to dryness at 70 ºC and finally
reconstituted in 200 µL Methanol.
2
Preparation of matrix matched plasma by liquid-liquid extraction method
using diethyl ether and hexane mixture (1:1 v/v)
To 500 µL plasma, 100 µL sodium carbonate (1.00 mol/L)
and 5 mL of diethyl ether : hexane (1:1 v/v) was added. It
was placed in rotary shaker at 20 rpm for 15 minutes for
uniform mixing and centrifuged at 12000 rpm for 15
minutes. Supernatant was collected and evaporated to
dryness at 60 ºC. It was finally reconstitute in 1000 µL
Methanol.
Preparation of calibration standards in matrix matched plasma
Response of Felodipine and Hydrochlorothiazide were
checked in both above mentioned matrices. It was found
that cold acetonitrile treated plasma and diethyl ether:
hexane (1:1 v/v) treated plasma were suitable for
• Felodipine Calibration Std
• HCTZ Calibration Std
Felodipine and Hydrochlorothiazide molecules respectively.
Calibration standards were thus prepared in respective
matrix matched plasma.
: 5 ppt, 10 ppt, 50 ppt, 100 ppt, 500 ppt, 1 ppb and 10 ppb
: 2 ppt, 5 ppt, 10 ppt, 50 ppt, 100 ppt, and 500 ppt
Figure 3. LCMS-8050 triple quadrupole mass spectrometer by Shimadzu
LCMS-8050 triple quadrupole mass spectrometer by
Shimadzu (shown in Figure 3), sets a new benchmark in
triple quadrupole technology with an unsurpassed
sensitivity (UFsensitivity), Ultra fast scanning speed of
30,000 u/sec (UFscanning) and polarity switching speed of
5 msecs (UFswitching). This system ensures highest quality
of data, with very high degree of reliability.
Figure 4. Heated ESI probe
In order to improve ionization efficiency, the newly
developed heated ESI probe (shown in Figure 4) combines
high-temperature gas with the nebulizer spray, assisting in
the desolvation of large droplets and enhancing ionization.
This development allows high-sensitivity analysis of a wide
range of target compounds with considerable reduction in
background.
LC/MS/MS analysis
Compounds were analyzed using Ultra High Performance
Liquid Chromatography (UHPLC) Nexera coupled with
LCMS-8050 triple quadrupole system (Shimadzu
Corporation, Japan), The details of analytical conditions are
given in Table 1 and Table 2.
3
Table 1. LC/MS/MS conditions for Felodipine
• Column
• Flow rate
• Oven temperature
• Mobile phase
:
:
:
:
• Gradient program (%B)
:
• Injection volume
• MS interface
• Nitrogen gas flow
• Zero air flow
• MS temperature
:
:
:
:
:
Shim-pack XR-ODS (75 mm L x 3 mm I.D.; 2.2 µm)
0.3 mL/min
40 ºC
A: 10 mM ammonium acetate in water
B: methanol
0.0 – 3.0 min → 90 (%); 3.0 – 3.1 min → 90 – 100 (%);
3.1 – 4.0 min → 100 (%); 4.0– 4.1 min → 100 – 90 (%)
4.1 – 6.5 min → 90 (%)
10 µL
ESI
Nebulizing gas 1.5 L/min; Drying gas 10 L/min;
Heating gas 10 L/min
Desolvation line 200 ºC; Heating block 400 ºC
Interface 200 ºC
Table 2. LC/MS/MS conditions for Hydrochlorothiazide
• Column
• Flow rate
• Mobile phase
:
:
:
:
• Gradient program (%B)
:
• MS interface
• Zero air flow
• MS temperature
:
:
:
:
:
Shim-pack XR-ODS (100 mm L x 3 mm I.D.; 2.2 µm)
0.2 mL/min
40 ºC
A: 0.1% formic acid in water
B: acetonitrile
0.0 – 1.0 min → 80 (%); 1.0 – 3.5 min → 40 – 100 (%);
3.5 – 4.5 min → 100 (%); 4.5– 4.51min → 100 – 80 (%)
4.51 – 8.0 min → 90 (%)
25 µL
ESI
Nebulizing gas 2.0 L/min; Drying gas 10 L/min;
Heating gas 15 L/min
Desolvation line 250 ºC; Heating block 500 ºC
Interface 300 ºC
Results
LC/MS/MS analysis results of Felodipine
LC/MS/MS method for Felodipine was developed on ESI
positive ionization mode and 383.90>338.25 MRM
transition was optimized for it. Checked matrix matched
plasma standards for highest (10 ppb) as well as lowest
concentrations (5 ppt) as seen in Figure 5 and Figure 6
respectively. Calibration curves as mentioned with R2 =
0.998 were plotted (shown in Figure 7). Also as seen in
Table 3, % Accuracy was studied to confirm the reliability
of method. Also, LOD as 2 ppt and LOQ as 5 ppt was
obtained.
4
(x100,000)
(x1,000)
5.0 383.90>338.25(+)
FELODIPINE
2.0 383.90>338.25(+)
1.0
FELODIPINE
2.5
1.5
0.5
0.0
0.0
0.0
2.5
5.0
0.0
Figure 5. Felodipine at 10 ppb in matrix matched plasma
2.5
5.0
Figure 6. Felodipine at 5 ppt in matrix matched plasma
Table 3: Results of Felodipine calibration curve
Sr. No.
Standard
Nominal Concentration
(ppb)
Measured Concentration
(ppb)
% Accuracy
(n=3)
% RSD for area counts
(n=3)
1
STD-FEL-01
0.005
0.005
97.43
9.87
2
STD-FEL-02
0.01
0.010
103.80
8.76
3
STD-FEL-03
0.05
0.053
104.47
2.24
4
STD-FEL-04
0.1
0.103
103.13
1.23
5
STD-FEL-05
0.5
0.469
94.88
1.33
6
STD-FEL-06
1
0.977
97.33
0.95
7
STD-FEL-07
10
10.023
100.90
0.60
2.0
Area (x1,000,000)
7
3.0
1.5
Area (x10,000)
2.5
4
2.0
1.0
1.5
3
1.0
0.5
0.5
1
134
2
5
0.0
0.0
6
2
0.0
2.5
5.0
7.5
0.05
0.10
Conc.
Conc.
Figure 7. Calibration curve of Felodipine
LC/MS/MS analysis results of Hydrochlorothiazide
LC/MS/MS method for Hydrochlorothiazide was developed
on ESI negative ionization mode and 296.10>204.90 MRM
transition was optimized for it. Checked matrix matched
plasma standards for highest (500 ppt) as well as lowest (2
ppt) concentrations as seen in Figures 8 and 9 respectively.
Calibration curves as mentioned with R2=0.998 were
plotted (shown in Figure 10). Also as seen in Table 4, %
Accuracy was studied to confirm the reliability of method.
Also, LOD as 1 ppt and LOQ as 2 ppt were obtained.
5
(x10,000)
2.5 296.10>204.90(-)
HCTZ
2.0
1.0
HCTZ
1.5
(x100)
296.10>204.90(-)
1.5
1.0
0.5
0.5
0.0
0.0
0.0
2.5
5.0
7.5
0.0
Figure 8. Hydrochlorothiazide at 500 ppt in matrix matched plasma
2.5
5.0
7.5
Figure 9. Hydrochlorothiazide at 2 ppt in matrix matched plasma
Table 4. Results of Hydrochlorothiazide calibration curve
Sr. No.
Standard
(ppb)
(ppb)
% Accuracy
(n=3)
(n=3)
1
STD-HCTZ-01
0.002
0.0020
102.03
6.65
2
STD-HCTZ-02
0.005
0.0048
95.50
3.53
3
STD-HCTZ-03
0.01
0.0099
100.07
3.80
4
STD-HCTZ-04
0.05
0.0512
102.67
1.60
5
STD-HCTZ-05
0.1
0.1019
102.11
3.58
6
STD-HCTZ-06
0.5
0.4944
102.13
1.68
Area (x100,000)
6
1.00
Area (x10,000)
0.75
1.5
4
1.0
0.50
0.5
0.25
5
1
4
0.00
2
3
0.0
0.000
0.025
0.050
Conc.
3
12
0.0
0.1
0.2
0.3
0.4
Conc.
Figure 10. Calibration curve of Hydrochlorothiazide
Conclusion
• Highly sensitive LC/MS/MS method for Felodipine and Hydrochlorothiazide was developed on LCMS-8050 system.
• LOD of 2 ppt and LOQ of 5 ppt was achieved for Felodipine and LOD of 1 ppt and LOQ of 2 ppt was achieved for
Hydrochlorothiazide by matrix matched methods.
• Heated ESI probe of LCMS-8050 system enables drastic augment in sensitivity with considerable reduction in
background. Hence, LCMS-8050 system from Shimadzu is an all rounder solution for bioanalysis.
6
References
[1] YU Peng; CHENG Hang, Chinese Journal of Pharmaceutical Analysis, Volume 32, Number 1, (2012), 35-39(5).
[2] Hiten Janardan Shah, Naresh B. Kataria, Chromatographia, Volume 69, Issue 9-10, (2009), 1055-1060.
PO-CON1467E
Highly sensitive quantitative estimation
of genotoxic impurities from API
and drug formulation using LC/MS/MS
ASMS 2014
TP496
Shruti Raju, Deepti Bhandarkar, Rashi Kochhar,
Shailesh Damale, Shailendra Rane, Ajit Datar,
Shimadzu Analytical (India) Pvt. Ltd.,
1 A/B Rushabh Chambers, Makwana Road, Marol,
Andheri (E), Mumbai-400059, Maharashtra, India.
Highly sensitive quantitative estimation of genotoxic
impurities from API and drug formulation using LC/MS/MS
Introduction
The toxicological assessment of Genotoxic Impurities (GTI)
and the determination of acceptable limits for such
impurities in Active Pharmaceutical Ingredients (API) is a
difficult issue. As per European Medicines Agency (EMEA)
guidance, a Threshold of Toxicological Concern (TTC) value
of 1.5 µg/day intake of a genotoxic impurity is considered
to be acceptable for most pharmaceuticals[1].
Dronedarone is a drug mainly used for indications of
cardiac arrhythmias. GTI of this drug has been
quantitated here. Method has been optimized for
simultaneous analysis of DRN-IA
{2-n-butyl-3-[4-(3-di-n-butylamino-propoxy)benzoyl]-5-nitro
benzofuran}, DRN-IB
{5-amino-3-[4-(3-di-n-butylamino-propoxy)benzoyl}-2-n-but
yl benzofuran} and BHBNB {2-n-butyl-3-(4-hydroxy
benzoyl)-5-nitro benzofuran}. Structures of Dronedarone
and its GTI are shown in Figure 1.
As literature references available on GTI analysis are
minimal, the feature of automatic MRM optimisation in
LCMS-8040 makes method development process less
tedious. In addition, the lowest dwell time and pause time
and ultrafast polarity switching of LCMS-8040 ensures
uncompromised and high sensitive quantitation.
C4H9
C4H9
N
N
O
O
O
O
C4H9
C4H9
NO2
NHSO2Me
C4H9
C4H9
O
O
Dronedarone
DRN-IA
C 4H 9
N
O
O
O
OH
C 4H 9
NO 2
NH 2
C 4H 9
C 4H 9
O
O
DRN-IB
BHBNB
Figure 1. Structures of Dronedarone and its GTI
2
Method of Analysis
Sample Preparation
• Preparation of DRN-IA and DRN-IB and BHBNB stock solutions
20 mg of each impurity standard was weighed separately and dissolved in 20 mL of methanol to prepare stock solutions
of each standard.
• Preparation of calibration levels
GTI mix standards (DRN-IA, DRN-IB and BHBNB) at concentration levels of 0.5 ppb, 1 ppb, 5 ppb, 10 ppb, 40 ppb, 50
ppb and 100 ppb were prepared in methanol using stock solutions of all the three standards.
• Preparation of blank sample
400 mg of Dronedarone powder sample was weighed and mixed with 20 mL of methanol. Mixture was sonicated to
dissolve sample completely.
• Preparation of spiked (at 12 ppb level) sample
400 mg of sample was weighed and spiked with 60 µL of 1 ppm stock solution. Solution was then mixed with 20 mL of
methanol. Mixture was sonicated to dissolve sample completely.
LC/MS/MS Analytical Conditions
Analysis was performed using Ultra High Performance
Corporation, Japan), shown in Figure 2. Limit of GTI for
Dronedarone is 2 mg/kg. However, general dosage of
Dronedarone is 400 mg, hence, limit for GTI is 0.8 µg/400
mg. This when reconstituted in 20 mL system, gives an
effective concentration of 40 ppb. For analytical method
development it is desirable to have LOQ at least 30 % of
limit value, which in this case corresponds to 12 ppb. The
developed method gives provision for measuring GTI at
much lower level. However, recovery studies have been
done at 12 ppb level.
Figure 2. Nexera with LCMS-8040 triple quadrupole system by Shimadzu
3
Below mentioned table shows the analytical conditions used for analysis of GTI.
Table 1. LC/MS/MS analytical conditions
• Column
• Mobile phase
• Flow rate
• Gradient program (B%)
• MS interface
• MS analysis mode
• Polarity
• MS gas flow
• MS temperature
: Shim-pack XR-ODS II (75 mm L x 3 mm I.D.; 2.2 µm)
: A: 0.1% formic acid in water
B: acetonitrile
: 0.3 mL/min
: 40 ºC
: 0.0–2.0 min → 35 (%); 2.0–2.1 min → 35-40 (%);
2.1–7.0 min → 40-60 (%); 7.0–8.0 min → 60-100 (%);
8.0–10.0 min → 100 (%); 10.0–10.01 min → 100-35 (%);
10.01–13.0 min → 35 (%)
: 1 µL
: Electro Spray Ionization (ESI)
: MRM
: Positive and negative
: Nebulizing gas 2 L/min; Drying gas 15 L/min
: Desolvation line 250 ºC; Heat block 400 ºC
Note: Flow Control Valve (FCV) was used for the analysis to divert HPLC flow towards waste during elution
of Dronedarone so as to prevent contamination of Mass Spectrometer.
Results
LC/MS/MS analysis
LC/MS/MS method was developed for simultaneous
quantitation of GTI mix standards. MRM transitions used
for all GTI are given in Table 2. No peak was seen in diluent
(methanol) at the retention times of GTI for selected MRM
transitions which confirms the absence of any interference
from diluent (shown in Figure 3). MRM chromatogram of
GTI mix standard at 5 ppb level is shown in Figure 4.
Linearity studies were carried out using external standard
calibration method. Calibration graphs of each GTI are
shown in Figure 5. LOQ was determined for each GTI
based on the following criteria – (1) % RSD for area < 15
%, (2) % Accuracy between 80-120 % and (3) Signal to
noise ratio (S/N) > 10. LOQ of 0.5 ppb was achieved for
DRN-IB and BHBNB whereas 1 ppb was achieved for
DRN-IA. Results of accuracy and repeatability for all GTI are
given in Table 3.
Table 2: MRM transitions selected for all GTI
Name of GTI
MRM transition
Retention time (min)
Mode of ionization
DRN-IB
479.15>170.15
1.83
Positive ESI
DRN-IA
509.10>114.10
5.85
Positive ESI
BHBNB
338.20>244.05
8.77
Negative ESI
4
1000 1:DRA-IB 479.15>170.15(+) CE: -29.0
2:DRA-IA 509.10>114.10(+) CE: -41.0
3:BHBNB 338.20>244.05(-) CE: 20.0
750
500
250
0
0.0
2.5
5.0
7.5
10.0
min
10.0
min
Figure 3. MRM chromatogram of diluent (methanol)
1:DRA-IB 479.15>170.15(+) CE: -29.0
30000
25000
20000
15000
BHBNB 338.20>244.05
DRN-IB 479.15>170.15
35000
DRN-IA 509.10>114.10
509.10>114.10(+) CE: -41.0
40000 2:DRA-IA
3:BHBNB 338.20>244.05(-) CE: 20.0
10000
5000
0
0.0
2.5
5.0
7.5
Figure 4. MRM chromatogram of GTI mix standard at 5 ppb level
750000
Area
Area
DRN-IB
R2-0.9989
1250000
1000000
500000
Area
DRN-IA
R2-0.9943
750000
100000
500000
250000
50000
250000
0
BHBNB
R2-0.9922
150000
0.0
25.0
50.0
75.0
Conc.
0
0.0
25.0
50.0
75.0
Conc.
0
0.0
25.0
50.0
75.0
Conc.
Figure 5. Calibration graphs for GTI
5
Table 3: Results of accuracy and repeatability for all GTI
Sr. No.
1
2
3
Name of GTI
Standard concentration
(ppb)
Calculated concentration
from calibration graph
(ppb) (n=6)
0.5
1
DRN-IB
DRN-IA
BHBNB
% Accuracy
(n=6)
(n=6)
0.492
98.40
9.50
1.044
104.40
6.62
5
4.961
99.22
3.10
12
12.014
100.12
2.97
40
38.360
95.90
1.17
50
49.913
99.83
1.08
100
103.071
103.07
0.86
1
0.994
99.40
5.02
5
4.916
98.32
2.82
12
11.596
96.63
2.43
40
37.631
94.08
1.27
50
48.605
97.21
1.40
100
100.138
100.14
0.99
0.5
0.486
97.20
4.88
1
1.062
106.20
6.97
5
4.912
98.24
2.16
12
11.907
99.23
1.31
40
37.378
93.45
0.37
50
48.518
97.04
0.43
100
96.747
96.75
0.91
Recovery studies
For recovery studies, samples were prepared as described
previously. MRM chromatogram of blank and spiked
samples are shown in Figures 6 and 7 respectively. Results
of recovery studies have been shown in Table 4. Recovery
could not be calculated for DRN-IB as blank sample
showed higher concentration than spiked concentration.
1:DRA-IB 479.15>170.15(+) CE: -29.0
400000 2:DRA-IA 509.10>114.10(+) CE: -41.0
3:BHBNB 338.20>244.05(-) CE: 20.0
250000
200000
150000
100000
50000
BHBNB 338.20>244.05
300000
DRN-IA 509.10>114.10
DRN-IB 479.15>170.15
350000
0
0.0
2.5
5.0
7.5
10.0
min
Figure 6. MRM chromatogram of blank sample
6
125000
1:DRA-IB 479.15>170.15(+) CE: -29.0
2:DRA-IA 509.10>114.10(+) CE: -41.0
3:BHBNB 338.20>244.05(-) CE: 20.0
50000
25000
BHBNB 338.20>244.05
75000
DRN-IA 509.10>114.10
DRN-IB 479.15>170.15
100000
0
0.0
2.5
5.0
7.5
10.0
min
Figure 7. MRM chromatogram of spiked sample
Table 4. Results of the recovery studies
Name of
Impurity
Concentration of
GTI mix standard spiked
in blank sample (ppb)
Average concentration
obtained from calibration graph
for blank sample (ppb) (A) (n=3)
Average concentration obtained
from calibration graph
for spiked sample (ppb) (B) (n=3)
% Recovery =
(B-A)/ 12 * 100
DRN-IB
12
94.210
NA
NA
DRN-IA
12
3.279
12.840
79.678
BHBNB
12
1.241
12.723
95.689
Conclusion
• A highly sensitive method was developed for analysis of GTI of Dronedarone.
• Ultra high sensitivity, ultra fast polarity switching (UFswitching) enabled sensitive, selective, accurate and reproducible
analysis of GTI from Dronedarone powder sample.
References
[1] Guideline on The Limits of Genotoxic Impurities, (2006), European Medicines Agency (EMEA).
PO-CON1470E
Development of 2D-LC/MS/MS
Method for Quantitative Analysis of
1α,25-Dihydroxylvitamin D3
in Human Serum
ASMS 2014
WP449
Daryl Kim Hor Hee1, Lawrence Soon-U Lee1,
Zhi Wei Edwin Ting2, Jie Xing2, Sandhya Nargund2,
Miho Kawashima3 & Zhaoqi Zhan2
1
Department of Medicine Research Laboratories,
National University of Singapore, 6 Science Drive 2,
Singapore 117546
2
Customer Support Centre, Shimadzu (Asia Pacific) Pte
Ltd, 79 Science Park Drive, #02-01/08, Singapore 118264
3
Global Application Development Centre, Shimadzu
Corporation, 1-3 Kanda Nishihiki-cho, Chiyoda-ku,
Tokyo 101-8448, Japan
Development of 2D-LC/MS/MS Method for Quantitative
Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum
Introduction
Developments of LC/MS/MS methods for accurate
quantitation of low pg/mL levels of 1α,25-dihydroxy
vitamin D2/D3 in serum were reported in recent years,
because their levels in serum were found to be important
indications of several diseases associated with vitamin D
metabolic disorder in clinical research and diagnosis [1].
However, it has been a challenge to achieve the required
sensitivity directly, due to the intrinsic difficulty of
ionization of the compounds and interference from matrix
[2,3]. Sample extraction and clean-up with SPE and
immunoaffinity methods were applied to remove the
interferences [4] prior to LC/MS/MS analysis. However, the
amount of serum required was normally rather high from
0.5mL to 2mL, which is not favourite in the clinical
applications. Direct analysis methods with using smaller
amount of serum are in demand. Research efforts have
been reported in literatures to enhance ionization
efficiency by using different interfaces such as ESI, APCI or
APPI and ionization reagents to form purposely NH3
adduct or lithium adduct [4,5]. Here, we present a novel
2D-LC/MS/MS method with APCI interface for direct
analysis of 1α,25-diOH-VD3 in serum. The method
achieved a detection limit of 3.1 pg/mL in spiked serum
samples with 100 uL injection.
Experimental
High purity 1α,25-dihydroxyl Vitamin D3 and deuterated
1α,25-dihydroxyl-d6 Vitamin D3 (as internal standard) were
obtained from Toronto Research Chemicals.
Charcoal-stripped pooled human serum obtained from
Bioworld was used as blank and matrix to prepare spiked
samples in this study. A 2D-LC/MS/MS system was set up
on LCMS-8050 (Shimadzu Corporation) with a column
switching valve installed in the column oven and controlled
by LabSolutions workstation. The details of columns,
mobile phases and gradient programs of 1st-D and 2nd-D LC
separations and MS conditions are compiled into Table 1.
The procedure of sample preparation of spiked serum
samples is shown in Figure 1. It includes protein
precipitation by adding ACN-MeOH solvent into the serum
in 3 to 1 ratio followed by vortex and centrifuge at high
speed. The supernatant collected was filtered before
standards with IS were added (post-addition). The clear
samples obtained were then injected into the 2-D
LC/MS/MS system.
Table 1: 2D-LC/MS/MS analytical conditions
LC condition
Column
Mobile Phase
of 1st D
Mobile Phase
of 2nd D
MS Interface condition
Interface
APCI, 400ºC
MS mode
Positive, MRM
A: Water with 0.1% formic acid
B: Acetontrile
Heat Block & DL Temp.
300ºC & 200ºC
C: Water with 0.1% formic acid
D: MeOH with 0.1% formic acid
Nebulizing Gas Flow
N2, 2.5 L/min
Drying Gas Flow
N2, 7.0 L/min
1st D: FC-ODS (2.0mml.D. x 75mm L, 3μm)
2nd D: VP-ODS (2.0mmI.D. x 150mm L, 4.6μm)
1st D gradient program & flow rate
B: 40% (0 to 0.1min) → 90% (5 to 7.5min)
→ 15% (11 to 12min) → 40% (14 to 25min);
Total flow rate: 0.5mL/min
2nd D gradient program & flow rate
D: 15% (0min) → 80% (20 to 22.5min) →
15% (23 to 25min); Peak cutting: 3.15 to 3.40;
Total flow rate: 0.5 mL/min
Oven Temp.
45ºC
Injection Vol.
100 uL
CID Gas
Ar (270kPa)
2
150µL of serum
450µL of ACN/MeOH (1:1)
Shake and Vortex 10mins
Centrifuge for 10 minutes at 13000rpm
480µL of Supernatant
0.2µm nylon filter
400µL of filtered protein precipitated Serum
50µL of of Std stock
50µL of IS stock
500µL of calibrate
Figure 1: Flow chart of serum sample pre-treatment method
Development of 2D-LC/MS/MS method
An APCI interference was employed for effective ionization
of 1α,25-diOH-VitD3 (C27H44O3, MW 416.7). A MRM
quantitation method for 1α,25-diOH-VitD3 with its
deuterated form as internal standard (IS) was developed.
MRM optimization was performed using an automated
MRM optimization program with LabSolutions workstation.
Two MRM transitions for each compound were selected
(Table 2), the first one for quantitation and the second one
for confirmation. The parent ion of 1α,25-diOH-VitD3 was
the dehydrated ion, as it underwent neutral lost easily in
ionization with ESI and APCI [2,3]. The MRM used for
quantitation (399.3>381.3) was dehydration of the second
OH group in the molecule.
Table 2: MRM transitions and CID parameters of 1α,25-diOH-VitD3 and deuterated IS
Name
RT1 (min)
1α,25-dihydroxyl Vitamin D3
22.74
1α,25-dihydroxyl-d6 Vitamin D3 (IS)
22.71
Transition (m/z)
CID Voltage (V)
Q1 Pre Bias
CE
Q3 Pre Bias
399.3 > 381.3
-20
-13
-14
399.3 > 157.0
-20
-29
-17
402.3 > 366.3
-20
-12
-18
402.3 > 383.3
-20
-15
-27
1, Retention time by 2D-LC/MS/MS method
3
5000
1:OH2D3 399.30>381.30(+) CE: -13.0
1:OH2D3 399.30>157.00(+) CE: -29.0
1:OH2D3 399.30>105.00(+) CE: -44.0
OH2-VD3
4000
3000
2000
1000
0
0.0
700
2.5
5.0
7.5
10.0
min
5.0
7.5
10.0
min
2:OH2D3-D6 402.30>383.30(+) CE: -15.0
2:OH2D3-D6 402.30>366.30(+) CE: -12.0
OH2-VD3-D3
The reason to develop a 2-D LC separation for a LC/MS/MS
method was the high background and interferences
occurred with 1D LC/MS/MS observed in this study and
also reported in literatures. Figure 2 shows the MRM
chromatograms of 1D-LC/MS/MS of spiked serum sample.
It can be seen that the baseline of the quantitation MRM
(399.3>381.3) rose to a rather high level and interference
peaks also appeared at the same retention time.
The 2-D LC/MS/MS method developed in this study
involves “cutting the targeted peak” in the 1st-D separation
precisely (3.1~3.4 min) and the portion retained in a
stainless steel sample loop (200 uL) was transferred into
the 2nd-D column for further separation. The operation was
accomplished by switching the 6-way valve in and out by a
time program. Both 1st-D and 2nd-D separations were
carried out in gradient elution mode. The organic mobile
phase of 2nd-D (MeOH with 0.1% formic acid) was
different from that of 1st-D (pure ACN). The interference
peaks co-eluted with the analyte in 1st-D were separated
from the analyte peak (22.6 min) as shown in Figure 3.
600
500
400
300
200
100
0
2.5
Peak cutting (125 uL) in 1st D separation
and transferred to 2nd D LC
Figure 2: 1D-LC/MS/MS chromatograms of 22.7 pg/mL
1α,25-diOH-VitD3 (top) and 182 pg/mL internal
standard (bottom) in serum (injection volume: 50uL)
Calibration curve (IS), linearity and accuracy
Two sets of standard samples were prepared in serum and
in clear solution (diluent). Each set included seven levels of
1α,25-diOH-VitD3 from 3.13 pg/mL to 200 pg/mL, each
added with 200 pg/mL of IS (See Table 3). The
chromatograms of the seven spiked standard samples in
serum are shown in Figure 3. A linear IS calibration curve
(R2 > 0.996) was established from these 2D-LC/MS/MS
analysis results, which is shown in Figure 4. It is worth to
note that the calibration curve has a non-zero Y-intercept,
indicating that the blank (serum) contains either residual 1
α,25-diOH-VitD3 or other interference which must be
deducted in the quantitation method. The peak area ratios
shown in Table 3 are the results after deduction of the
background peaks. The accuracy of the method after this
correction is between 92% and 117%.
Area Ratio
4000
4000
3000
5.0
1α,25-diOH-VitD3
3000
4.0
2000
3.0
1000
2.0
R2 = 0.9967
2000
1000
Non-zero intercept
1.0
22.0
23.0
min
0
0.0
0
10
20
Figure 3: Overlay of 2nd-D chromatograms of 7 levels
from 3.13 pg/mL to 200 pg/mL spiked in serum.
min
0.00
0.25
0.50
0.75 Conc. Ratio
Figure 4: Calibration curves of
1α,25-diOH VD3 in serum by IS method.
4
Table 3: Seven levels of standard samples for calibration curve and performance evaluation
Conc. Level
of Std.
1α,25-diOH VD3
(pg/mL)
Conc. Ratio1
(Target/IS)
Area Ratio2
(in serum)
Area Ratio2
(in clear solu)
Accuracy3
(%)
Matrix Effect
(%)
L1
3.13
0.0156
0.243
0.414
103.8
58.7
L2
6.25
0.0313
0.321
0.481
100.0
66.8
L3
12.5
0.0625
0.456
0.603
117.3
75.6
L4
25.0
0.1250
0.757
0.914
115.9
82.9
L5
50.0
0.2500
1.188
1.354
95.5
87.7
L6
100.0
0.5000
2.168
2.580
92.15
84.0
L7
200.0
1.0000
4.531
4.740
102.0
95.6
1, Target = 1α,25-diOH VD3; 2, Area ratio = area of target / area of IS; 3, Based on the data of spiked serum samples
Matrix effect, repeatability, LOD/LOQ and specificity
Matrix effect of the 2D-LC/MS/MS method was determined by
comparison of peak area ratios of standard samples in diluent
and in serum at the seven levels. The results are compiled into
Table 3. The matrix effect of the method are between 58%
and 95%. It seems that the matrix effect is stronger at lower
concentrations than at higher concentrations. Repeatability of
peak area of the method was evaluated with L2 and L3 spiked
serum samples for both target and IS. The Results of RSD (n=6)
are displayed in Table 4.
The MRM peaks of 1α,25-diOH VD3 in clear solution and in
serum are displayed in pairs (top and bottom) in Figure 5. It
can be seen from the first pair (diluent and serum blank)
that a peak appeared at the same retention of 1α,25-diOH
VD3 in the blank serum. As pointed out above, this peak is
250
250
0
0
0
22.5
24.7
1:399.30>157.00(+)
500
OH2VD3/22.595
1:399.30>157.00(+)
Serum
blank
500
250
250
500
22.5
24.7
750 1:399.30>381.30(+)
OH2VD3/22.565
22.5
750 1:399.30>381.30(+)
750
0
0
24.7
L3
22.5
24.7
1000
22.5
24.7
1:399.30>381.30(+)
1:399.30>381.30(+)
1:399.30>157.00(+)
500
250
2000
1000
1:399.30>157.00(+)
500
L7
3000
250
750 1:399.30>381.30(+)
L1
L5
1:399.30>381.30(+)
4000 1:399.30>157.00(+)
OH2VD3/22.630
250
L3
1:399.30>381.30(+)
1:399.30>157.00(+)
4000 1:399.30>157.00(+)
L5
3000
2000
OH2VD3/22.598
500
500
1000
OH2VD3/22.573
L1
OH2VD3/22.602
Diluent
500
OH2VD3/22.622
1:399.30>157.00(+)
1:399.30>157.00(+)
OH2VD3/22.619
750 1:399.30>381.30(+)
750 1:399.30>381.30(+)
1:399.30>157.00(+)
OH2VD3/22.565
750 1:399.30>381.30(+)
from either the residue of 1α,25-diOH VD3 or other
interference present in the serum. Due to this background
peak, the actual S/N ratio could not be calculated. Therefore,
it is difficult to determine the LOD and LOQ based on the
S/N method. Tentatively, we propose a reference LOD and
LOQ of the method for 1α,25-diOH VD3 to be 3.1 pg/mL
and 10 pg/mL, respectively.
The specificity of the method relies on several criteria: two
MRMs (399>381 and 399>157), their ratio and RT in 2nd-D
chromatogram. The MRM chromatograms shown in Figure
5 demonstrate the specificity of the method from L1 (3.1
pg/mL) to L7 (200 pg/mL). It can be seen that the results of
spiked serum samples (bottom) meet the criteria if
compared with the results of samples in the diluent (top).
L7
1000
0
0
22.5
24.7
22.5
24.7
0
0
0
22.5
24.7
22.5
24.7
22.5
24.7
Figure 5: MRM peaks of 1α,25-diOH-VitD3 spiked in pure diluent (top) and in serum (bottom) of L1, L3, L5 and L7 (spiked conc. refer to Table 3)
5
Table 4: Repeatability Test Results (n=6)
Sample
L2
L3
Compound
Spiked Conc. (pg/mL)
%RSD
1α,25-diOH VD3
6.25
10.10
IS
200
7.66
1α,25-diOH VD3
12.5
9.33
IS
200
6.28
Conclusions
A 2D-LC/MS/MS method with APCI interface has been
developed for quantitative analysis of
1α,25-dihydroxylvitamin D3 in human serum without
offline extraction and cleanup. The detection and
quantitation range of the method is from 3.1 pg/mL to 200
pg/mL, which meets the diagnosis requirements in clinical
applications. The performance of the method was
evaluated thoroughly, including linearity, accuracy,
repeatability, matrix effect, LOD/LOQ and specificity. The
results indicate that the 2D-LC/MS/MS method is sensitive
and reliable in detection and quantitation of trace
1α,25-dihydroxylvitamin D3 in serum. Further studies to
enable the method for simultaneous analysis of both
1α,25-dihydroxylvitamin D3 and 1α,25-dihydroxylvitamin
D2 are needed.
References
1. S. Wang. Nutr. Res. Rev. 22, 188 (2009).
2. T. Higashi, K. Shimada, T. Toyo’oka. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. (2010) 878, 1654.
3. J. M. El‐Khoury, E. Z. Reineks, S. Wang. Clin. Biochem. 2010. DOI: 10.1002/jssc.20200911.
4. Chao Yuan, Justin Kosewick, Xiang He, Marta Kozak and Sihe Wang, Rapid Commun. Mass Spectrom. 2011, 25,
1241–1249
5. Casetta, I. Jans, J. Billen, D. Vanderschueren, R. Bouillon. Eur. J. Mass Spectrom. 2010, 16, 81.
PO-CON1450E
Analysis of polysorbates in biotherapeutic
products using two-dimensional
HPLC coupled with mass spectrometer
ASMS 2014
WP 182
William Hedgepeth, Kenichiro Tanaka
Shimadzu Scientific Instruments, Inc., Columbia MD
Analysis of polysorbates in biotherapeutic products
using two-dimensional HPLC coupled with mass spectrometer
Introduction
Polysorbate 80 is commonly used for biotherapeutic
products to prevent aggregation and surface adsorption, as
well as to increase the solubility of biotherapeutic
compounds. A reliable method to quantitate and
characterize polysorbates is required to evaluate the quality
and stability of biotherapeutic products. Several methods
for polysorbate analysis have been reported, but most of
them require time-consuming sample pretreatment such as
derivatization and alkaline hydrolysis because polysorbates
do not have sufficient chromophores. Those methods also
require an additional step to remove biotherapeutic
compounds. Here we report a simple and reliable method
for quantitation and characterization of polysorbate 80 in
biotherapeutic products using two-dimensional HPLC.
Materials
Reagents and standards
Reagents: Tween® 80 (Polysorbate 80), IgG from human
serum, potassium phosphate monobasic, potassium
phosphate dibasic, and ammnonium formate were
purchased from Sigma-Aldrich. Water was made in house
using a Millipore Milli-Q Advantage A10 Ultrapure Water
Purification System. Isopropanol was purchased from
Honeywell.
Standard solutions: 10 mmol/L phosphate buffer (pH 6.8)
was prepared by dissolving 680 mg of potassium
phosphate monobasic and 871 mg of potassium
phosphate dibasic in 1 L of water.
Polysorbate 80 was diluted with 10 mmol/L phosphate
buffer (pH 6.8) to 200, 100, 50, 20, 10 mg/L and
transferred to 1.5 mL vials for analysis.
Sample solutions: A model sample was prepared by
dissolving 2 mg of IgG in 0.1 mL of a 100 mg/L polysorbate
80 standard solution. The sample was centrifuged and
transferred to a 1.5 mL vial for analysis.
O
O
O
HO
O
z
wO
OH
O
O
OH
x
y
w+x+y+z=approx. 20
CH3
Fig.1 Typical structure of polysorbate 80
2
System
The standard and sample solutions were injected into a
Shimadzu Co-Sense for BA system consisting of two
LC-20AD pumps and a LC-20AD pump equipped with a
solvent switching valve, DGU-20A5R degassing unit,
SIL-20AC autosampler, CTO-20AC column oven equipped
with a 6-port 2-position valve, and a CBM-20A system
controller. Polysorbate 80 was detected by a LCMS-2020
single quadrupole mass spectrometer or a LCMS-8050
triple quadrupole mass spectrometer because polysorbates
do not have any chromophores and are present at low
concentrations in antibody drugs. A SPD-20AV UV-VIS
detector was used to check protein removal.
Fig. 2 shows the flow diagram of the Co-Sense for BA
system. In step 1, a sample pretreatment column
“Shim-pack MAYI-ODS” traps polysorbate 80 in the
sample. Proteins (antibody) cannot enter the pore interior
that is blocked by a hydrophilic polymer bound on the
outer surface. Other additives and excipients such as
sugars, salts, and amino acids cannot be retained by the
ODS phase of the inner surface due to their polarity. In
step 2, the trapped polysorbate 80 is introduced to the
analytical column by valve switching.
Step 1 : Protein removal
Mass spectrometer
Pump 2
Mobile phase C
Analytical column
Valve
(Position 0)
Mobile phase A
(solution for sample injection)
Autosampler
Mobile phase D
Protein,
Salts,
Amino acids,
Sugars
Polysorbate
80
UV-VIS detector
Pump 1
Sample pretreatment column
Mobile phase B
(solution for rinse)
Step 2 : Analyzing the trapped polysorbate
Polysorbate
80
Mass spectrometer
Pump 2
Mobile phase C
Analytical column
Valve
Mobile phase A
(Position 1)
(solution for sample injection)
Autosampler
Mobile phase D
UV-VIS detector
Pump 1
Sample pretreatment column
Mobile phase B
(solution for rinse)
Fig.2 Flow diagram of Co-Sense for BA
3
Results
Quantitation method
A fast analysis for quantitation will be shown here. Table 1
shows the analytical conditions and Fig. 3 shows the TIC
chromatogram of a 100 mg/L polysorbate 80 standard
solution and the mass spectrum of the peak at 4.4 min.
Polysorbates contain many by-products, so several peaks
appeared on the TIC chromatogram. The peak at 4.4 min
was identified as polyoxyethylene sorbitan monooleate
(typical structure of polysorbate 80) based on E. Hvattum
et al 2011. The ion at 783 was used as a marker for
detection in selected ion mode (SIM). This ion is
attributable to the 2NH4+ adduct of polyoxyethylene
sorbitan monooleate containing 25 polyoxyethylene
groups. Fig. 4 shows the SIM chromatogram of the model
sample (20 g/L of IgG, 100 mg/L of polysorbate 80 in 10
mmol/L phosphate buffer pH6.8). Polysorbate 80 in the
model sample was successfully analyzed. The peak at 4.4
min was used for quantitation.
Six replicate injections for the model sample were made to
evaluate the reproducibility. The relative standard
deviations of retention time and peak area were 0.034 %
and 1.11 %, respectively. The recovery ratio was obtained
by comparing the peak area of the model sample and a
100 mg/L polysorbate 80 standard solution and was 99 %.
Five different levels of polysorbate 80 standard solutions
ranging from 10 to 200 mg/L were used for the linearity
evaluation. The correlation coefficient (R2) of determination
was higher than 0.999.
Table 1 Analytical Conditions
System
[Sample Injection]
Column
Mobile Phase
Solvent Switching
Flow Rate
Valve Position
Injection Volume
[Separation]
Column
Mobile Phase
: Co-Sense for BA equipped with LCMS-2020
: Shim-pack MAYI-ODS (5 mm L. x 2.0 mm I.D., 50 μm)
: A: 10 mmol/L ammonium formate in water
B: Isopropanol
: A (0-1.5 min), B (1.5-3.5 min), A (3.5-9 min)
: 0.6 mL/min
: 0 (0-1 min, 7-9 min), 1 (1-7 min)
: 1 µL
: Kinetex 5u C18 100A (50 mm L. x 2.1 mm I.D., 5 μm)
B: Isopropanol
Time Program
: B. Conc 5 % (0-1 min) - 100 % (6-7 min) -5 % (7.01-9 min)
Flow Rate
: 0.3 mL/min
Column Temperature : 40 ºC
[UV Detection]
Detection
Flow Cell
[MS Detection]
Ionization Mode
Applied Voltage
Nebulizer Gas Flow
DL Temperature
Block Heater Temp.
Scan
SIM
: 280 nm
: Semi-micro cell
: ESI Positive
: 4.5 kV
: 1.5 mL/min
: 250 ºC
: 400 ºC
: m/z 300-2000
: m/z 783
4
4000000
3000000
2000000
1000000
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
min
950
m/z
Inten.(x100,000)
Triply charged ions
1.5
587 601 616
572
1.0
631
Doubly charged ions
645
660
557
783
675
543
0.5
689
528
704717
739
761
805
827
849
871
893
915
0.0
500
550
600
650
700
750
800
850
900
Fig.3 TIC Chromatogram of 100 mg/L polysorbate 80 standard solution and mass spectrum of the peak at 4.4 min
100000
75000
50000
25000
0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
min
Fig.4 SIM chromatogram of the model sample
Characterization method
An analysis for characterization will be shown here. Table 2
shows the analytical conditions and Fig. 5 shows the TIC
chromatogram of the model sample and mass spectra of
the peaks from 10 to 30 min. A longer column and
gradient were applied to obtain better resolution.
Polysorbate 80 consists of not only monooleate (typical
structure of polysorbate 80), but also many by-products
such as polyoxyethylene, polyoxyethylene sorbitan,
polyoxyethylene isosorbide, dioleate, trioleate, tetraoleate
and others. The peaks on the TIC chromatogram are
assumed to correspond to those by-products. For example,
the peaks from 10 to 22 min correspond to
polyoxyethylene and polyoxyethylene isosorbide and the
peaks from 22 to 30 min correspond to polyoxyethylene
sorbitan. This method is helpful for characterization as well
as checking degradation such as auto-oxidation and
hydrolysis.
5
Table 2 Analytical Conditions
System
[Sample Injection]
Column
Mobile Phase
: Co-Sense for BA equipped with LCMS-8050
: Shim-pack MAYI-ODS (5 mm L. x 2.0 mm I.D., 50 μm)
B: Isopropanol
: A (0-1.5 min), B (1.5-3.5 min), A (3.5-9 min)
: 0.6 mL/min (0-10 min, 95.01-110 min), 0.1 mL/min (10.01-95 min)
: 0 (0-3 min, 100-110 min), 1 (3-100 min)
: 5 µL
Solvent Switching
Flow Rate
Valve Position
Injection Volume
[Separation]
Column
Mobile Phase
: Kinetex 5u C18 100A (100 mm L. x 2.1 mm I.D., 5 μm)
B: Isopropanol
Time Program
: B. Conc 5 % % (0-3min) – 35% (15min) – 100% (100min) – 5% (100.01-110min)
Flow Rate
: 0.2 mL/min
Column Temperature : 40 ºC
[UV Detection]
Detection
Flow Cell
[MS Detection]
Ionization Mode
Applied Voltage
Nebulizer Gas Flow
Drying Gas Flow
Heating Gas Flow
DL Temperature
Block Heater Temp.
Q1 Scan
: 280 nm
: Semi-micro cell
: ESI Positive
: 4.5 kV
: 2 mL/min
: 10 mL/min
: 10 mL/min
: 300 ºC
: 250 ºC
: 400 ºC
: m/z 300-2000
(x100,000,000)
1:TIC(+)
(x10,000,000)
1:TIC(+)
4.0
7.5
5.0
3.0
2.5
2.0
0.0
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
min
1.0
0.0
0
10
20
30
40
50
Inten.(x100,000)
6.0
3.0
648.8
736.8
1.0
0.0
560.7
421.7
443.8
399.7
465.8
377.6
520.7
516.6
564.7 608.8 652.8
300
HO
784.9
500
O
O
y
O
O
OH
z
600
700
Polyoxyethylene
isosorbide
800
O
H
x
min
557.6
869.0
900
m/z
0.0
587.0606.9
440.2
1.0
913.0
Polyoxyethylene
628.9651.0673.0695.0
717.1
739.0
761.1
783.1
805.1
827.1
572.3
454.8
2.0
425.4
400
500
600
O
HO
100
469.5
740.9
400
90
513.6
528.3
498.9
543.0
3.0
824.9
696.9
80
484.2
4.0
780.9
445.4
355.6 401.6
423.5
379.5
70
5.0
692.8
604.7
2.0
60
Inten.(x100,000)
w
O
HO
O
x
OH
O
z
OH
OH
O
700
800
m/z
Polyoxyethylene
sorbitan
y
Fig.5 TIC chromatogram of the model sample
6
Confirmation of protein removal
Fig. 6 shows the chromatogram of elution from the sample pretreatment column. Protein (IgG) was successfully removed
from the sample by using the MAYI-ODS column.
uV
5uL injection of model sample
1250000
1uL injection of model sample
1000000
750000
500000
250000
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
min
Fig.6 Chromatogram of elution from the sample pretreatment column
Conclusions
1. Co-Sense for BA system automatically removed protein from the sample and enabled quantitation and characterization
of polysorbate 80 in a protein formulation.
2. The quantitation method was successfully applied to the model sample with excellent reproducibility and recovery.
3. The high-resolution chromatogram was obtained by the characterization method. This method is helpful for
characterization as well as checking degradation such as auto-oxidation and hydrolysis.
Reference
E. Hvattum, W.L. Yip, D. Grace, K. Dyrstad, Characterization of polysorbate 80 with liquid chromatography mass
spectrometry and nuclear magnetic resonance spectroscopy: Specific determination of oxidation products of thermally
oxidized polysorbate 80, J Pharm Biomed Anal 62, (2012) 7-16
PO-CON1457E
A Rapid and Reproducible Immuno-MS
Platform from Sample Collection to
Quantitation of IgG
ASMS 2014
WP161
Rachel Lieberman1, David Colquhoun1, Jeremy Post1,
Brian Feild1, Scott Kuzdzal1, Fred Regnier2,
1
Shimadzu Scientific Instruments, Columbia, MD, USA
2
Novilytic L.L.C, North Webster, IN, USA
A Rapid and Reproducible Immuno-MS Platform from Sample
Collection to Quantitation of IgG
Novel Aspect
Using rapid, automated processing, coupled to the speed and sensitivity of the LCMS-8050 allows for improved analysis of
Immunoglobulin G.
Introduction
Dried blood spot analysis (DBS) has provided clinical
laboratories a simple method to collect, store and transport
samples for a wide variety of analyses. However, sample
stability, hematocrit effects and inconsistent spotting
techniques have limited the ability for wide spread
adoption in clinical applications. Dried plasma spots (DPS)
offer new opportunities by providing stable samples that
avoid variability caused by the hematocrit. This
presentation focuses on an ultra-fast-immuno-MS platform
that combines next generation plasma separator cards
(Novilytic L.L.C., North Webster, IN) with fully automated
immuno-affinity enrichment and rapid digestion as an
upfront sample preparation strategy for mass spectrometric
analysis of immunoglobulins.
Sample Workflow
Plasma
Generation
Affinity
Selection
NoviplexTM Card
Rapid plasma extraction technology
from whole blood (~ 18 minutes)
- 2.5 uL of plasma collected (3 min)
- Air dry for 15 minutes
- Extract plamsa disc for analysis
Buffer
Exchange
Enzyme
Digestion
Desalting
LC/MS/MS
Perfinity Workstation
LCMS-8050 Triple Quadrupole MS
Automates and integrates key
proteomic workflow steps:
- Affinity Selection (15 min)
- Trypsin digestion (1-8 min)
- Online Desalting
- Reversed phase LC
Exceptional reproducibility
(CV less than 10%)
- Ultrafast MRM methods
- Up to 555 MRM transitions per run
- Heated electrospray source
- Scan speeds up to 30,000 u/sec
- Polarity switching 5 msec
2
Methods
IgG was weighed out and then diluted in 500 μL of 0.5%
BSA solution. Approximately15 uL of IgG standard was
spiked into mouse whole blood and processed using the
Noviplex card. The resulting plasma collection disc was
extracted with 30 uL of buffer and each sample was
reduced and alkylated to yield a total sample volume of
100 uL. IgG standards and QC samples were directly
injected onto the Perfinity-LCMS-8050 platform for affinity
pulldown with a Protein G column followed by trypsin
digestion and LC/MS/MS analysis.
Level
Conc.
(μg/mL)
Amount on
column (μg)
Amount on
column (pmol)
Time (min)
%B
0
2
1
465
34.88
581.25
80
0.2
2
60
2
315
23.63
393.75
8
50
3
142.5
10.69
178.13
9.5
50
4
127.5
9.56
159.37
10
90
5
102
7.65
127.50
12.5
90
6
60
4.50
75.00
12.51
2
7
22.5
1.69
28.12
16
2
IgG concentrations for calibration levels.
%B
40
20
0
0
2
4
6
8
10 12 14 16
Time (minutes)
LCMS gradient conditions.
Transitions
+/-
Q1 Rod Bias
(V)
CE (V)
Q3 Rod Bias
(V)
937.70>836.25
+
-27
-28
-26
937.70>723.95
+
-27
-30
-22
603.70>805.7
+
-22
-16
-13
Compound Name
TTPPVLDSDGSFFLYSK
100
VVSVLTVLHQDWLNGK
MRM transitions on LCMS-8050 for two IgG peptides monitored.
Noviplex Cards
(2)
(3)
(4)
(1)
Approximately 50 uL of the spiked whole blood was
pipetted onto the Noviplex card test area (1). The spot was
allowed to dry for 3 minutes (2). The top layer of the card
was then peeled back (3) to reveal the plamsa collection
disc. The plasma collection disc was allowed to dry for an
additional 15 minutes. Once the disc was dry (4), it was
placed into an eppendorf tube for solvent extraction.
3
Results - Chromatograms
300000000
275000000
250000000
225000000
200000000
175000000
150000000
125000000
Optimization of Collision Energies for peptides of interest
100000000
75000000
50000000
25000000
Range CE: -50 to -10 V
TTPPVLDSDGFFLYSK
0
1250000
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
min
1000000
Total Ion Chromatogram for IgG
750000
500000
250000
0
6.200
6.225
6.250
6.275
6.300
6.325
6.350
6.375
6.400
6.425
6.450
6.475
Inten.
6.500
6.525
6.550
6.575
6.600
6.625
6.650
6.675
min
938
2.00
[M+2H]+2
1.75
1.50
[P1+2H]+2
1.25
5000
TTPPVLDSDGSFFLYSK
4500
1.00
VVSVLTVLHQDWLNGK
4000
937
[P2+2H]+2
0.75
0.50
3500
938
837
836
397
0.25
3000
352
407
337 369 397
407
379 397
295
283
283
0.00
2500
300
466
443
449
400
524
510
561
724
591
500
724
723
640 658
600
756
700
836
836
836
851
809
800
915
1163
1046
891
900
1000
1100
1200
1300
1400
m/z
2000
1500
1000
500
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
11.5
min
MRM Chromatogram for Level 4 standard of spiked IgG in whole blood.
Carryover Assessment
1100
90
Control - Mouse blood
1000
Blank Injection
80
900
70
800
700
60
600
50
500
40
400
30
300
20
200
10
100
0
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
11.5
min
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
11.5
min
4
Results - Calibration Curves
Calibration Curve and MS Chromatograms
TTPPVLDSDGSFFLYSK
25000
937.70>836.25(+)
937.70>723.95(+)
Level 1
2000
937.70>836.25(+)
937.70>723.95(+)
VVSVLTVLHQDWLNGK
603.70>805.70(+)
Level 7
10000
Level 1
603.70>805.70(+)
600
20000
7500
1500
400
15000
5000
1000
10000
Level 7
500
300
200
5000
500
2500
0
0
0
5.50
5.75
6.00
6.25
6.50
100
5.50
5.75
6.00
6.25
6.50
0
6.00
6.25
6.50
6.00
6.75
6.25
6.50
6.75
Area
30000
2
Area
r = 0.989
r2 = 0.979
25000
50000
20000
15000
25000
10000
5000
0
0
100
200
300
400
0
Conc .
0
100
200
300
400
Conc .
Results - Tables and Replicates
QC data and Calculations for IgG Peptides
VVSVLTVLHQDWLNGK
Sample
Ret. Time
Area
Calc. Conc.
QC 1
6.49
32,492
QC 2
6.516
11,726
QC 3
6.514
QC 4
Std. Conc.
% Accuracy
502.804
465
108.1
167.189
142.5
117.3
8,507
115.155
102
112.9
6.492
2,727
21.745
22.5
96.6
Sample
Ret. Time
Area
Calc. Conc.
Std. Conc.
% Accuracy
QC 1
6.029
61,525
416.447
465
89.6
QC 2
6.052
25,355
155.568
142.5
109.2
QC 3
6.047
16,900
94.58
102
92.7
QC 4
6.029
6,502
19.587
22.5
87.1
TTPPVLDSDGSFFLYSK
5
Skyline Data - Retention Time Replicates
VVSVLTVLHQDWLNGK
TTPPVLDSDGSFFLYSK
y15 - 836.4169++
6.60
6.15
1433 P M_2252014...L7...004
1433 P M_2252014...L6...006
1433 P M_2252014...L7...004
1433 P M_2252014...L6...006
839 AM_2262014...L4...002
Replicate
1433 P M_2252014...L5...008
5.90
839 AM_2262014...L3...003
6.35
839 AM_2262014...L2...004
5.95
1433 P M_2252014...L5...008
6.00
6.40
839 AM_2262014...L4...002
6.45
6.05
839 AM_2262014...L3...003
6.50
6.10
839 AM_2262014...L2...004
6.55
839 AM_2262014...L1...005
Retention Time
6.20
839 AM_2262014...L1...005
Retention Time
y14 - 805.4385++
6.65
Replicate
Integration of Skyline Software into LabSolutions allows for further interrogation of data. Here are representative figures
showing the retention time reproducibility for each peptide monitored during the analysis.
Conclusions
Combining the sample collection technique of next generation plasma separator Noviplex cards for quick plamsa collection
from whole blood, with the automated affinity selection and trypsin digestion of the Perfinity workstation coupled to
LCMS-8050, provides a very rapid and reproducible Immuno-MS platform for quantitation of IgG peptides. Furthermore,
this rapid immuno-MS platform can be applied to many other peptide/protein applications.
PO-CON1473E
Simultaneous Determinations of 20 kinds
of common drugs and pesticides
in human blood by GPC-GC-MS/MS
ASMS 2014
TP 757
Qian Sun, Jun Fan, Taohong Huang,
Shin-ichi Kawano, Yuki Hashi,
Shimadzu Global COE, Shanghai, China
Simultaneous Determinations of 20 kinds of common
drugs and pesticides in human blood by GPC-GC-MS/MS
Introduction
On-line gel permeation chromatography-gas
chromatography/mass spectrometry (GPC-GC-MS) is a
unique technique to cleanup sample that reduce the time
of sample preparation. GPC can efficiently separates fats,
protein and pigments from samples, due to this advantage,
on-line GPC is widely used for pesticide analysis.
Meanwhile, compared to widely used GC-MS, GC-MS/MS
techniques provide much better selectivity thus significantly
lower detection limits. In this work, a new method was
developed for rapid determination of 20 common drugs
and pesticides in human blood by GPC-GC-MS/MS. The
modified QuEChERS method was used for sample
preparation.
Experimental
The human blood samples were extracted with acetonitrile,
then was purified by PSA, C18 and MgSO4 to remove most
of the fats, protein and pigments in samples, then after
on-line GPC-GC-MS/MS analysis which further removed
macromolecular interference material, such as protein and
cholesterol, the background interference brought about by
the complex matrix in samples was effectively reduced.
Sample pretreament
human blood 2 mL
CH3CN
vortex
PSA/C18/MgSO4
vortex
centrifuge
supernatant
evaporate
set volume using moblie phase
GPC-GC-MS/MS
Figure 1 Schematic flow diagram of the sample preparation
2
Instrument
GPC
Mobile phase
Flow rate
Column
Oven temperature
Injection volume
:
:
:
:
:
acetone/cyclohexane (3/7, v/v)
0.1mL/min
Shodex CLNpak EV-200 (2 mmI.D. x 150 mmL.)
40 ºC
10 μL
GCMS-TQ8030
Column
: deactivated silica tubing [0.53 mm(ID) x 5 m(L)]
+pre-column Rtx-5ms [0.25 mm(ID) x 5 m(L)]
Rtx-5ms [0.25mm(ID) x 30 m(L), Thickness: 0.25 μm]
Injector
: PTV
Injector time program
: 120 ºC(4.5min)-(80 ºC/min)-280 ºC(33.7 min)
Oven temperature program : 82 ºC(5min)-(8 ºC/min)-300 ºC(7.75 min)
Linear velocity
: 48.8 cm/sec
Ion Source temperature
: 210 ºC
Interface temperature
: 300 ºC
Results
For all of analytes, recoveries in the acceptable range of
70~120% and repeatability (relative standard deviations,
RSD)≤5% (n=3) were achieved for matrices at spiking levels
of 0.01 µg/mL. The limitis of detection were 0.03~4.4 µg/L.
The method is simple, rapid and characterized with
acceptable sensitivity and accuracy to meet the
requirements for the analysis of common drugs and
pesticides in the human blood.
(x10,000,000)
1.00
0.75
0.50
0.25
0.00
15.0
17.5
20.0
22.5
25.0
27.5
30.0
32.5
35.0
37.5
Figure 2 MRM chromatograms of standard mixture
3
Table 1 Results of method validation for drugs and pesticides
(Concentration range: 5-100 μg/L, LODs: S/N≥3, LOQs: S/N≥10, RSDs: n=3)
0.01 µg/mL
No.
Compound Name
tR
(min)
Correlation
Coefficient*
LOD
(µg/L)
LOQ
(µg/L)
Recovery (%)
RSD (%)
1
Dichlorvos
10.795
0.9993
0.103
0.345
72.9
2.99
2
Methamidophos
11.800
0.9994
0.023
0.076
85.3
3.58
3
Barbital
15.210
0.9994
0.018
0.058
72.4
1.72
4
Sulfotep
17.580
0.9995
0.011
0.037
110.7
2.27
5
Dimethoate
18.310
0.9993
0.400
1.333
103.7
3.10
6
Malathion
21.555
0.9997
0.005
0.016
82.7
2.52
7
Chlorpyrifos
21.715
0.9996
0.010
0.033
85.7
3.57
8
Phenobarbital
22.000
0.9995
0.353
1.177
79.6
3.25
9
Parathion
22.180
0.9993
0.003
0.009
92.3
3.17
10
Triazophos
25.675
0.9994
0.046
0.155
87.7
1.32
11
Zopiclone deg.
26.025
0.9993
0.189
0.631
83.5
1.28
12
Diazepam
27.635
0.9992
0.007
0.022
98.3
1.55
13
Midazolam
29.250
0.9994
0.048
0.160
87.1
2.01
14
Zolpidem
31.225
0.9993
1.298
4.325
99.3
1.01
15
Clonazepam
31.795
0.9995
0.432
1.440
110.0
1.57
16
Estazolam
32.335
0.9994
0.092
0.305
103.7
1.37
17
Clozapine
32.400
0.9991
0.050
0.167
100.6
3.12
18
Alprazolam
32.730
0.9993
0.028
0.095
103.3
1.48
19
Zolpidem
33.095
0.9995
1.027
3.425
87.3
1.75
20
Triazolam
33.700
0.9992
0.027
0.091
81.3
2.56
Conclusion
A very quick, easy, effective, reliable method in human
blood based on modified QuEChERS method was
developed using GPC-GCMS-TQ8030. The performance of
the method was very satisfactory with results meeting
validation criteria. The method has been successfully
applied for determination of human blood samples and
ostensibly has further application opportunities, e.g.
biological samples.
PO-CON1466E
Low level quantitation of Loratadine
from plasma using LC/MS/MS
ASMS 2014
TP498
Shailesh Damale, Deepti Bhandarkar, Shruti Raju,
Rashi Kochhar, Shailendra Rane, Ajit Datar,
Low level quantitation of Loratadine from plasma
using LC/MS/MS
Introduction
Loratadine is a histamine antagonist drug used for the
treatment of itching, runny nose, hay fever and such other
allergies. Here, an LC/MS/MS method has been developed
for high sensitive quantitation of this molecule from
plasma using LCMS-8050, a triple quadrupole mass
spectrometer from Shimadzu Corporation, Japan. Presence
of heated Electro Spray Ionization (ESI) interface in
LCMS-8050 ensured good quantitation and repeatability
even in the presence of a complex matrix like plasma. Ultra
high sensitivity of LCMS-8050 enabled development of a
low ppt level quantitation method for Loratadine.
Loratadine
Ethyl 4- (8-chloro-5, 6-dihydro-11H-benzo [5, 6]
cyclohepta [1, 2-b] pyridin-11-ylidene)
-1-piperidinecarboxylate
Figure 1. Structure of Loratadine
Loratadine, a piperidine derivative, is a potent long-acting,
non-sedating tricyclic antihistamine with selective
peripheral H1-receptor antagonist activity. It is used for
relief of nasal and non-nasal symptoms of seasonal
allergies and skin rashes[1,2,3]. Due to partial distribution in
central nervous system, it has less sedating power
compared to traditional H1 blockers. Loratadine is given
orally, is well absorbed from the gastrointestinal tract, and
has rapid first-pass hepatic metabolism; it is metabolized by
isoenzymes of the cytochrome P450 system, including
CYP3A4, CYP2D6, and, to a lesser extent, several others.
Loratadine is almost totally (97–99 %) bound to plasma
proteins and reaches peak plasma concentration (Tmax) in ~
1–2 h[4,5].
Method of Analysis
This bioanalytical method was developed for measuring
Loratadine in therapeutic concentration range for the
analysis of routine samples. It was important to develop a
simple and accurate method for estimation of Loratadine in
human plasma.
Preparation of matrix matched plasma by protein precipitation method
using cold acetonitrile
To 100 µL of plasma 500 µL cold acetonitrile was added
for protein precipitation. It was placed in rotary shaker at
20 rpm for 15 minutes for uniform mixing. This solution
was centrifuged at 12000 rpm for 15 minutes. Supernatant
was taken and evaporated to dryness at 70 ºC . The
residue was reconstituted in 200 µL Methanol.
Preparation of calibration standards in matrix matched plasma
1 ppt, 5 ppt, 50 ppt, 100ppt, 500 ppt, 1 ppb, 5 ppb and
10 ppb of Loratadine calibration standards were prepared
in cold acetonitrile treated matrix matched plasma.
2
using LC/MS/MS
LC/MS/MS analysis
LCMS-8050 triple quadrupole mass spectrometer by
Shimadzu Corporation, Japan (shown in Figure 2A), sets a
new benchmark in triple quadrupole technology with an
unsurpassed sensitivity (UFsensitivity) with Scanning speed
of 30,000 u/sec (UFscanning) and polarity switching
speed of 5 msecs (UFswitching). This system ensures
highest quality of data, with very high degree of
reliability.
In order to improve ionization efficiency, the newly
developed heated ESI probe combines high-temperature
gas with the nebulizer spray, assisting in the desolvation
of large droplets and enhancing ionization. This
development allows high-sensitivity analysis of a wide
range of target compounds with considerable reduction
in background.
Presence of heated Electro spray interface in LCMS-8050
(shown in Figure 2B) ensured good quantitative sensitivity
even in presence of a complex matrix like plasma.
The parent m/z of 382.90 giving the daughter m/z of
337.10 in the positive mode was the MRM transition used
for quantitation of Loratadine. MS voltages and collision
energy were optimized to achieve maximum transmission
of mentioned precursor and product ion. Gas flow rates,
source temperature conditions and collision gas were
optimized, and linearity graph was plotted for 4 orders of
magnitude.
Figure 2A. LCMS-8050 triple quadrupole mass spectrometer by Shimadzu
Table 1. LC conditions
Column
Mobile Phase
Table 2. LCMS conditions
Shim-pack XR-ODS (100 mm L x 2.0 mm ID ; 2.2 µm)
Time (min)
A conc. (%)
B conc. (%)
0.01
40
60
1.50
0
100
4.00
0
100
4.10
40
60
13.00
Flow Rate
MS Interface
Polarity
A : 0.1% formic acid in water
B : acetonitrile
Gradient Program
Figure 2B. Heated ESI probe
Stop
ESI
Positive
Nebulizing Gas Flow
2.0 L / min (nitrogen)
Drying Gas Flow
10.0 L / min (nitrogen)
Heating Gas Flow
15.0 L / min (zero air)
Interface Temp.
300 ºC
Desolvation Line Temp.
250 ºC
Heater Block Temp.
400 ºC
MRM Transition
382.90 > 337.10
0.15 mL/min
Oven Temperature
40 ºC
Injection Volume
20 µL
3
using LC/MS/MS
Results
LC/MS/MS Analysis
LC/MS/MS method for Loratadine was developed on ESI
+ve ionization mode and 382.90>337.10 MRM transition
was optimized for Loratadine. Checked matrix matched
plasma standards for highest (10 ppb) as well as lowest
(0.001 ppb) concentrations as seen in Figures 4A and 4B
respectively. Optimized MS method to ensure no plasma
interference at the retention time of Loratadine (Figure 5).
Calibration curve was plotted for Loratadine concentration
range. Also as seen in Table 3, % Accuracy was studied to
confirm the reliability of method.
Linear calibration curves were obtained with regression
coefficients R2 > 0.998. % RSD of area was within 15 %
and accuracy was within 80-120 % for all calibration levels.
(x1,000,000)
(x10,000)
3.5 382.90>337.10(+)
LORATADINE/3.391
382.90>337.10(+)
3.0
2.5
5.0
4.0
2.0
3.0
1.5
LORATADINE/3.377
2.0
1.0
1.0
0.5
0.0
0.0
-0.5
-1.0
0.0
2.5
5.0
7.5
0.0
Figure 4A. Mass chromatogram 10 ppb
2.5
5.0
7.5
Figure 4B. Mass chromatogram 0.001 ppb
Specificity and interference
1.2
(x10,000)
1:LORATIDINE 382.90>337.10(+) CE: -23.0 LORA_PLASMA_003.lcd
1:LORATIDINE 382.90>337.10(+) CE: -23.0 LORA_PLASMA_002.lcd
-----------
1.1
1.0
0.9
LOQ Level
Blank
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
-0.2
-0.3
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
min
Figure 5. Overlay chromatogram
4
using LC/MS/MS
Area (x10,000,000)
8
2.0
Area (x100,000)
4
2.0
7
1.0
3
1.0
0.0
234
1
5
1
6
0.0
2
0.0
0.05
2.5
5.0
7.5
0.10
Conc.
Conc.
Figure 6. Loratadine calibration curve
Result Table
Table 3. Results of Loratadine calibration curve
Sr. No.
Standard
(ppb)
(ppb)
(n=3)
% Accuracy
(n=3)
1
STD-01
0.001 0.00096
0.62
95.83 2
STD-02
0.005
0.0050
5.24
100.73 3
STD-03
0.05 0.057
0.98
114.83 4
STD-04
0.1 0.095 1.81
95.40
5
STD-05
0.5
0.048
1.40
95.70
6
STD-06
1.0
0.986
0.11
98.53
7
STD-07
5.0
5.077 1.07
101.53
8
STD-08
10.0
9.983
1.96
99.37
Conclusion
• Highly sensitive LC/MS/MS method for Loaratadine was developed on LCMS-8050 system.
• Calibration was plotted from 10 ppb to 0.001 ppb, and LOQ was computed as 0.001 ppb.
5
using LC/MS/MS
References
[1] Bhavin N. Patel, Naveen Sharma, Mallika Sanyal, and Pranav S. Shrivastav, Journal of chromatographic Sciences,
Volume 48, (2010), 35-44.
[2] J. Chen, YZ. Zha, KP. Gao, ZQ. Shi, XG. Jiang, WM. Jiang, XL. Gao, Pharmazie, Volume 59, (2004), 600-603.
[3] M. Haria, A. Fitton, and D.H. Peters, Drugs, Volume 48, (1994), 617-637.
[4] J. Hibert, E. Radwanski, R. Weglein, V. Luc, G. Perentesis, S. Symchowicz, and N. Zampaglione, J.clin. Pharmacol,
Volume 27, (1987), 694-698.
[5] S.P.Clissold, E.M. Sorkin, and K.L. Goa, Drugs, Volume 37,(1989), 42-57.
Food
• Page 111
An LCMS method for the detection of cocoa
butter substitutes, replacers, and equivalents
in commercial chocolate-like products
• Page 141
High sensitivity quantitation method of dicyandiamide and melamine in milk powders by liquid
chromatography tandem mass spectrometry
• Page 116
Highly sensitive and robust LC/MS/MS method
for quantitative analysis of articial sweeteners
in beverages
• Page 147
Multiresidue pesticide analysis from dried chili
powder using LC/MS/MS
• Page 122
Highly sensitive and rapid simultaneous method
for 45 mycotoxins in baby food samples by
HPLC-MS/MS using fast polarity switching
• Page 129
High sensitivity analysis of acrylamide in potato
chips by LC/MS/MS with modified QuEChERS
sample pre-treatment procedure
• Page 135
Determination of benzimidazole residues in
animal tissue by ultra high performance liquid
chromatography tandem mass spectrometry
• Page 154
Multi pesticide residue analysis in tobacco by
GCMS/MS using QuEChERS as an extraction
method
• Page 161
Simultaneous quantitative analysis of 20 amino
acids in food samples without derivatization
using LC-MS/MS
PO-CON1458E
An LCMS Method for the Detection
of Cocoa Butter Substitutes,
Replacers, and Equivalents in
Commercial Chocolate-like Products
ASMS 2014
ThP632
Jared Russell, Liling Fang and Willard Bankert
Shimadzu Scientific Instruments., Columbia, MD
An LCMS Method for the Detection of Cocoa Butter Substitutes,
Replacers, and Equivalents in Commercial Chocolate-like Products
Introduction
There is increasing demand for genuine cocoa butter (CB)
in chocolate products in developed nations, however, this
demand has created a shortage of CB and raised its costs.
To overcome this, chocolate manufactures sometimes add
vegetable-derived fats to some chocolate products to
reduce costs while still maintaining desirable physical
characteristics. It is of current interest to have a reliable
method to detect, identify, and quantify the triacylglycerol
(TAG) components of cocoa butter substitutes, replacers,
and equivalents (CBEs) in chocolate products. Traditionally
GC was used for this task, but due to the low volatility of
triacylglycerides and their susceptibility to thermal
decomposition, retention time is the only identifying factor
for the TAGs and typical GC analyses of this type can take
40 minutes. LCMS is able to not only provide faster
throughput, but also has the additional advantage of
allowing characterization of the TAG, including qualitative
regiospecific analysis. We have developed a single, UHPLC
column-based LCMS method to analyze the TAG
components in commercial chocolate and chocolate-like
products. This analysis has a runtime of 17minutes, making
it suitable for relatively high throughput. Additionally, the
method was very repeatable, with an interday variability of
<7% for the absolute area counts of the three major TAGs
in CB (POP,POS,SOS).
Materials and Method
A Shimadzu Nexera UHPLC coupled to a Shimadzu
LCMS-8040 triple quadrupole mass spectrometer was
utilized for this analysis. A pure CB standard was used as a
reference. Chocolate and chocolatey products were
purchased in retail stores over a range of cocoa content.
Sample Preparation
For analysis, we slightly modified a sample preparation
method originally used for algal oils. For analysis, 5mg of
sample was weighed and then dissolved in a 3:1
Toluene-Isopropyl Alcohol solution. We then sonicated the
mixture for 5 minutes. The solution was filtered through a
Thomson filter vial (P/N 35538-100) to remove sugars and
other insoluble materials and diluted 5-fold using 3:1
Toluene-IPA and injected into the UHPLC-MS system.
Chromatography
Instrument
Column
Mobile Phase A
Mobile Phase B
Gradient Program
Flow Rate
Column Temperature
Injection Volume
:
:
:
:
:
Shimadzu Nexera UHPLC system
Shimadzu Shim-Pack XR-ODSIII (200x2.1mm,)
LC/MS Acetonitrile
1:1 Dichloromethane-Isopropyl Alcohol
48% B (initially) – gradient to 51% B (0-8.0 min) – gradient to 54% B
(8.0 – 11.0 min) – gradient to 74% B (11.0-14.0 min) – hold at 74% B
(14.0-15.0 min) – reequilibrate at 48% B (15.1-17 min)
: 0.33 mL/min
: 30°C
: 1 μL
Mass Spectrometry
Instrument
Ionization
Polarity
Scan Mode
:
:
:
:
Shimadzu LCMS-8040 Triple Quadrupole Mass Spectrometer
APCI
Positive
Q3 Scan
2
Results
Retail Chocolates from Hershey’s, Lindt and Tcho, as well
as a chocolatey candy - Charleston Chew - were compared
against pure cocoa butter. The chocolates used were
selected to cover a range of Cocoa content and purity. We
specifically chose to use Hershey’s Mr. Goodbar and
Charleston Chews because they listed the use of vegetable
oils in their ingredients list. As you can see in the
chromatograms, the products that market themselves as
pure chocolate have similar chromatograms in comparison
to the pure CB.
We used an MS library that was provided to us by Dr. John
Carney and Mona Koutchekinia to identify the types of
TAGs contained in the chocolates using the spectral
information captured in the Q3 scans. A minimum
similarity of 70 was required for a result to be considered a
match. In order to identify usage of CBEs, we applied the
equation: %POP<44.025-0.733*%SOS, which was
determined by the European Commission Joint Research
Centre, which can detect around 2% CBE usage in CB
content, or approximately 0.4% CBE content in chocolate.
The chocolate products we tested all agreed with the
expected results: All of the dark chocolate products we
tested passed this specification, as well as Hershey’s Milk
Chocolate. The two products which had a higher %POP
than is allowable, Mr. Goodbar and Charleston Chew,
were selected specifically for the inclusion of vegetable
oils. It may be informative to further test the accuracy of
this testing method by adulterating cocoa butter with
known quantities of CBEs. The data has been summarized
in Table 1.
Table 1: Percentage of the major TAGs in CB in various chocolate products
Product
%POP
%POS
%SOS
%POP needs to
be less than
Cocoa Butter
23.7%
46.9%
29.5%
43.8
Lindt 85% Cocoa
16.9%
46.4%
36.6%
43.8
TCHO 70% from Ghana
17.8%
46.1%
36.1%
43.8
TCHO 65% from Ecuador
20.9%
46.2%
32.9%
43.8
Hershey's Special Dark
20.0%
47.1%
32.9%
43.8
Hershey's Milk Chocolate
18.6%
46.6%
34.8%
43.8
Hershey's Mr Goodbar
44.8%
21.1%
34.1%
43.8
Charleston Chew
100.0%
0.0%
0.0%
44.0
3
SOS*
POS
(x100,000,000)
1:TIC(+) Cocoa Butter.lcd
1:TIC(+) Lindt 85% Cocoa.lcd
1.5 1:TIC(+) TCHO 70% from Ghana.lcd
1:TIC(+) TCHO 65% from Ecuador.lcd
1.4 1:TIC(+) Hershey's Special Dark 45% Cacao.lcd
1:TIC(+) Hershey's Milk Chocolate.lcd
POP
1.3
1.2
1.1
OOP
PLP
0.9
OOS
1.0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
13.0
14.0
15.0
16.0
min
14.0
15.0
16.0
min
POS
(x10,000,000)
9.0 1:TIC(+) Cocoa Butter.lcd
1:TIC(+) Hershey's Mr. Goodbar.lcd
8.5 1:TIC(+) Charleston Chew.lcd
12.0
8.0
SOS*
7.5
7.0
6.5
POP
6.0
5.5
5.0
4.5
4.0
PLP
OOP
3.0
2.5
OOS
3.5
2.0
1.5
1.0
0.5
0.0
0.0
1.0
2.0
9.0
10.0
11.0
12.0
13.0
Figure 1. Chromatograms of the various chocolate products analyzed versus pure cocoa butter
4
Conclusions
We have developed a 17 minute method for the rapid determination of CBE usage in chocolate products by using a
UHPLC column and Q3 ion scans to analyze samples and then matching spectral information with an MS library of ion
ratios for identifying TAGs.
Further studies could add a calibration curve to enable quantification of TAGs. This method should also provide a base
method which can be modified to support TAG analysis in other product types.
References
Co ED, Koutchekinia M, Carney J et al. Matching the Functionality of Single-Cell Algal Oils with Different Molecular
Compositions. 2014.
Buchgraber M and Anklam E. Validation of a Method for the Detection of Cocoa Butter Equivalents in Cocoa Butter and
Plain Chocolate. 2003.
Acknowledgements
Dr. John Carney and Mona Koutchekinia for the invaluable information they provided.
PO-CON1471E
Highly Sensitive and Robust LC/MS/MS
Method for Quantitative Analysis
of Artificial Sweeteners in Beverages
ASMS 2014
MP351
Jie Xing1, Wantung Liw1, Zhi Wei Edwin Ting1,
Yin Ling Chew*2 & Zhaoqi Zhan1
1
Customer Support Centre, Shimadzu (Asia Pacific)
Pte Ltd, 79 Science Park Drive, #02-01/08, SINTECH IV,
Singapore Science Park 1, Singapore 118264
2
Department of Chemistry, Faculty of Science,
National University of Singapore, 21 Lower Kent
Ridge Road, Singapore 119077, *Student
Highly Sensitive and Robust LC/MS/MS Method for
Quantitative Analysis of Artificial Sweeteners in Beverages
Introduction
Artificial sweeteners described as intense, low-calorie and
non-nutritive are widely used as sugar substitutes in
beverages and foods to satisfy consumers’ desire to sweet
taste while concerning about obesity and diabetes. As
synthetic additives in food, the use of artificial sweeteners
must be approved by authority for health and safety
concerns. For example, Aspartame, Acesulfame-K,
Saccharin, Sucralose and Neotame are the FDA approved
artificial sweeteners on the US market. However, there are
also many other artificial sweeteners allowed to use in EU
and many other countries (Table 2), but not in the US. In
this regard, analysis of artificial sweeteners in beverages
and foods has become essential due to the relevant
regulations in protection of consumers’ benefits and safety
concerns in many countries [1, 2]. Recently, artificial
sweeteners are found as emerging environmental
contaminants in surface water and waste water [3].
Initially, HPLC analysis method with ELSD detection was
adopted, because many artificial sweeteners are non-UV
absorption compounds [2]. Recently, LC/MS/MS methods
have been developed and used for identification and
quantitation of artificial sweeteners in food and beverages
as well as water for its high sensitivity and selectivity [3, 4].
Here we report a high sensitivity LC/MS/MS method for
identification and quantitation of ten artificial sweeteners
(Table 2) in beverage samples. An ultra-small injection
volume was adopted in this study to develop a very robust
LC/MS/MS method suitable for direct injection of beverage
samples without any sample pre-treatment except dilution
with solvent.
Experimental
Ten artificial sweeteners of high purity as listed in Table 2
were obtained from chemicals suppliers. Stock standard
solutions and a set of calibrants were prepared from the
chemicals with methanol/water (50/50) solvent as the
diluent. Three brand soft-drinks and a mouthwash bought
from local supermarket were used as testing samples in
this study. The samples were not pretreated by any means
except dilution with the diluent prior to injection into
LCMS-8040 (Shimadzu Corporation, Japan), a triple
quadrupole LC/MS/MS system. The front-end LC system
connected to the LCMS-8040 is a high pressure binary
gradient Nexera UHPLC. The details of analytical conditions
of LC/MS/MS method are shown in Table 1.
Table 1: LC/MC/MS analytical conditions of artificial sweeteners on LCMS-8040
Column
Flow Rate
Mobile Phase
Gradient program
MS mode
ESI condition
Inj. Vol.
Synergi, Polar-RP C18 (100 x 2 mm, 2.5µm )
0.25 mL/min
A: water with 0.1% Formic acid - 0.03% TA
B: MeOH with 0.1% FA - 0.03% Trimethylamine
B: 10% (0.01 to 0.5 min) → 95% (8 to 9 min) → 10% (9.01 to 11min)
ESI, MRM, positive-negative switching
Nebulizing gas: 3L/min, Drying gas: 15L/min, Heating block: 400ºC, DL: 250ºC
0.1uL, 0.5uL, 1uL, 5uL and 10uL
2
Method development
First, precursor selection and MRM optimization of the ten
sweeteners studied was carried out using an automated
MRM optimization program of the LabSolutions. Six
compounds were ionized in negative mode and four in
positive mode as shown in Table2. For each compound,
two optimized MRM transitions were selected and used,
with the first one for quantitation and the second one for
confirmation.
The ten compounds were well-separated as sharp peaks
between 2 min and 8.2 min as shown in Figure 1. Linear
calibration curves of wide concentration ranges were
established with mixed standards in diluent as summarized
in Table 2. We also investigated the performance of the
LC/MS/MS method established by employing very small
injection volumes (0.1, 0.5, 1 and 5 uL). This is because
actual beverages usually contain very high contents of
sweeteners (>>1ppm) to MS detection. Analysts normally
dilute the samples before injection into LC/MS/MS. An
alternative way is to inject a very small volume of samples
even without dilution. Figs 2 & 3 show a chromatogram
and calibration curves established with 0.1uL injection,
which demonstrates the feasibility of an ultra-small
injection volume combined with high sensitivity LC/MS/MS.
Table 2: Artificial Sweeteners, MRM transitions and calibration curves on LCMS-8040
Cat1
Compd. & Abbr. Name
A2
Acesulfame K (Ace-K)
A5
Cyclamate (CYC)3
A3
Saccharin (SAC)
A4
Sucralose2 (SUC)
A1
Aspartame (ASP)
A6
Neotame (NEO)
B1
Alitame (ALI)
B3
Dulcin (DUL)
B2
Neohespiridin
Dihydrochalcone (NHDC)
C1
Glycyrrhi-Zinate (GLY)
MRM parameter
RT & Calibration Curve4
Trans. (m/z)
Pola. (+/-)
Q1 (V)
CE (V)
Q3 (V)
161.9 >82.1
-
11
14
29
161.9 >78.0
-
11
32
28
178.3 >80.1
-
19
24
30
178.3 >79.0
-
12
27
10
181.9 >106.1
-
13
20
15
181.9 >42.1
-
13
36
13
441.0 >395.1
-
20
11
25
441.0 >359.1
-
20
15
23
295.1 >120.1
+
-19
-25
-25
295.1 >180.1
+
-19
-14
-20
379.3 >172.2
+
-18
-23
-20
379.3 >319.3
+
-18
-18
-24
332.2 >129.1
+
-23
-19
-26
332.2 >187.1
+
-23
-16
-21
181.1 >108.1
+
-22
-25
-21
181.1 >136.1
+
-21
-18
-26
611.3 >303.1
-
30
38
30
611.3 >125.3
-
30
47
20
821.5 >351.2
-
22
46
20
821.5 >193.2
-
22
52
19
RT (min)
Conc. R. (ug/L)
R2
1.99
1 - 20000
0.9999
2.87
5 - 20000
0.9996
3.28
1 - 20000
0.9984
4.61
5 - 20000
0.9983
5.15
0.1 - 2000
0.9999
7.51
0.05 - 1000
0.9998
5.44
0.1 - 2000
0.9995
5.58
5 - 10000
0.999
6.71
0.5 - 2000
0.9988
8.19
5 - 1000
0.9996
1. A1~A6: US FDA, EU and others approval; B1~B3: only EU and other countries approval. C1: natural sweetener, info not available.
2. Sucralose precursor ion m/z 441.0 is formic acid adduct ion.
3. Sodium cyclamate known as “magic sugar” was initially banned in the US in 2000. FDA lifted the ban in 2013.
4. Injection volume: 10 uL
3
Glycyrrhizic
1.0
NHDC
Cyclamate
2.0
Aspartame
Alitame
3.0
Saccharin
4.0
Sucralose
Acesulfame K
5.0
Neotame
Dulcin
(x10,000)
0.0
0.0
2.5
5.0
7.5
10.0
min
Figure 1: MRM Chromatogram of ten sweeteners by LC/MS/MS with 10uL injection:
Asp & Ali 1ppb, Neo 0.5ppb, Dul, Gly, Ace-K, Sac, Suc and Cyc 10ppb, NHDC 1ppb.
Area (x10,000)
Area (x100,000)
4.0
Ace-K
r2=0.9977
3.0
Area(x10,000)
1.0
1.0
1.0
Conc.
Conc.
0.0
NEO
r2=0.9982
Conc.
10000
Conc.
Conc.
0.0
0
0
10000
Conc.
Conc.
Area (x10,000)
DUL
r2=0.9987
3.0
0.0
0.5
5.0
2.0
0
0
Conc.
1000
Conc.
0.0
0.0
0
0
500 Conc.
Conc.
0.0
0
1000
Conc.
Conc.
Area (x10,000)
GLY
r2=0.9997
5.0
0.0
0.0
0
1000
Area(x1,000)
2.5
2.5
0.5
1.0
10000
0.0
0
Area(x1,000)
1.0
2.0
1.0
1.0
0.5
0.0
Area(x10,000)
0.0
7.5
NHDC
r2=0.9991
4.0
25.0 Conc.
Conc.
0.0
0.0
0
0
10000
500 Conc.
Conc.
Figure 3: Calibration curves of artificial sweeteners on LCMS-8040 with
an ultra-small injection volume (0.1 uL) of same set of calibrants as shown in Table 2.
(x1,000)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
Glycyrrhizic
4.0
Neotame
4.5
NHDC
500
0.0
0.0
24
0
Area (x100,000)
Area(x10,000)
1.0
0.5
25.0 Conc.
0.0
2.5
Dulcin
0.0
0.0
0
Conc.
0.5
Area(x1,000)
3.0
1.0
1.0
0.0
500 Conc.
ALI
r2=0.9990
1.5
Area(x10,000)
2.5
0
10000
Area (x100,000)
Area (x100,000)
5.0
0
Area(x1,000)
2.5
Aspartame
Alitame
10000
500
Saccharin
0
0.0
0
2.5
0.5
ASP
r2=0.9983
1.0
1.0
Sucralose
0.0
0.0
Area (x100,000)
1.5
SUC
r2=0.9991
1.5
Area(x1,000)
5.0
Cyclamate
1.0
5.0
Area(x1,000)
2.0
Area (x100,000)
SAC
r2=0.9977
Acesulfame K
2.0
Area (x10,000)
CYC
r2=0.9948
0.0
0.0
2.5
5.0
7.5
10.0
min
Figure 2: MRM Chromatogram of ten sweeteners by LC/MS/MS with 0.1uL injection:
Asp & Ali 0.1ppm, Neo 0.05ppm, Dul, Gly, Ace-K, Sac, Suc and Cyc 1ppm, NHDC 0.1ppm.
4
Method performance
Table 3 summarizes the results of repeatability and
sensitivity of the method with mixed standards. The method
was not evaluated with beverage spiked samples. However,
because beverage samples are normally diluted many times,
matrix effect and interferences can be ignored for high
sensitivity LC/MS/MS analysis. The results indicate that the
method with ultra-small injection volume exhibits good
linearity, repeatability and sensitivity.
Table 3: Repeatability and Sensitivity of LC/MS/MS method of artificial sweeteners
Name
Repeatability (peak area), 10uL
Sensitivity (ug/L)
Conc. (ug/L)
RSD%
Conc. (ug/L)
RSD%
Ace-K
20
5.1
100
5.2
LOQ/LOD (0.1 µL inj)
200
50
40
10
CYC
20
11.7
100
8.1
800
500
200
SAC
20
8.0
100
5.8
250
100
50
SUC
20
7.5
100
2.7
200
100
ASP
2
7.8
10
3.0
80
20
NEO
1
5.3
5
1.0
5
ALI
2
8.6
10
1.7
DUL
20
7.5
100
3.1
NHDC
2
9.2
10
4.6
GLY
20
8.2
100
5.4
LOQ/LOD (0.5 µL inj)
LOQ/LOD 10 (µL inj)
4.0
1.33
90
14
4.5
20
4.5
1.5
50
15
2.4
0.8
20
4
0.5
0.17
3
2
1
0.03
N.A.
40
25
10
5
0.2
N.A.
160
50
30
10
1.4
0.5
100
25
40
6
0.5
0.18
400
150
15
5
5.0
1.8
Analysis of beverage samples
The LC/MS/MS method established was applied for
screening and quantitation of the targeted sweeteners in
three brand beverages: S1, S2 and S3, and a mouthwash
S4. The results are shown in Figure 4 and Table 4. It is
interested to note that glycyrrizinate was found in the
mouthwash.
Table 4: Screening and quantitation results for ten artificial sweeteners in beverages and mouthwash (mg/L)
Artificial Sweetener
S1
S2
S3
S4
ASP
116.9
127.9
ND
ND
Ace-K
143.9
165.9
97.2
ND
Saccharin
ND
ND
ND
208.7
SUC
55.1
ND
183.4
ND
GLY
ND
ND
ND
449.3
Others
ND
ND
ND
ND
1. S2 was diluted 100 times, the rests were diluted 10 times. 1 uL injection.
2. ND = not detected.
5
S2
4.0
3.0
2.0
1.0
Acesulfame K (x10)
2.0
Aspartame
3.0
Sucralose (x10)
S1
4.0
5.0
Aspartame
(x100,000)
(x1,000,000)
Acesulfame K (x10)
1.0
0.0
0.0
0.0
2.5
5.0
7.5
10.0 min
0.0
7.5
5.0
7.5
10.0 min
1.5
S4
1.0
1.0
0.5
0.0
0.0
0.0
Glycyrrhizic
Sucralose
Acesulfame K
2.0
S3
5.0
(x100,000)
(x100,000)
3.0
2.5
Saccharin
5.0
2.5
5.0
7.5
10.0 min
0.0
2.5
10.0 min
Figure 4: Screening and quantitation for 10 targeted artificial sweeteners in beverage and mouthwash samples by LC/MS/MS with 1uL injection.
Conclusions
A MRM-based LC/MS/MS method was developed and
evaluated for screening and quantitation of ten artificial
sweeteners in beverages. This high sensitivity LC/MS/MS
method combined with small or ultra-small injection
volume (0.1~1.0 uL) was proven to be feasible and reliable
in actual samples analysis of the targeted sweeteners in
beverages, achieving high throughput and free of sample
pre-treatment (except dilution). The method is expected to
be applicable to surface water and drinking water samples.
For wastewater and various foods, sample pre-treatment is
usually required. However, the advantages of the method
in high sensitivity and ultra-small injection volume are
expected to enable it tolerates relatively simple sample
pre-treatment procedures.
References
1. http://en.wikipedia.org/wiki/Sugar_substitute and EU directive 93/35/EC, 96/83/EC, 2003/115/EC, 2006/52/EC and
2009/163/EU.
2. Buchgraber and A. Wasik, Report EUR 22726 EN (2007).
3. F.T. Large, M. Scheurer and H.-J Brauch, Anal Bioanal Chem, 403: 2503-2518 (2012)
4. Ho-Soo Lim, Sung-Kwan Park, In-Shim Kwak, Hyung-Ll Kim, Jun-Hyun Sung, Mi-Youn Byun and So-Hee Kim, Food Sci,
Biotechnol, 22(S):233-240 (2013)
PO-CON1480E
Highly sensitive and rapid
simultaneous method for 45 mycotoxins
in baby food samples by HPLC-MS/MS
using fast polarity switching
ASMS 2014
MP345
Stéphane MOREAU1 and Mikaël LEVI2
Shimadzu Europe, Albert-Hahn Strasse 6-10,
Duisburg, Germany
2
Allende, 77448 Marne la Vallée Cedex 2, France
1
Highly sensitive and rapid simultaneous method for 45 mycotoxins
in baby food samples by HPLC-MS/MS using fast polarity switching
Introduction
Mycotoxins are toxic metabolites produced by fungal
molds on food crops. For consumer food safety, quality
control of food and beverages has to assay such
contaminants. Depending on the potency of the mycotoxin
and the use of the food, the maximum allowed level is
defined by legislation. Baby food is particularly critical. For
example, European Commission has fixed the maximum
level of Aflatoxin B1 and M1 to 0.1 and 0.025 µg/kg,
respectively, in baby food or milk.
Therefore, a sensitive method to assay mycotoxins in
complex matrices is mandatory. In order to ensure
productivity of laboratory performing such assays, a unique
rapid method able to measure as much mycotoxins as
possible independently of the sample origin is also needed.
In this study, we tested three kind of samples: baby milk
powder, milk thickening cereals (flour, rice and tapioca)
and a vegetable puree mixed with cereals.
Sample preparation
Sample preparation was performed by homogenization
followed by solid phase extraction using specific cartridges
(Isolute® Myco, Biotage, Sweden) covering a large
spectrum of mycotoxins.
Sample (5g) was mixed with 20 mL of water/acetonitrile
(1/1 v/v), sonicated for 5 min and agitated for 30 min at
room temperature. After centrifugation at 3000 g for 10
min, the supernatant was diluted with water (1/4 v/v).
Columns (60mg/3 mL) were conditioned with 2 mL of
acetonitrile then 2 mL of water. 3 mL of the diluted
supernatant were loaded at the lowest possible flow rate.
Then column was washed with 3 mL of water followed by
3 mL of water/acetonitrile (9/1 v/v). After drying,
compounds were successively eluted with 2 mL of
acetonitrile with 0.1% of formic acid and 2 mL of
methanol.
The eluate was evaporated under nitrogen flow at 35 ºC
until complete drying (Turbovap, Biotage, Sweden).
The sample was reconstituted in 150 µL of a mixture of
water/methanol/acetonitrile 80/10/10 v/v with 0.1% of
formic acid.
LC-MS/MS analysis
Extracts were analysed on a Nexera X2 (Shimadzu, Japan)
UHPLC system and coupled to a triple quadrupole mass
spectrometer (LCMS-8050, Shimadzu, Japan). Analysis was
carried out using selected reaction monitoring acquiring 2
transitions for each compound.
2
Table 1 – LC conditions
Analytical column
Mobile phase
Gradient
Column temperature
Injection volume
Flow rate
: Shimadzu GLC Mastro™ C18 150x2.1 mm 3µm
: A = Water 2mM ammonium acetate and 0.5% acetic acid
B = Methanol/Isopropanol 1/1 + 2mM ammonium acetate
and 0.5% acetic acid
: 2%B (0.0min), 10%B (0.01min), 55%B (3.0min), 80%B (7.0 -8.0min),
2%B (8.01min), Stop (11.0min)
: 50ºC
: 10 µL
: 0.4 mL/min
Table 2 – MS/MS conditions
Ionization mode
Temperatures
Gas flows
CID gas pressure
Polarity switching time
Pause time
Dwell time
: Heated ESI (+/-)
: HESI: 400ºC
Desolvation line: 250ºC
Heat block: 300ºC
: Nebulizing gas (N2): 2 L/min
Heating gas (Air): 15 L/min
Drying gas (N2): 5 L/min
: 270 kPa (Ar)
: 5 ms
: 1 ms
: 6 to 62 ms depending on the number of concomitant transitions
to ensure a minimum of 30 points per peak in a maximum loop time
of 200 ms (including pause time and polarity switching)
3
Table 3 – MRM transitions
Name
Ret. Time (min)
MRM Quan
MRM Qual
15-acetyldeoxynivalenol (15ADON) [M+H]+
3.37
339 > 297.1
339 > 261
3-acetyldeoxynivalenol (3ADON) [M+H]+
3.37
339 > 231.1
339 > 231.1
Aflatoxine B1 (AFB1) [M+H]+
3.78
312.6 > 284.9
312.6 > 240.9
Aflatoxine B2 (AFB2) [M+H]+
3.57
315.1 > 259
315.1 > 286.9
Aflatoxine G1 (AFG1) [M+H]+
3.46
329.1 > 242.9
329.1 > 199.9
Aflatoxine G2 (AFG2) [M+H]+
3.26
330.9 > 244.9
330.9 > 313.1
Aflatoxine M1 (AFM1) [M+H]+
3.30
329.1 > 273
329.1 > 229
Alternariol [M-H]-
4.78
257 > 214.9
257 > 213.1
Alternariol monomethyl ether [M-H]-
5.81
271.1 > 255.9
271.1 > 228
Beauvericin (BEA) [M+H]+
8.03
784 > 244.1
784 > 262
Citrinin (CIT) [M+H]+
4.16
251.3 > 233.1
251.3 > 205.1
D5-OTA (ISTD)
5.22
409.2 > 239.1
N/A
Deepoxy-Deoxynivalenol (DOM-1) [M-H]-
3.02
279.2 > 249.3
279.2 > 178.4
Deoxynivalenol (DON) [M-CH3COO]-
2.61
355.3 > 295.2
355.3 > 265.1
Deoxynivalenol 3-Glucoside (D3G) [M+CH3COO]-
2.45
517.5 > 457.1
517.5 > 427.1
Deoxynivalenol 3-Glucoside (D3G) [M+CH3COO]-
2.45
517.5 > 457.1
517.5 > 427.1
Diacetoxyscirpenol (DAS) [M+NH4]+
1.20
384 > 283.3
384 > 343
Enniatin A (ENN A) [M+H]+
8.51
699.2 > 682.2
699.3 > 210
Enniatin A1 (ENN A1) [M+H]+
8.22
685.3 > 668.3
685.3 > 210.1
Enniatin B (ENN B) [M+H]+
7.57
657 > 640.4
657 > 195.9
Enniatin B1 (ENN B1) [M+H]+
7.92
671.2 > 654.2
671.2 > 196
Fumagillin (FUM) [M+H]+
6.16
459.2 > 131.1
459.2 > 338.7
Fumonisine B1 (FB1) [M+H]+
4.10
722.1 > 334.2
722.1 > 352.2
Fumonisine B2 (FB2) [M+H]+
4.71
706.2 > 336.3
706.2 > 318.1
Fumonisine B3
4.38
706.2 > 336.2
706.2 > 688.1
Fusarenone-X (FUS-X) [M+H]+
2.84
355.1 > 247
355.1 > 175
HT2 Toxin [M+Na]+
4.58
446.9 > 344.9
446.9 > 285
Moniliformin (MON) [M-H]-
1.16
97.2 > 40.9
N/A
Neosolaniol (NEO) [M+NH4]+
2.90
400.2 > 215
400.2 > 185
Nivalenol (NIV) [M+CH3COO]-
2.41
371.2 > 280.9
371.2 > 311.1
Ochratoxin A (OTA) [M+H]+
5.53
404.2 > 239
404.2 > 358.1
Ochratoxin B (OTB) [M+H]+
4.83
370.2 > 205.1
370.2 > 187
Patulin (PAT) [M-H]-
2.35
153 > 81.2
153 > 53
Sterigmatocystin (M+H]+
5.60
325.3 > 310
325.3 > 281.1
T2 Tetraol [M+CH3COO]-
1.64
356.8 > 297.1
356.8 > 59.1
T2 Toxin [M+NH4]+
4.94
484.2 > 215
484.2 > 305
Tentoxin [M-H]-
4.77
413.1 > 140.9
413.1 > 271.1
Tenuazonic acid (TEN) [M-H]-
4.51
196.1 > 138.8
196.1 > 112
Wortmannin (M-H)
3.95
426.9 > 384
426.9 > 282.1
Zearalanol (alpha) (ZANOL) [M-H]-
5.17
321.3 > 277.2
321.3 > 303.2
Zearalanol (beta) (ZANOL) [M-H]-
4.85
321.3 > 277.2
321.3 > 303.1
Zearalanone (ZOAN) [M-H]-
5.43
319 > 275.1
319 > 301.1
Zearalenol (alpha) (ZENOL) [M-H]-
5.25
319.2 > 275.2
319.2 > 160.1
Zearalenol (beta) (ZENOL) [M-H]-
4.94
319.2 > 275.2
319.2 > 160.1
Zearalenone (ZON) [M-H]-
5.52
316.8 > 174.9
316.8 > 131.1
4
Results and discussion
Method development
LC conditions were transferred from a previously described
method (Tamura et al., Poster TP-739, 61st ASMS). In
particularly, the column was chosen to provide very good
peak shape for chelating compounds like fumonisins
thanks to its inner PEEK lining.
Stainless steel Body
Polymer frit
Small adjustments in the mobile phase and in the gradient
program were made to handle more mycotoxins, especially
the isobaric ones. These modifications are reported in the
Table 1.
Polymer lining
Stationary phase
Figure 1 – Structure of the Mastro™ column
Also, autosampler rinsing conditions were kept to ensure
carry-over minimisation of some difficult compounds.
Electrospray parameters (gas flows and temperatures) were
cautiously optimized to find the optimal combination for
the most critical mycotoxins (aflatoxins). Since these
parameters act in a synergistic way, a factorial design
experiment is needed to find it. Manually testing all
combinations in the chromatographic conditions is very
time consuming. Therefore, new assistant software
(Interface Setting Support) was used to generate all
possible combinations and generate a rational batch
analysis. Optimal combination was found in
chromatographic conditions. The difference observed
between optimum and default or worst parameters was of
200 and 350%, respectively.
Figure 2 – Parameters selection view in the Interface Setting Support Software
5
Results
Extraction and ionisation recovery for aflatoxins was
measured in the three matrices by comparing peak areas of
the raw sample extract to extract spiked at 50 ppb after or
before extraction and to standard solution. Results in table
4 showed that the total recovery was quite acceptable to
ensure accurate quantification. Results from other matrices
were not significatively different.
Table 4 – Extraction and ionisation recoveries in puree
AFB1
AFB2
AFG1
AFG2
AFM1
Extraction recovery
101%
109%
104%
114%
118%
Ionisation recovery
49%
90%
96%
106%
91%
Total recovery
49%
98%
100%
121%
108%
Repeatability was evaluated at low level for aflatoxins. Figure 3 shows an overlaid chromatogram (n=4) for aflatoxins.
(x10,000)
2.50
2.25
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
3.0
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
4.0
min
Figure 3 – Chromatogram of aflatoxins at 0.1 ppb in milk thickening cereals
12000000
11500000
11000000
10500000
10000000
9500000
9000000
8500000
8000000
7500000
7000000
6500000
6000000
5500000
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
-500000
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
min
Figure 4 – Chromatogram of the 45 mycotoxins in standard at 50 ppb (2 ppb for aflatoxins and ochratoxines)
6
Conclusion
• A very sensitive method for multiple mycotoxines was set up to ensure low LOQ in baby food sample,
• Thanks to high speed polarity switching, a high number of mycotoxines can be assayed using the same method in a
short time,
• The extraction method demonstrate good recoveries to ensure accurate quantification.
PO-CON1461E
High Sensitivity Analysis of Acrylamide
in Potato Chips by LC/MS/MS
with Modified QuEChERS Sample
Pre-treatment Procedure
ASMS 2014
MP342
Zhi Wei Edwin Ting1; Yin Ling Chew*2;
Jing Cheng Ng*2; Jie Xing1; Zhaoqi Zhan1
1
Shimadzu (Asia Pacific) Pte Ltd, Singapore, SINGAPORE;
2
Ridge Road, Singapore119077, *Student
High Sensitivity Analysis of Acrylamide in Potato Chips by
LC/MS/MS with Modified QuEChERS Sample Pre-treatment Procedure
Introduction
Acrylamide was found to form in fried foods like
potato-chips via the so-called Maillard reaction of
asparagine and glucose (reducing sugar) at higher
temperature (120ºC) in 2002 [1,2]. The health risk of
acrylamide present in many processing foods became a
concern immediately, because it is known that the
compound is a neurotoxin and a potential carcinogen to
humans [3]. Various analytical methods, mainly LC/MS/MS
and GC/MS based methods, were established and used in
analysis of acrylamide in foods in recent years [4]. We
present a novel LC/MS/MS method for quantitative
determination of acrylamide in potato chips with using a
modified QuEChERS procedure for sample extraction and
clean-up, achieving high sensitivity and high recovery. A
small sample injection volume (1uL) was adopted purposely
to reduce the potential contamination of samples to the
interface of MS system, so as to enhance the operation
stability in a laboratory handling food samples with high
matrix contents.
Experimental
Acrylamide and isotope labelled acrylamide-d3 (as internal
standard) were obtained from Sigma-Aldrich. The
QuEChERS kits were obtained from RESTEK. A modified
procedure of the QuEChERS was optimized and used in the
sample extraction of acrylamide (Q-sep Q100 packet,
original unbuffered) in potato chips and clean-up of matrix
with d-SPE tube (Q-sep Q250, AOAC 2007.01). Acrylamide
and acrylamide-d3 (IS) stock solutions and diluted
calibrants were prepared using water as the solvent.
Method development and performance evaluation were
carried out using spiked acrylamide samples in the
extracted potato chip matrix. A LCMS-8040 triple
quadrupole LC/MS/MS (Shimadzu Corporation, Japan) was
used in this work. A polar-C18 column of 2.5µm particle
size was used for fast UHPLC separation with a gradient
elution method. Table 1 shows the details of analytical
conditions on LCMS-8040 system,.
Table 1: LC/MS/MS analytical conditions of LCMS-8040 for acrylamide
LC condition
Column
Flow Rate
MS Interface condition
Phenomenex Synergi 2.5u Polar-Rp 100A
(100 x 2.00mm)
0.2 mL/min
Mobile Phase
A: water
B: 0.1% formic acid in Methanol
Elution Mode
Gradient elution, B%: 1% (0 to 1 min) →
80% (3 to 4.5 min) → 1% (5.5 to 10min)
Oven Temp.
40ºC
Injection Vol.
1.0 µL
Interface
ESI
MS mode
Positive, MRM, 2 transitions each compound
Block Temp.
400ºC
DL Temp.
200ºC
CID Gas
Ar (230kPa)
Nebulizing Gas Flow
N2, 1.5L/min
Drying Gas Flow
N2, 10.0L/min
2
QuEChERS Sample Pre-treatment
The details of a modified QuEChERS procedure for potato
chips are shown in Figure 1. Hexane was used to defat
potato chips, removing oils and non-polar components. In
the extraction step with Q-sep Q100Packet extraction salt
(contain 4g MgSO4 & 0.5g NaCl), additional 4g of MgSO4
was added to absorb the water completely (aqueous phase
disappeared). Acrylamide is soluble in both aqueous and
organic phases. With this modification, high recovery of
acrylamide was obtained. It is believed that this is because
complete removal of water in the mixed extract solution
could promote acrylamide transferring into the organic
phase. Dispersive SPE tube was used as PSA to remove
organic acids which may decompose acrylamide in the
process.
[1]
[3]
Weigh 2.0g of sample in a 50mL centrifuge tube
Add 5mL hexane, 10mL water
and 10mL acetonitrile
Vortex and shake vigorously for 1min
Add Q-sep Q100Packet salt
Additional 4g MgSO4 (anhydrous)
Vortex and shake vigorously for 5min
[4]
Discard the hexane (top layer)
[5]
Transfer the solution into a 20mL volumetric flask
wash extraction salt with ACN
in the centrifuge tube
Combine the washing solution into the
volumetric flask (above)
[2]
[6]
[7]
Transfer 1mL of solution into the 2mL Q-sep Q250
QuEChERS dSPE tube
[8]
Vortex and centrifuge for 10min at 13000rpm
[9]
Transfer 500uL extract to a 1.5mL vial
Evaporate to dryness by N2 blow
[10] Reconstitute with 250uL of Milli Q water
Method Development
As acrylamide is a more polar compound, a Polar-RP
type column was selected. Isotope labeled internal
standard (acrylamide-d3) was used to compensate the
variation of acrylamide peak area caused by system
fluctuation and inconsistency in sample preparation of
different batches.
The precursor ions of acrylamide and acrylamide-d3 (IS)
were their protonated ions (m/z72.1 and m/z75.1). The
MRM optimization was carried out using an automated
program of the LabSolutions workstation, which could
generate a list of all MRM transitions with optimized CID
voltages accurate to (+/-) 1 volt in minutes. Two MRM
transitions of acrylamide and acryl-amide-d3 were
selected as quantifier and confirmation ion as shown in
Table 2.
The obtained extract solution of potato chips was used
as “blank” and also matrix for preparation of
post-spiked calibrants for establishment of calibration
curve with IS (acrylamide-d3). To obtain reliable results,
the blank and each post-spiked calibrant as shown in
Table 3 were injected three times and the average peak
area ratios were calculated and used.
[11] Analyze by Shimadzu LCMS-8040
Figure 1: Flow chart of sample pre-treatment with modified QuEChERS.
Table 2: MRM transitions and CID voltages
Name
MRM (m/z)
Acrylamide-d3
Acrylamide
CID Voltage (V)
Q1
CE
Q3
75.1 > 58.0*
-29
-15
-22
75.1 > 30.1
-29
-24
-30
72.1 > 55.0*
-17
-16
-24
72.1 > 27.1
-17
-22
-30
*MRM transition as quantifier
Table 3: Acrylamide spiked samples and peak
area ratios of measured by IS method
Acrylamide
post-spiked
IS postspiked
Conc. Ratio
Calculated
Area Ratio
measured*
L0, Blank
0
0.6033
L1, 1ppb
0.02
0.6120
0.10
0.6786
0.20
0.8239
L2, 5ppb
L3, 10ppb
50ppb
L4, 50ppb
1.00
1.7686
L5 100ppb
2.00
2.8196
L6, 500ppb
10.00
11.8330
*= Area (acrylamide) / Area (IS)
3
It was found that the potato chips used as “blank” in this
study was not free of acrylamide. Instead, it contained 27.1
ng/mL of acrylamide in the extract solution. A linear
calibration curve was established with an intercept of
1.25
Area Ratio (x10)
2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 1ppb 01a.lcd
2:Acrylamide
15000072.10>55.00(+) CE: -15.0 Acrylamide 50ppb 01a.lcd
300000 2:Acrylamide
72.10>55.00(+) CE: -15.0 Acrylamide 100ppb 01a.lcd
Y= 1.1239X + 0.594168
R2 = 0.9999
1.00
0.75
0.594 at zero spiked concentration (L0) as shown in Figure
2. Good linearity with correlation coefficient (R2) greater
than 0.9999 across the range of 1.0 ng/mL– 500.0 ng/mL
was obtained.
Area Ratio
200000
100000
1.0
0.50
100000
0.5
50000
0.25
0.00
0.0
0.00
0.0
2.5
Conc. Ratio
5.0
7.5
0
Conc. Ratio
0
2.5
1.5
5.0
2.0
7.5
2.5
min
min
Figure 2: Calibration curve (left) and MRM peaks (right) of acrylamide spiked into potato chips matrix, 1-500 ppb with 50 ppb IS added.
Method Performance Evaluation
It was hard to estimate the LOD and LOQ of the analytical
method due to the presence of acrylamide (27.1 ng/mL) in
the “blank” (extract of potato chips). However, as reported
also by other researchers, it is difficult to obtain potato
chips free of acrylamide actually. To obtain actual
concentration, it is normally subtracting the background
content of acrylamide of a “blank” sample used as
reference from a measurement of testing sample. The
same way was used to estimate actual S/N value in this
work. As a result, the LOD and LOD of acrylamide of this
method with 1ul injection volume were estimated to be
lower than 1ng/mL and 3ng/mL, respectively. This is
consistence with the results estimated with the IS.
The repeatability of the method was evaluated with L2 and
L4 spiked samples. The results are shown in Table 4 and
Figure 3. The peak area %RSD of acrylamide and IS were
below 4%.
The matrix effect (M.E.), recovery efficiency (R.E.) and
process efficiency (P.E.) of the method were determined
with a duplicate set of spiked samples of 50 ng/mL level
except for the non-spiked sample. The chromatograms of
“set 2”, i.e., non-spiked extract, pre-spiked, post-spiked
and the standard in neat solution are shown in Figure 4.
Noted that, the existing acrylamide in the extract of the
potato chips used as reference was accounted for 27.1
ng/mL, corresponding to 135.5 ng per gram of potato
chips. The average R.E, M.E and P.E of the method for
extraction and analysis of acrylamide obtained are shown
in Table 6.
Table 4: Repeatability Test Results (n=6)
spiked Sample
L2
L4
Compound
Conc. (ng/mL)
%RSD
Acrylamide
5
3.5
Acrylamide-d3
50
3.8
Acrylamide
50
3.9
Acrylamide-d3
50
3.6
4
2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R01.lcd
30000 2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R04.lcd
20000
10000
1.00
1.5
2.0
2.5
2.5
5.0
7.5
min
Figure 3: Overlay MRM chromatograms of 5 ng/mL acrylamide spiked in potato chips extract (total: 27.1+5 = 32.1 ng/mL)
50000
50000
(a) Extract
(non-spiked)
50000
(b) standard
50000
(c) post-spiked
40000
40000
40000
40000
30000
30000
30000
30000
20000
20000
20000
20000
10000
10000
10000
10000
0
0
1.5
2.0
2.5
0
1.5
2.0
2.5
(d) pre-spiked
0
1.5
2.0
2.5
1.5
2.0
2.5
Figure 4: The MRM peaks of acrylamide detected in “blank” extract of potato chips (a),
neat standard of 50ppb (b) post-spiked sample of 50ppb (c) and pre-spiked sample of 50ppb.
Table 6: Method evaluation of at 50.0ng/mL concentration in potato chips matrix
Parameter
Set 1
Set 2
Average
R.E.
104.7%
112.0%
108.4%
M.E.
96.5%
84.6%
90.5%
P.E.
100.8%
94.5%
97.6%
Conclusions
Acrylamide is formed unavoidably in starch-rich food in
cooking and processing at high temperature like potato
chips, French fries, cereals and roasted coffee etc. The
analysis method established in this work can be used to
monitor the levels of acrylamide in processing food
accurately and reliably. The QuEChERS method is proven to
be fast and effective in extraction of acrylamide from
potato chips. The excellent performance of the method in
terms of sensitivity, linearity, repeatability and recovery are
related to the outstanding performance of the LC/MS/MS
used which features ultra fast mass spectrometry (UFMS)
technology. The high sensitivity of the method allows the
analysis to be performed with a very small injection volume
(1µL or below), which would be a great advantage in
running heavily food samples with high matrix contents
and strong matrix effects. Maintenance of the interface of
a mass spectrometer could also be reduced significantly.
5
References
[1] Swedish National Food Administration. “Information about acrylamide in food, 24 April 2002”, http://www.slv.se
[2] Mottram, D.S., & Wedzicha, B.L., Nature, 419 (2002), 448-449.
[3] Ahn, J.S., Castle, J., Clarke, D.B., Lloyd, A.S., Philo, M.R., & Speck, D.R., Food Additives and Contaminants, 19 (2002),
1116-1124.
[4] Mastovska, K., & Lehotary, S.J., J. Food Chem., 54 (2006), 7001-7998.
PO-CON1472E
Determination of Benzimidazole Residues
in Animal Tissue by Ultra High Performance
Liquid Chromatography Tandem
Mass Spectrometry
ASMS 2014
TP 281
Yin Huo, Jinting Yao, Changkun Li, Taohong Huang,
Shin-ichi Kawano, Yuki Hashi
Shimadzu Global COE, Shimadzu (China) Co., Ltd., China
Determination of Benzimidazole Residues in Animal Tissue by
Ultra High Performance Liquid Chromatography Tandem
Mass Spectrometry
Introduction
Benzimidazoles are broad-spectrum, high efficiency, low
toxicity anthelmintic. Because some benzimidazoles and
their metabolites showed teratogenic and mutagenic
effects in animal and target animal safety evaluation
experiment, many countries have already put
benzimidazoles and metabolites as the monitoring object.
This poster employed a liquid chromatography-electrospray
ionization tandem mass spectrometry (LC-ESI-MS/MS)
method to determinate 16 benzimidazole residues in
animal tissue. The method is simple, rapid and high
sensitivity, which meets the requirements for the analysis
of veterinary drug residue in animal tissue.
Method
Sample Preparation
(1) Animal tissue samples were extracted with ethyl acetate-50% potassium hydroxide-1% BHT
(2) The samples were treated with n-hexane for defatting and further cleaned-up on MCX solid phase (SPE) cartridge.
(3) The separation of benzimidazoles and their metabolites was performed on LC-MS/MS instrument.
LC/MS/MS Analysis
The analysis was performed on a Shimadzu Nexera UHPLC instrument (Kyoto, Japan) equipped with LC-30AD pumps, a
CTO-30A column oven, a DGU-30A5 degasser, and an SIL-30AC autosampler. The separation was carried out on a
Shim-pack XR-ODS III (2.0 mmI.D. x 50 mmL., 1.6 μm, Shimadzu) with the column temperature at 30 ºC. A triple
quadrupole mass spectrometer (Shimadzu LCMS-8040, Kyoto, Japan) was connected to the UHPLC instrument via an ESI
interface.
UHPLC (Nexera system)
Column
Mobile phase A
Mobile phase B
Gradient program
Flow rate
Column temperature
Injection volume
:
:
:
:
:
:
:
Shim-pack XR-ODS III (2.0 mmI.D. x 50 mmL., 1.6 μm)
water with 0.1% formic acid
acetonitrile
as in Table 1
0.4 mL/min
30 ºC
20 µL
Table 1 Time program
Time (min)
Module
Command
Value
0.01
Pumps
Pump B Conc.
5
3.50
Pumps
Pump B Conc.
80
4.00
Pumps
Pump B Conc.
80
4.01
Pumps
Pump B Conc.
5
6.00
Controller
Stop
2
Mass Spectrometry
MS/MS (LCMS-8040 triple quadrupole mass spectrometer)
Ionization
Polarity
Ionization voltage
Nebulizing gas flow
Heating gas pressure
DL temperature
Heat block temperature
Mode
:
:
:
:
:
:
:
:
ESI
Positive
+4.5 kV
3.0 L/min
15.0 L/min
200 ºC
350 ºC
MRM
Table 2 MRM parameters of 16 benzimidazoles (*: for quantitation)
Compound
Precursor
m/z
Fenbendazole
300.10
Albendazole sulfoxide
282.00
Thiabendazole
202.00
Thiabendazole-5-hydroxy
218.00
Oxfendazole
316.20
Albendazole
266.30
Albendazole -2-aminosulfone
Albendazole sulfone
Mebendazole
Mebendazole-amine
5-Hydroxymebendazole
Flubendazole
240.30
298.30
296.30
238.30
298.30
314.30
2-Aminoflubendazole
256.30
Cambendazole
303.20
Oxibendazole
250.30
Oxfendazole
332.20
Product
m/z
Dwell Time
(ms)
Q1 Pre Bias
(V)
CE (V)
Q3 Pre Bias
(V)
268.05*
50
-15.0
-21.0
-18.0
159.05
50
-15.0
-36.0
-30.0
240.10*
10
-14.0
-12.0
-17.0
208.05
10
-14.0
-23.0
-22.0
175.10*
10
-30.0
-24.0
-18.0
131.15
10
-30.0
-31.0
-25.0
191.05*
50
-30.0
-23.0
-13.0
147.10
50
-30.0
-32.0
-27.0
159.15*
20
-11.0
-34.0
-30.0
191.15
20
-11.0
-22.0
-20.0
234.10*
8
-30.0
-19.0
-25.0
-20.0
191.10
8
-30.0
-33.0
133.20*
50
-15.0
-27.0
-24.0
198.10
50
-15.0
-18.0
-21.0
159.10*
20
-13.0
-37.0
-30.0
224.05
20
-13.0
-27.0
-23.0
264.15*
10
-13.0
-21.0
-27.0
105.25
10
-13.0
-35.0
-19.0
105.20*
10
-15.0
-26.0
-20.0
133.20
10
-15.0
-36.0
-25.0
266.10*
10
-30.0
-22.0
-18.0
160.15
10
-30.0
-35.0
-30.0
282.15*
10
-14.0
-22.0
-19.0
123.15
10
-14.0
-35.0
-24.0
123.20*
10
-16.0
-26.0
-22.0
95.20
10
-16.0
-41.0
-18.0
217.15*
5
-30.0
-28.0
-23.0
261.10
5
-30.0
-17.0
-28.0
218.15*
5
-30.0
-17.0
-23.0
176.15
5
-30.0
-27.0
-18.0
300.10*
10
-15.0
-22.0
-21.0
159.05
10
-15.0
-39.0
-30.0
3
Mass Spectrometry
6
8
4
12
7
40000
5
50000
3
60000
1:218.00>191.05(+)(10.00)
2:240.30>133.20(+)(2.00)
3:202.00>175.10(+)
4:238.30>105.20(+)(3.00)
5:256.30>123.20(+)(2.00)
6:298.30>266.10(+)
7:282.00>240.10(+)
8:303.20>217.15(+)
9:250.30>218.15(+)
10:316.20>159.15(+)(2.00)
11:298.30>159.10(+)(2.00)
12:266.30>234.10(+)
13:296.30>264.15(+)
14:332.20>300.10(+)(2.00)
15:314.30>282.15(+)
16:300.10>268.05(+)
2 1
70000
16 drugs mixture are presented in Fig.1. The correlation
coefficients for 16 drugs (0.5 – 50 ng/mL) were found to
0.9993~0.9999. MRM chromatograms of pork samples
and pork samples spiked with standards are shown in
Fig.2. By analyzing 16 drugs at three levels including 0.5
ng/mL, 5 ng/mL, 50 ng/mL, excellent repeatability was
demonstrated with the %RSD being better than 5% for all
the compound within six injections as shown in Table 3.
Results of recovery test were good as shown in Table 4.
9
A liquid chromatography-electrospray ionization tandem
mass spectrometry (LC-ESI-MS/MS) method has been
developed to identify and quantify trace levels of 16
benzimidazoles residue (fenbendazole, albendazole
sulfoxide, thiabendazole, thiabendazole- 5-hydroxy,
oxfendazole, albendazole, albendazole-2-aminosulfone,
albendazole sulfone, mebendazole, mebendazole-amine,
5-hydroxymebendazole, flubendazole,
2-aminoflubendazole, cambendazole, oxibendazole,
oxfendazole) in animal tissue. The MRM chromatograms of
10000
16
14
15
20000
13
11 10
30000
0
0.0
1.0
2.0
3.0
4.0
min
Figure 1 MRM chromatograms of standard 16 drugs (1 ng/mL)
(1: Thiabendazole-5-hydroxy; 2: Albendazole -2-Aminosulfone; 3: Thiabendazole;
4: Mebendazole-amine; 5: 2-Aminoflubendazole;6: 5-Hydroxymebendazole;
7: Albendazole Sulfoxide; 8: Cambendazole; 9: Oxibendazole; 10: Oxfendazole;
11: Albendazole sulfone; 12: Albendazole; 13: Mebendazole; 14: Oxfendazole;
15: Flubendazole; 16: Fenbendazole)
4
Mass Spectrometry
Table 3 Repeatability of 16 drugs in pork sample (n=6)
%RSD (0.5 ng/mL)
Compound
%RSD (5.0 ng/mL)
%RSD (50 ng/mL)
R.T.
Area
R.T.
Area
R.T.
Area
Fenbendazole
0.059
3.01
0.064
1.48
0.082
0.34
Albendazole Sulfoxide
0.202
4.26
0.084
2.86
0.153
0.92
Thiabendazole
0.272
4.52
0.180
2.85
0.132
2.58
0.526
4.44
0.249
3.91
0.158
1.41
Oxfendazole
0.121
2.71
0.089
2.91
0.105
0.97
Albendazole
0.073
2.07
0.090
1.29
0.099
0.92
Albendazole -2-Aminosulfone
0.392
4.36
0.162
2.08
0.177
1.72
Albendazole sulfone
0.103
3.95
0.126
0.63
0.113
0.64
1.69
0.094
0.74
0.149
2.72
0.243
0.94
0.091
2.31
0.099
0.79
0.140
1.17
Flubendazole
0.107
4.22
0.058
1.52
0.091
1.00
2-Aminoflubendazole
0.339
4.30
0.177
2.53
0.166
1.43
Cambendazole
0.150
4.90
0.123
3.38
0.121
1.87
Oxibendazole
0.091
3.46
0.108
1.31
0.125
1.20
Oxfendazole
0.170
3.23
0.044
3.09
0.084
0.80
6
12
30000
8
40000
1:218.00>191.05(+)(10.00)
2:240.30>133.20(+)
3:202.00>175.10(+)
4:238.30>105.20(+)
5:256.30>123.20(+)
6:298.30>266.10(+)
7:282.00>240.10(+)
8:303.20>217.15(+)
9:250.30>218.15(+)
10:316.20>159.15(+)
11:298.30>159.10(+)
12:266.30>234.10(+)
13:296.30>264.15(+)
14:332.20>300.10(+)
15:314.30>282.15(+)
16:300.10>268.05(+)
4
50000
1
1:218.00>191.05(+)
2:240.30>133.20(+)
3:202.00>175.10(+)
4:238.30>105.20(+)
5:256.30>123.20(+)
6:298.30>266.10(+)
7:282.00>240.10(+)
8:303.20>217.15(+)
9:250.30>218.15(+)
10:316.20>159.15(+)
11:298.30>159.10(+)
12:266.30>234.10(+)
13:296.30>264.15(+)
14:332.20>300.10(+)
15:314.30>282.15(+)
16:300.10>268.05(+)
9
0.095
3.95
10000
10000
16
20000
0
0.0
1.0
2.0
3.0
4.0
min
0
0.0
14
15
11 10
20000
13
7
5
30000
4.95
0.363
3
40000
0.093
2
50000
Mebendazole
Mebendazole-amine
1.0
2.0
3.0
4.0
min
Figure 2 MRM chromatograms of pork sample (left) and spiked pork sample (right)
(1: Thiabendazole-5-hydroxy; 2: Albendazole -2-Aminosulfone; 3: Thiabendazole;
4: Mebendazole-amine; 5: 2-Aminoflubendazole;6: 5-Hydroxymebendazole;
7: Albendazole Sulfoxide; 8: Cambendazole; 9: Oxibendazole; 10: Oxfendazole;
11: Albendazole sulfone; 12: Albendazole; 13: Mebendazole; 14: Oxfendazole;
15: Flubendazole; 16: Fenbendazole)
5
Mass Spectrometry
Table 4 Recovery of 16 drugs in pork sample
Compound
Sample Conc.
(µg/kg)
Spike Conc.
(µg/kg)
Measured Conc.
(µg/kg)
Recovery
(%)
Fenbendazole
N.D.
10.0
9.5
94.5
Albendazole Sulfoxide
N.D.
10.0
8.1
80.9
Thiabendazole
N.D.
10.0
9.8
98.2
N.D.
10.0
10.0
99.8
Oxfendazole
N.D.
10.0
11.4
113.8
Albendazole
N.D.
10.0
9.6
96.3
Albendazole -2-Aminosulfone
N.D.
10.0
9.6
96.1
Albendazole sulfone
N.D.
10.0
11.8
118.5
Mebendazole
N.D.
10.0
11.3
112.8
Mebendazole-amine
N.D.
10.0
11.8
118.3
N.D.
10.0
9.8
97.8
Flubendazole
N.D.
10.0
10.4
103.6
2-Aminoflubendazole
N.D.
10.0
9.3
92.6
Cambendazole
N.D.
10.0
10.8
107.8
Oxibendazole
N.D.
10.0
9.6
96.1
Oxfendazole
N.D.
10.0
9.1
90.7
Conclusion
The sensitive and reliable LC/MS/MS technique was
successfully applied for determination of 16
benzimidazoles residue. The calibration curves of 16
benzimidazoles ranging from 0.5 to 50 ng/mL were
established and the correlation coefficients were
0.9993~0.9999. The LODs of the 16 benzimidazoles
were 1 -2.2 µg/kg. The recoveries were in the range of
80.9%~118.5% for pork samples, with relative standard
deviations less than 5%.
PO-CON1459E
High Sensitivity Quantitation Method
of Dicyandiamide and Melamine in
Milk Powders by Liquid Chromatography
Tandem Mass Spectrometry
ASMS 2014
TP275
Zhi Wei Edwin Ting1, Jing Cheng Ng2*,
Jie Xing1 & Zhaoqi Zhan1
1
Customer Support Centre, Shimadzu (Asia Pacific)
Pte Ltd, 79 Science Park Drive, #02-01/08, SINTECH IV,
Singapore Science Park 1, Singapore 118264
2
Ridge Road, Singapore 119077, *Student
High Sensitivity Quantitation Method of Dicyandiamide
and Melamine in Milk Powders by Liquid Chromatography
Introduction
Melamine was found to be used as a protein-rich
adulterant first in pet-food in 2007, and then in infant
formula in 2008 in China [1]. The outbreak of the
melamine scandal that killed many dogs and cats as well as
led to death of six infants and illness of many had caused
panic in publics and great concerns in food safety
worldwide. Melamine was added into raw milk because of
its high nitrogen content (66%) and the limitation of the
Kjeldahl method for determination of protein level
indirectly by measuring the nitrogen content. In fact, in
addition to melamine and its analogues (cyanuric acid etc),
a number of other nitrogen-rich compounds was reported
also to be potentially used as protein-rich adulterants,
including amidinourea, biuret, cyromazine, dicyandiamide,
triuret and urea [2]. Recently, low levels of dicyandiamide
(DCD) residues were found in milk products from New
Zealand [3]. Instead of addition directly as an adulterant,
the trace DCD found in milk products was explained to be
relating to the grass “contaminated by DCD”.
Dicyandiamide has been used to promote the growth of
pastures for cows grazing. We report here an LC/MS/MS
method for sensitive detection and quantification of both
dicyandiamide (DCD) and melamine in infant milk powder
samples.
Experimental
High purity dicyandiamide (DCD) and melamine were
obtained from Sigma Aldrich. Amicon Ultra-4 (MWCO 5K)
centrifuge filtration tube (15 mL) obtained from Millipore
was used in sample pre-tretment. The milk powder sample
was pre-treated according to a FDA method [1] with some
Weigh 2.0g of milk powder sample
modification as illustrated in Figure 1. The final clear
sample solution was injected into LC/MS/MS for analysis.
Stock solutions of DCD and melamine were prepared in
pure water.
Table 1: Analytical conditions of DCD and melamine
in milk powders on LCMS-8040
LC conditions
Add 14mL of 2.5% formic acid
(1) Sonicate for 1hr
(2) Centrifuge at 6000rpm for 10min
Transfer 4mL of supernatant to Amicon Ultra-4
(MWCO 5K) centrifuge filtration tube (15mL)
Centrifuge at 7500rpm for 10min
Collect clear filtrate
To 50uL of filtrate added 950uL of ACN
Filter the filtrate by a 0.2um PTFE syringe filter
Further 10x dilution with ACN
LC/MS/MS analysis
Fig 1: Sample pre-treatment workflow
Column
Flow Rate
Alltima HP HILIC 3µ, 150 x 2.10mm
0.2 mL/min
Mobile Phase
A: 0.1 % formic acid in H2O/ACN (5:95 v/v)
B: 20mM Ammonium Formate in H2O/ACN (50:50 v/v)
Elution Mode
Gradient elution: 5% (0.01 to 3.0 min) →
95% (3.5 to 5.0 min) → 5% (5.5 to 9.0 min)
Oven Temperature
40ºC
Injection Volume
5 µL
MS conditions
Interface
ESI
MS mode
Positive
Block Temperature
400ºC
DL Temperature
300ºC
CID Gas
Ar (230kPa)
Nebulizing Gas Flow
N2, 2.0L/min
Drying Gas Flow
N2, 15.0L/min
2
An LCMS-8040 triple quadrupole LC/MS/MS (Shimadzu
Corporation, Japan) was used in this work. The system is
consisted of a high pressure binary gradient Nexera UHPLC
coupled with a LCMS-8040 MS system. An Alltima HP
HILIC column was used for separation of DCD and
melamine with a gradient program developed (Table 1).
The details of the LC and MS conditions are shown in
Table 1. A set of calibrants (0.5, 1.0, 2.5, 5 and 10 ppb)
was prepared from the stock solutions using of ACN/water
(90/10) as diluent.
MRM optimization
Table 2: MRM transitions and optimized parameters
MRM optimization of DCD and melamine were performed
using an automated MRM optimization program of the
LabSolutions. The precursors were the protonated ions of
DCD and melamine. Two optimized MRM transitions of
each compound were selected and used for quantitation
and confirmation. The MRM transitions and parameters are
shown in Table 2.
Name
RT (min) Transition (m/z)
DCD
2.55
MEL
6.29
Voltage (V)
Q1 Pre Bias
CE
Q3 Pre Bias
85.1 > 68.1
-15
-21
-26
85.1 > 43.0
-15
-17
-17
127.1 > 85.1
-26
-20
-17
127.1 > 68.1
-26
-27
-26
Method Development
A LC/MS/MS method was developed for quantitation of
DCD and melamine based on the MRM transitions in Table
2. Under the HILIC separation conditions (Table 1), DCD
and melamine eluted at 2.55 min and 6.29 min as sharp
peaks (see Figures 4 & 5). Figures 2 and 3 show the
calibration curves of DCD and melamine standard in neat
solutions and in milk matrix solutions (spiked). The linearity
with correlation coefficient (R2) greater than 0.997 across
the calibration range of 0.5~10.0 ng/mL was obtained for
both compounds in both neat solution and matrix (spiked).
Area (x10,000)
Area (x100,000)
Melamine (127.1>85.1)
R2 = 0.999
3.5
DCD (85.1>68.1)
R2 = 0.997
7.5
3.0
2.5
5.0
2.0
1.5
2.5
1.0
0.5
0.0
0.0
0.0
2.5
5.0
7.5
Conc.
0.0
2.5
5.0
7.5
Conc.
Figure 2: Calibration curves of DCD and melamine in neat solution
3
Area (x100,000)
Area (x10,000)
2.5
DCD (85.1>68.1)
5.0
R2 = 0.998
Melamine (127.1>85.1)
R2 = 0.997
2.0
4.0
1.5
3.0
1.0
2.0
0.5
1.0
0.0
0.0
0.0
2.5
5.0
7.5
Conc.
0.0
2.5
5.0
7.5
Conc.
Figure 3: Calibration curves of DCD and melamine spiked in milk powder matrix
Performance Evaluation
The repeatability of the method was evaluated at the levels
of 0.5 ng/mL and 1.0 ng/mL. Figures 4 & 5 show the MRM
chromatograms of DCD and melamine of six consecutive
DCD
(85.1>68.1)
5.0
6.0
Melamine
(127.1>85.1)
5.0
4.0
0.75
0.25
0.00
2.25
2.50
2.75
4.5
DCD
(85.1>68.1)
4.0
3.5
(x1,000)
Melamine
(127.1>85.1)
3.0
4.0
2.5
3.0
0.50
2.00
(x100)
(x1,000)
(x1,000)
1.00
injections of 0.5 ng/mL level with and without matrix. The
peak area %RSD for the two analytes were lower than
9.2% (see Table 3).
3.0
2.0
2.0
1.0
1.0
2.0
1.5
1.0
0.5
0.0
0.0
5.5
min
6.0
6.5
min
2.00
2.25
Figure 4: Overlapping of six MRM peaks of 0.5 ng/mL
DCD and melamine in neat solution
2.50
2.75
min
0.0
5.5
6.0
6.5
min
Figure 5: Overlapping of six MRM peaks of 0.5 ng/mL
DCD and melamine in milk powder matrix
Table 3: Results of repeatability and sensitivity evaluation of DCD and melamine (n=6)
Sample
Compd.
DCD
In solvent
MEL
DCD
In matrix
MEL
Conc. (ng/mL)
%RSD
0.5
5.9
1.0
5.3
0.5
5.5
1.0
2.6
0.5
5.9
1.0
8.2
0.5
9.2
1.0
2.4
LOD (ng/mL)
LOQ (ng/mL)
0.03
0.10
0.03
0.09
0.05
0.16
0.05
0.15
4
The LOD and LOQ were estimated from the results of
0.5 ng/mL in both neat and matrix solution. The LOD
and LOQ results were summarized in Table 3. The
method achieved LOQs (in matrix) of 0.16 and 0.15
ng/mL (ppb) for DCD and melamine, respectively. Tables
4 & 5 show the results of matrix effect and recovery of
the method. The matrix effects for DCD and melamine
in the whole concentration ranges were at 64%~70%
and 62%~73%, respectively.
The recovery was determined by comparing the results
of pre-spiked and post-spiked mixed samples of DCD
and melamine in the milk powder matrix (2.5 ng/mL
each compound). The chromatograms of these samples
are shown in Figure 6. The recovery of DCD and
melamine were determined to be 103% and 105%
respectively.
Table 4: Matrix effect (%) of DCD and melamine
in milk powder matrix
Table 5: Recovery of DCD and melamine determined
with spiked sample of 2.5 ng/mL
Conc. (ng/mL)
0.5
1
2.5
5
10
Compound
Pre-spiked Area
Post-spiked Area
Recovery (%)
DCD
70.4
65.4
66.9
64.8
66.6
DCD
14,393
13,987
102.9
MEL
62.2
62.5
73.1
68.9
68.0
MEL
65,555
62,659
104.6
6000
6000
5000
5000
4000
4000
3000
Blank matrix of
milk powder
3000
1:85.10>68.05(+)
1:85.10>43.00(+)
7000
6000
DCD
Pre-spiked
5000
4000
3000
2000
2000
2000
1000
1000
1000
0
0
17500
2.25
2.50
2.75
3.00
2:127.10>85.10(+)
2:127.10>68.05(+)
17500
12500
10000
2.25
2.50
2:127.10>85.10(+)
2:127.10>68.05(+)
15000
15000
12500
Blank matrix of
milk powder
10000
2.75
3.00
2.00
17500
12500
Melamine
Pre-spiked
10000
7500
7500
5000
5000
5000
2500
2500
2500
0
0
6.25
6.50
6.75
2.25
2.50
2:127.10>85.10(+)
2:127.10>68.05(+)
15000
7500
6.00
DCD
Post-spiked
0
2.00
Melamine
2.00
1:85.10>68.05(+)
1:85.10>43.00(+)
Dicyandiamide
7000
2.75
3.00
Melamine
1:85.10>68.05(+)
1:85.10>43.00(+)
Dicyandiamide
7000
Melamine
Post-spiked
0
6.00
6.25
6.50
6.75
6.00
6.25
6.50
6.75
Figure 6: MRM peaks of DCD and melamine in pre- and post-spiked samples of 2.5 ng/mL (each).
DCD and melamine were not detected in blank matrix of milk powder.
5
Conclusions
A high sensitivity LC/MS/MS method was developed on
LCMS-8040 for detection and quantitation of
dicyandiamide (DCD) and melamine in milk powders. The
method performance was evaluated using infant milk
powders as the matrix. The method achieved LOQ of 0.16
ng/mL for both compounds in the matrix, allowing its
application in simultaneous analysis of melamine, a protein
adulterant in relatively high concentration, and
dicyandiamide residue in trace level in milk powders
samples.
References
1. S. Turnipseed, C. Casey, C. Nochetto, D. N. Heller, FDA Food, LIB No. 4421, Volume 24, October 2008.
2. S. MachMahon, T. H. Begley, G. W. Diachenko, S. A. Stromgren, Journal of Chromatography A, 1220, 101-107 (2012).
3. http://www.naturalnews.com/041834_Fonterra_milk_powder_dicyandiamide.html
PO-CON1465E
Multiresidue pesticide analysis from
dried chili powder using LC/MS/MS
ASMS 2014
WP350
Deepti Bhandarkar, Shruti Raju, Rashi Kochhar,
Shailesh Damale, Shailendra Rane, Ajit Datar,
Jitendra Kelkar, Pratap Rasam
Multiresidue pesticide analysis from dried chili powder
using LC/MS/MS
Introduction
Pesticide residues in foodstuffs can cause serious health
problems when consumed. LC/MS/MS methods have been
increasingly employed in sensitive quantification of
pesticide residues in foods and agriculture products.
However, matrix effect is a phenomenon seen in Electro
Spray Ionization (ESI) LC/MS/MS analysis that impacts the
data quality of the pesticide analysis, especially for complex
matrix like spice/herb.
Chili powder is one such complex matrix that can exhibit
matrix effect (either ion suppression or enhancement). A
calibration curve based on matrix matched standards can
demonstrate true sensitivity of analyte in presence of
matrix. Therefore, this approach was used to obtain more
reliable and accurate data as compared to quantitation
against neat (solvent) standards[1].
Multiresidue, trace level analysis in complex matrices is
challenging and tedious. Feature of automatic MRM
optimization in LCMS-8040 makes method development
process less tedious. In addition, the lowest dwell time and
pause time along with ultra fast polarity switching
(UFswitching) enables accurate, reliable and high sensitive
quantitation. UFsweeperTM II technology in the system
ensures least crosstalk, which is very crucial for
multiresidue pesticide analysis.
Method of Analysis
Sample Preparation
Commercially available red chili was powdered using mixer
grinder. To 1 g of this chili powder, 20 mL water:methanol
(1:1 v/v) was added and the mixture was sonicated for 10
mins. The mixture was centrifuged and supernatant was
collected. This supernatant was used as diluent to prepare
pesticide matrix matched standards at concentration levels
of 0.01 ppb, 0.02 ppb, 0.05 ppb, 0.1 ppb, 0.2 ppb, 0.5
ppb, 1 ppb, 2 ppb, 5 ppb, 10 ppb and 20 ppb. Each
concentration level was then filtered through 0.2 µ nylon
filter and used for the analysis.
LC/MS/MS Analytical Conditions
Pesticides were analyzed using Ultra High Performance
Corporation, Japan), shown in Figure 1. The details of
analytical conditions are given in Table 1.
Table 1. LC/MS/MS analytical conditions
• Column
• Guard column
• Mobile phase
• Flow rate
• Gradient program (B%)
• MS interface
• Polarity
• MS temperature
• MS analysis mode
: Shim-pack XR-ODS (75 mm L x 3 mm I.D.; 2.2 µm)
: Phenomenex SecurityGuard ULTRA Cartridge
: A: 5 mM ammonium formate in water:methanol (80:20 v/v)
B: 5 mM ammonium formate in water:methanol (10:90 v/v)
: 0.2 mL/min
: 40 ºC
: 0.0–1.0 min → 45 (%); 1.0–13.0 min → 45-100 (%);
13.0–18.0 min → 100 (%); 18.0–19.0 min → 100-45 (%);
19.0–23.0 min → 45 (%)
: 15 µL
: ESI
: Positive and negative
: Nebulizing gas 2 L/min; Drying gas 15 L/min
: Desolvation line 250 ºC; Heat block 400 ºC
: Staggered MRM
2
using LC/MS/MS
Figure 1. Nexera with LCMS-8040 triple quadrupole system by Shimadzu
Results
LC/MS/MS method was developed for analysis of 80
pesticides belonging to different classes like carbamate,
organophosphate, urea, triazines etc. in a single run[2]. LOQ
was determined for each pesticide based on the following
criteria – (1) % RSD for area < 16 % (n=3), (2) % Accuracy
between 80-120 % and (3) Signal to noise ratio (S/N) > 10.
LOQ achieved for 80 pesticides have been summarized in
Table 2 and results for LOQ and linearity for each pesticide
have been given in Table 3. Representative MRM
chromatogram of pesticide mixture at 1 ppb level is shown
in Figure 2. Representative MRM chromatograms at LOQ
level for different classes of pesticides are shown in Figure 3.
Table 2: Summary of LOQ achieved
LOQ (ppb)
0.01
0.02
0.05
0.1
0.2
0.5
1
Number of pesticides
1
1
3
8
17
24
26
Table 3. Results of LOQ and linearity for pesticide analysis
Sr. No.
Name of compound
MRM Transition
Polarity
LOQ (ppb)
Linearity (R2)
1
Spinosyn D
746.20>142.10
Positive
0.01
0.9987
2
Fenpyroximate
421.90>366.10
Positive
0.02
0.9915
3
Bifenazate
301.00>198.00
Positive
0.05
0.9947
4
Spinosyn A
732.20>142.10
Positive
0.05
0.9974
5
Spiromesifen
371.00>273.10
Positive
0.05
0.9957
6
Acetamiprid
222.90>126.00
Positive
0.1
0.9910
7
Carbofuran
221.70>123.00
Positive
0.1
0.9971
8
Dimethoate
229.80>198.90
Positive
0.1
0.9970
9
Dimethomorph I
387.90>301.00
Positive
0.1
0.9991
10
Dimethomorph II
387.90>301.00
Positive
0.1
0.9992
11
Isoproturon
207.00>72.10
Positive
0.1
0.9984
12
Pirimiphos methyl
305.70>108.00
Positive
0.1
0.9997
13
Trifloxystrobin
408.90>186.00
Positive
0.1
0.9989
3
using LC/MS/MS
Sr. No.
Name of compound
MRM Transition
Polarity
LOQ (ppb)
Linearity (R2)
14
Anilophos
367.70>198.85
Positive
0.2
0.9974
15
Atrazine
215.90>174.00
Positive
0.2
0.9985
16
Carboxin
235.90>143.00
Positive
0.2
0.9952
17
Cyazofamid
324.85>108.10
Positive
0.2
0.9971
18
Edifenphos
310.60>111.00
Positive
0.2
0.9997
19
Ethion
384.70>198.80
Positive
0.2
0.9957
20
Fipronil
434.70>330.00
Negative
0.2
0.9973
21
Linuron
248.80>159.90
Positive
0.2
0.9945
22
Metolachlor
283.90>252.00
Positive
0.2
0.9966
23
Oxycarboxin
267.90>174.90
Positive
0.2
0.9995
24
Phosalone
367.80>181.90
Positive
0.2
0.9987
25
Phosphamidon
299.90>173.90
Positive
0.2
0.9997
26
Thiacloprid
252.90>126.00
Positive
0.2
0.9976
27
Thiobencarb
257.90>125.10
Positive
0.2
0.9977
28
Thiodicarb
354.90>88.00
Positive
0.2
0.9906
29
Triadimefon
293.90>196.90
Positive
0.2
0.9994
0.9977
30
Tricyclazole
189.90>162.90
Positive
0.2
31
Aldicarb
208.10>116.05
Positive
0.5
0.9962
32
Benfuracarb
411.10>190.10
Positive
0.5
0.9981
33
Bitertanol
338.00>99.10
Positive
0.5
0.9935
34
Buprofezin
305.70>201.00
Positive
0.5
0.9933
35
Clodinafop propargyl
349.90>266.00
Positive
0.5
0.9978
36
Chlorantraniliprole
483.75>452.90
Positive
0.5
0.9994
37
Diclofop methyl
357.90>280.80
Positive
0.5
0.9976
38
Flufenacet
363.70>193.90
Positive
0.5
0.9997
39
Flusilazole
315.90>247.00
Positive
0.5
0.9983
40
Hexaconazole
313.90>70.10
Positive
0.5
0.9996
41
Hexythiazox
352.90>227.90
Positive
0.5
0.9909
42
Iodosulfuron methyl
507.70>167.00
Positive
0.5
0.9971
43
Iprobenfos
288.70>205.00
Positive
0.5
0.9981
44
Malaoxon
314.90>99.00
Positive
0.5
0.9996
45
Malathion
330.90>284.90
Positive
0.5
0.9997
46
Mandipropamid
411.90>356.20
Positive
0.5
0.9952
47
Metalaxyl
280.00>220.10
Positive
0.5
0.9996
48
Methabenzthiazuron
221.70>150.00
Positive
0.5
0.9957
49
Methomyl
162.90>88.00
Positive
0.5
0.9988
50
Oxadiazon
362.15>303.00
Positive
0.5
0.9963
51
Penconazole
283.90>70.10
Positive
0.5
0.9992
52
Phorate
260.80>75.00
Positive
0.5
0.9987
53
Phorate sulfoxide
276.80>96.90
Positive
0.5
0.9991
54
Thiophanate methyl
342.90>151.00
Positive
0.5
0.9996
55
Avermectin B1a
890.30>305.10
Positive
1
0.9990
56
Carpropamid
333.70>139.00
Positive
1
0.9985
4
using LC/MS/MS
Sr. No.
Name of compound
MRM Transition
Polarity
LOQ (ppb)
Linearity (R2)
57
Clomazone
241.90>127.00
Positive
1
0.9967
58
Clorimuron ethyl
415.30>186.00
Positive
1
0.9965
59
Cymoxanil
198.90>128.10
Positive
1
0.9949
60
Diafenthiuron
385.00>329.10
Positive
1
0.9961
61
Diflubenzuron
310.80>158.00
Positive
1
0.9982
62
Dodine
228.10>60.00
Positive
1
0.9980
63
Emamectin benzoate
886.30>158.10
Positive
1
0.9983
64
Fenamidone
311.90>236.10
Positive
1
0.9997
65
Fenarimol
330.70>268.00
Positive
1
0.9900
66
Fenazaquin
306.95>57.10
Positive
1
0.9992
67
Flonicamid
229.90>202.70
Positive
1
0.9971
68
Flubendiamide
680.90>254.05
Negative
1
0.9993
69
Forchlorfenuron
247.90>129.00
Positive
1
0.9956
70
Kresoxim methyl
331.00>116.00
Positive
1
0.9996
71
Paclobutrazol
293.90>70.10
Positive
1
0.9974
72
Pencycuron
328.90>125.00
Positive
1
0.9943
73
Pendimethalin
281.90>212.10
Positive
1
0.9932
74
Profenofos
372.70>302.70
Positive
1
0.9966
75
Propargite
368.00>231.10
Positive
1
0.9950
76
Propoxur
209.90>110.90
Positive
1
0.9987
77
Pyrazosulfuron ethyl
414.90>182.00
Positive
1
0.9992
78
Pyriproxyfen
321.90>96.10
Positive
1
0.9975
79
Simazine
201.90>103.90
Positive
1
0.9992
80
Thiomethon
246.80>89.10
Positive
1
0.9989
50000
40000
30000
20000
10000
0
5.0
10.0
15.0
min
Figure 2. MRM chromatogram of pesticide mixture at 1 ppb level
5
using LC/MS/MS
4000
750
500
N-Methyl
Carbamate
3000
4000
3000
2000
1000
1000
0
0
4.0
5.0
5.0
4000 42:215.90>174.00(+)
7.0
8.0
7.0
115:746.20>142.10(+)
Triazine
Macrocyclic
Lactone
300
Atrazine
3000
2000
6.0
200
8.0
9.0
10.0
121:421.90>366.10(+)
1250
Pyrazole
1000
750
Fenpyraoximate
3.0
Spinosyn D
2.0
500
100
250
1000
0
8.0
9.0
0
16.0
126:680.90>254.05(-)
3000
18.0
19.0
80:283.90>70.10(+)
6000
Flubendiamide
Anthranilic
Diamide
17.0
3000
5000
18.0
2500
2000
1000
0
10.0
17.0
Chloroacetanilide
4000
1000
16.0
7500 70:283.90>252.00(+)
Azole
5000
2000
15.0
Metolachlor
7.0
Penconazole
6.0
4000
Urea
2000
250
5000
44:207.00>72.10(+)
Isoproturon
Organophosphorus
Dimethoate
1000
33:221.70>123.00(+)
Carbofuran
15:229.80>198.90(+)
0
11.0
12.0
13.0
0
11.0
12.0
13.0
14.0
10.0
11.0
12.0
13.0
Figure 3. Representative MRM chromatograms at LOQ level from different classes of pesticides
Conclusion
• A highly sensitive method was developed for analysis of 80 pesticides belonging to different classes, from dried chili
powder in a single run.
• Ultra high sensitivity, ultra fast polarity switching (UFswitching), low pause time and dwell time along with UFsweeperTM
II technology enabled sensitive, selective, accurate and reproducible multiresidue pesticide analysis from complex matrix
like dried chili powder.
6
using LC/MS/MS
References
[1] Kwon H, Lehotay SJ, Geis-Asteggiante L., Journal of Chromatography A, Volume 1270, (2012), 235–245.
[2] Banerjee K, Oulkar DP et al., Journal of Chromatography A, Volume 1173, (2007), 98-109.
PO-CON1463E
Multi pesticide residue analysis in
tobacco by GCMS/MS using QuEChERS
as an extraction method
ASMS 2014
TP762
Durvesh Sawant(1), Dheeraj Handique(1), Ankush Bhone(1),
Prashant Hase(1), Sanket Chiplunkar(1), Ajit Datar(1),
Jitendra Kelkar(1), Pratap Rasam(1), Kaushik Banerjee(2),
Zareen Khan(2)
(1) Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh
(2) National Referral Laboratory, National Research
Centre for Grapes, P.O. Manjri Farm, Pune-412307,
Maharashtra, India.
Multi pesticide residue analysis in tobacco
by GCMS/MS using QuEChERS as an extraction method
Introduction
India is the world’s second largest producer (after China)
and consumer (after Brazil) of tobacco with nearly $
1001.54 million revenue generated annually from its
export.[1] In countries like India, with tropical-humid
climate, the incidences of insect attacks and disease
infestations are frequent and application of pesticides for
their management is almost obligatory. Like any other
crop, tobacco (Nicotiana tabacum Linn.), one of the
world’s leading high-value crops, is also prone to pest
attacks, and the farmers do apply various pesticides as a
control measure.
The residues of pesticides applied on tobacco during its
cultivation may remain in the leaves at harvest that may
even sustain post harvest processing treatments and could
appear in the final product. Thus, monitoring of pesticide
residues in tobacco is an important issue of critical concern
from public health and safety point of view demanding
implementation of stringent regulatory policies.[2]
To protect the consumers by controlling pesticide residue
levels in tobacco, the Guidance Residue Levels (GRL) of 118
pesticides have been issued by the Agro-Chemical Advisory
Committee (ACAC) of the Cooperation Center for
Scientific Research Relative to Tobacco (CORESTA).
Tobacco is a complex matrix and hence requires selective
extraction and extensive cleanup such as QuEChERS (Quick
Easy Cheap Effective Rugged Safe) to ensure trace level
detection with adequate precision and accuracy. The
objective of the present study was to develop an effective,
sensitive and economical multi-pesticide residue analysis
method for 203 pesticides in tobacco as listed in Table 1.
Figure 1. Dried tobacco
Method of Analysis
Extraction of pesticides from tobacco
Extraction of pesticides was done using QuEChERS method, as described below.[3]
Take 2 g of dry powdered tobacco leaves (Figure 1).
Add 18 mL of water containing 0.5 % acetic acid.
Homogenize the sample and Keep it for 30 min.
Add 10 mL ethyl acetate. Immediately, put 10 g sodium sulfate.
Homogenize it thoroughly at 15000 rpm for 2 min.
Centrifuge at 5000 rpm for 5 min for phase separation.
Draw 3 mL of ethyl acetate upper layer from the extract for further cleanup.
2
Add 1 mL toluene to it and vortex for 0.5 min.
Add cleanup mixture [PSA (150 mg), C18 (150 mg), GCB (75 mg) and
anhydrous MgSO4 (300 mg)] and vortex for 2 min.
Centrifuge the mixture at 7000 rpm for 7 min.
Collect the supernatant and filter through a 0.2 µm PTFE membrane filter.
Inject 2.0 µL of the clean extract into GCMS-TQ8030 (Figure 2).
Figure 2. GCMS-TQ8030 Triple quadrupole system by Shimadzu
Key Features of GCMS-TQ8030
• ASSP™ (Advanced Scanning Speed Protocol) enables high-speed scan and data acquisition for accurate quantitation at
20,000 u/sec
• Capable of performing simultaneous Scan/MRM
• UFsweeper® technology efficiently sweeps residual ions from the collision cell for fast, efficient ion transport ensuring no
cross-talk
• Two overdrive lenses reduce random noise from helium, high-speed electrons and other factors to improve S/N ratio
• Flexible platform with EI (Electron Ionization), CI (Chemical Ionization), and NCI (Negative Chemical Ionization)
techniques
• Full complement of acquisition modes including MRM, Scan/MRM, Precursor Ion, Product Ion and Neutral Loss Scan
3
Table 1. List of pesticides
Sr. No.
Pesticide
Sr. No.
Pesticide
Sr. No.
Pesticide
Sr. No.
Pesticide
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
2,6-Dichlorobenzamide
2-Phenylphenol
3,4-Dichloraniline
3-Chloroaniline
4-Bromo 2-Chloro phenol
4,4-Dichlorobenzophenone
Acetochlor
Acrinathrin
Alachlor
Aldrin
Azinphos-ethyl
Azinphos-methyl
Azoxystrobin
Barban
Beflubutamid
Benfluralin
Benoxacor
Beta-endosulfan
Bifenox
Bifenthrin
Bitertanol
Boscalid
Bromacil
Bromophos-ethyl
Bromopropylate
Bromuconazole-1
Bromuconazole-2
Butralin
Butylate
Carbaryl
Carbofuran
Carfentrazone
Chlordane-trans
Chlordecone
Chlorfenvinphos
Chlormephos
Chlorobenzilate
Chloroneb
Chlorothalonil
Chlorpyriphos-ethyl
Chlorpyriphos-methyl
Chlorpyriphos-oxon
Chlorthal-dimethyl
Cinidon-ethyl
Cis-1,2,3,6 tetrahydrophthalimide
Clodinafop propargyl
Clomazone
Crimidine
Cyanophos
Cyfluthrin-1
Cyfluthrin-2
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
Cyfluthrin-3
Cyfluthrin-4
Cyhalofop-butyl
Cypermethrin-2
Cypermethrin-3
Cypermethrin-4
Cyprodinil
Delta-HCH
Demeton-s-methyl
Demeton-S-methyl sulphone
Dialifos
Diazinon
Dichlobenil
Dichlofluanid
Diclofop
Dicloran
Dieldrin
Diethofencarb
Difenoconazole-1
Difenoconazole-2
Diflubenzuron
Diflufenican
Dimethipin
Dimethomorph-1
Dimethomorph-2
Dimoxystrobin
Diniconazole
Dinoseb
Dinoterb
Dioxathion
Edifenfos
Endosulfan sulphate
Endrin
Epoxiconazole
Ethalfluralin
Ethoprophos
Etoxazole
Etridiazole
Etrimfos
Famoxadone
Fenamidone
Fenarimol
Fenbuconazole
Fenchlorphos
Fenchlorphos oxon
Fenhexamid
Fenobucarb
Fenoxycarb
Fenthion sulphoxide
Fenvalerate
Fipronil
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
Fipronil sulphone
Flucythrinate-1
Flucythrinate-2
Flufenacet
Flumoixazine
Fluquinconazole
Flurochloridone-1
Flurochloridone-2
Flutolanil
Flutriafol
Fluxapyoxad
Folpet
Fuberidazole
Heptachlor
Hexaconazole
Iprobenfos
Isoprocarb
Isoprothiolane
Isopyrazam
Isoxaben
Lactofen
Lambda-cyhalothrin
Malaoxon
Malathion
Mepanipyrim
Mepronil
Metalaxyl
Metalaxyl M
Metazachlor
Metconazole
Methabenzthiazuron
Methacrifos
Methidathion
Methiocarb
Metholachlor-s
Methoxychlor
Metribuzin
Mevinphos
Monolinuron
Myclobutanyl
Napropamide
Nitrapyrin
Oxadiargyl
Oxadiazon
Oxycarboxin
p,p-DDE
Parathion-ethyl
Parathion-methyl
Penconazole
Pencycuron (Deg.)
Pendimethalin
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
Permethrin-1
Permethrin-2
Pethoxamid
Phosalone
Phosmet
Pirimicarb
Pretilachlor
Procymidone
Profenofos
Propanil
Propaquizafop
Propazine
Propham
Propiconazole-1
Propisoclor
Propyzamide
Proquinazid
Pyraflufen-ethyl
Pyrazophos
Pyrimethanil
Pyriprooxyfen
Pyroquilon
Quinoxyfen
Simazine
Spirodiclofen
Sulfotep
Swep
Tebufenpyrad
Tebupirimfos
Tebuthiuron
Tefluthrin
Terbacil
Tetraconazole
Tetradifon
Thiobencarb
Tolylfluanid
Tralkoxydim
Triadimefon
Tri-allate
Triazophos
Tricyclazole
Trifloxystrobin
Triflumizole
Triflumuron
Trifluralin
Triflusulfuron
Triticonazole
Valifenalate
Vinclozolin
Zoxamide (Deg.)
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
4
GCMS/MS Analytical Conditions
The analysis was carried out on Shimadzu GCMS-TQ8030 as per the conditions given below.
Chromatographic parameters
• Column
• Injection Mode
• Sampling Time
• Split Ratio
• Carrier Gas
• Flow Control Mode
• Linear Velocity
• Column Flow
• Injection Volume
• Injection Type
• Total Program Time
• Column Temp. Program
:
:
:
:
:
:
:
:
:
:
:
:
Rxi-5Sil MS (30 m L x 0.25 mm I.D.; 0.25 µm)
Splitless
2.0 min
5.0
Helium
Linear Velocity
40.2 cm/sec
1.2 mL/min
2.0 µL
High Pressure Injection
41.87 min
Rate (ºC /min)
Temperature (ºC)
70.0
25.00
150.0
3.00
200.0
8.00
280.0
Hold time (min)
2.00
0.00
0.00
10.00
Mass Spectrometry parameters
• Ion Source Temp.
• Interface Temp.
• Ionization Mode
• Acquisition Mode
:
:
:
:
230.0 ºC
280.0 ºC
EI
MRM
Results
For MRM optimisation, well resolved pesticides were
grouped together. Standard solution mixture of
approximately 1 ppm concentration was prepared and
analyzed in Q3 scan mode to determine the precursor ion
for individual pesticides. Selected precursor ions were
allowed to pass through Q1 & enter Q2, also called as
Collision cell. In Collision cell, each precursor ion was
bombarded with collision gas (Argon) at different energies
(called as Collision Energy-CE) to produce fragments
(product ions). These product ions were further scanned in
Q3 to obtain their mass to charge ratio. For each precursor
ion, product ion with highest intensity and its
corresponding CE value was selected, thereby assigning a
characteristic MRM transition to every pesticide. Based on
MRM transitions, the mixture of 203 pesticides was
analyzed in a single run (Figure 3).
Method was partly validated for each pesticide with
respect to linearity (0.5 to 25 ppb), reproducibility, LOQ
and recovery. The validation summary for two pesticides
namely Mevinphos and Parathion-ethyl (Sr. Nos.140 and
149 in Table 1) is shown in Figures 4 and 5. The summary
data of linearity and LOQ for 203 pesticides is given in
Table 2 and 3 respectively.
5
(x100,000)
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
32.5
35.0
37.5
40.0
min
Figure 3. MRM Chromatogram for 203 pesticides mixture
Calibration overlay
Linearity curve
2.5
(x10,000)
MRM : 192.00>127.00
5.0
Recovery overlay
Area (x100,000)
(x10,000)
MRM : 192.00>127.00
2.0
1.00
1.5
0.75
Post extraction spike
Pre extraction spike
4.0
3.0
0.50
1.0
2.0
1.0
0.25
0.5
0.0
7.0
7.5
8.0
8.5
9.0 min
0.00
0.0
0.0
5.0
10.0
15.0
20.0
7.25
Conc.
7.50
7.75
8.00
8.25
min
8.50
Linearity (R2)
LOD (ppb)
LOQ (ppb)
S/N at LOQ
% RSD at LOQ
(n=6)
% Recovery
at LOQ
0.9999
0.3
1
173
6.93
89.28
Figure 4. Summary data for mevinphos
Calibration overlay
Linearity curve
Recovery overlay
Area (x100,000)
(x10,000)
3.5 MRM : 291.10>137.00
1.50
3.0
1.25
2.5
8.0
5.0
4.0
0.75
3.0
2.0
0.50
0.5
1.0
0.25
0.0
0.0
15.0
15.5
16.0
16.5
min
Post extraction spike
Pre extraction spike
6.0
1.00
1.0
MRM : 291.10>137.00
7.0
2.0
1.5
(x1,000)
0.00
0.0
5.0
10.0
15.0
20.0
15.0
Conc.
15.5
16.0
16.5
min
Linearity (R2)
LOD (ppb)
LOQ (ppb)
S/N at LOQ
% RSD at LOQ
(n=6)
% Recovery
at LOQ
0.9993
1.5
5
93
4.05
109.10
Figure 5. Summary data for parathion-ethyl
6
Table 2. Linearity Summary
Table 3. LOQ Summary
Sr. No.
Linearity (R2)
Number of
pesticides
Sr. No.
LOQ (ppb)
Number of
pesticides
% RSD range
(n=6)
S/N Ratio
range
1
0.9950 - 1.0000
193
1
1
15
6 – 15
16 – 181
2
0.9880 - 0.9950
10
2
5
18
3 – 15
19 – 502
3
10
158
0.95 – 15
10 – 14255
4
25
12
1 – 10
19 – 660
% Recovery
range
70 – 130
Conclusion
• A highly sensitive method was developed for quantitation of 203 pesticides in complex tobacco matrix by using
Shimadzu GCMS-TQ8030.
• The MRM method developed for 203 pesticides can be used for screening of pesticides in various food commodities. For
90 % of the pesticides, the LOQ of 10 ppb or below was achieved.
• Ultra Fast scanning, UFsweeper® and ASSP™ features enabled sensitive, selective, fast, reproducible, linear and accurate
method of analysis.
Reference
[1] Tobacco Board (Ministry of Commerce and Industry, Government of India), Exports performance during 2013-14,
(2014), 1.
http://tobaccoboard.com/admin/statisticsfiles/Exp_Perf_Currentyear.pdf
[2] CORESTA GUIDE Nº 1, The concept and implementation of cpa guidance residue levels, (2013), 4.
http://www.Coresta.org/Guides/Guide-No01-GRLs%283rd-Issue-July13%29.pdf
[3] Zareen S Khan, Kaushik Banerjee, Rushali Girame, Sagar C Utture et al., Journal of Chromatography A, Volume 1343,
(2014), 3.
PO-CON1453E
Simultaneous quantitative analysis of
20 amino acids in food samples without
derivatization using LC-MS/MS
ASMS 2014
TP 510
Keiko Matsumoto1; Jun Watanabe1; Itaru Yazawa2
1 Shimadzu Corporation, Kyoto, Japan;
2 Imtakt Corporation, Kyoto, Japan
Simultaneous quantitative analysis of 20 amino acids in food
samples without derivatization using LC-MS/MS
Introduction
In order to detect many kinds of amino acids with high
selectivity in food samples, the LC/MS analysis have been
used widely. Amino acids are high polar compound, so
they are hard to be retained to reverse-phased column
such as ODS (typical method in LC/MS analysis). It needs
their derivartization or addition of ion pair reagent in
mobile phase to retain them. For easier analysis of amino
acids, it is expected to develop the method without using
reagents mentioned above.
This time, we tried to develop a simultaneous high sensitive
analysis method of 20 amino acids by LC/MS/MS with
mix-mode column (ion exchange, normal-phase) and the
typical volatile mobile phase suitable for LC/MS analysis.
Amino acid standard regents and food samples were
purchased from the market. Standards of 20 kinds of
amino acids were optimized on each
compound-dependent parameter and MRM transition.
As an LC-MS/MS system, HPLC was coupled to triple
quadrupole mass spectrometer (Nexera with LCMS-8050,
Shimadzu Corporation, Kyoto, Japan). Sample was eluted
with a binary gradient system and LC-MS/MS with
electrospray ionization was operated in
multiple-reaction-monitoring (MRM) mode.
High Speed Mass Spectrometer
UF-MRM
High-Speed MRM at 555ch/sec
UFswitching
High-Speed Polarity Switching 5msec
Result
Method development
First, MRM method of 20 amino acids was optimized. As a
result, all compounds were able to be detected high
sensitively and were detected in positive MRM transitions. As
the setting temperature of ESI heating gas was found to
affected on the sensitivity of amino acids, it was also
optimized. Even though amino acids were not derivartized
and ion-pairing reagent wasn’t used, 20 amino acids were
retained by using a mixed-mode stationary phase structure
and separated excellently on the below-mentioned
condition.
2
HPLC conditions (Nexera system)
Column
: Intrada Amino Acid (3.0mmI.D. x 50mm, 3um, Imtakt Corporation, Kyoto, Japan)
Mobile phase
Case1
A
: Acetonitrile / Formic acid = 100 / 0.1
B
: 100mM Ammonium formate
Time program
: B conc.14%(0-3 min) -100%(10min) - 14%(10.01-15min)
Case2 (High Resolution condition)
A
: Acetonitrile / Tetrahydrofuran / 25mM Ammonium formate / formic acid = 9 / 75 / 16 / 0.3
B
: 100mM Ammonium formate / Acetonitrile = 80 / 20
Time program
: B conc.0%(0-2 min) - 5%(3min) - 30%(6.5min) - 100%(12min)
- 0%(12.01-17min)
Flow rate
: 0.6 mL/min
Injection volume
: 2 uL
Column temperature
: 40 °C
MS conditions (LCMS-8050)
Ionization
: ESI, Positive MRM mode
MRM transition are shown in Table 1.
Case1
Mobile Phase
A: Acetonitrile / Formic acid = 100 / 0.1
B: 100mM Ammonium formate
Thr
Phe
Pro
4.4
Ile
Leu
Met
3.0
4.5
4.6
4.7
4.8
Asp
Ser
4.9
5.0
5.1
His
5.2 min
Lys
Gln
Arg
Val
Glu
Tyr
2.0
Gly
Ara
Trp
4.0
Asn
5.0
(Cys)2
6.0
7.0
8.0
9.0
min
Figure 2 Mass Chromatograms of 20 Amino acids (concentration of each compound : 10nmol/mL)
3
Case2 (High Resolution condition)
Mobile Phase
A: Acetonitrile / Tetrahydrofuran / 25mM Ammonium formate / formic acid = 9 / 75 / 16 / 0.3
B: 100mM Ammonium formate / Acetonitrile = 80 / 20
Thr
Pro
Asp
Ara
Ser
Gly
Ile
4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 min
Trp Phe
Arg
Leu
Tyr
Met Val
Glu
1.0
2.0
3.0
Thr
4.0
Gln
5.0
(Cys)2
Asn
6.0
7.0
His
8.0
Lys
9.0
10.0 min
Figure 3 Mass Chromatograms of 20 Amino acids (concentration of each compound : 10nmol/mL)
In this study, two conditions of mobile phase were
investigated. It was found that 20 amino acids were
separated with higher resolution in case2.
As the mobile phase condition of case1 is more simple and
the result of case1 was sufficiently well, case1 analytical
condition was used for quantitative analysis. The dilution
series of these compounds were analyzed. All amino acids
were detected with good linearity and repeatability (Table1).
4
Table1 Linearity and Repeatability of 20 amino acids
Linearity
MRM Transition
Repeatability*
Range (nmol/mL)
Coefficient (r2)
%RSD
Trp
205.10>188.10
0.01-100
0.9950
1.4
Phe
166.10>120.10
0.01-100
0.9971
1.2
Tyr
182.10>136.00
0.05-100
0.9900
1.7
Met
150.10>56.10
0.05-200
0.9963
0.1
Lue, Lle
132.10>86.15
0.01-100
0.9955
0.7
Val
118.10>72.05
0.05-100
0.9991
1.9
Glu
148.10>84.10
0.05-10
0.9965
4.5
Pro
116.10>70.10
0.01-50
0.9933
1.5
Asp
134.20>74.10
0.5-500
0.9953
1.4
Thr
120.10>74.00
0.1-50
0.9923
4.5
Ala
90.10>44.10
0.5-500
0.9989
16.2
Ser
106.10>60.20
0.5-500
0.9988
6.5
Gln
147.10>84.10
0.05-1
0.9959
3.9
Gly
76.20>29.90
5-200
0.9974
11.0
Asn
133.10>74.05
0.05-20
0.9939
6.1
(Cys)2
241.00>151.95
0.05-20
0.9909
2.3
His
156.10>110.10
0.05-200
0.9983
1.7
Lys
147.10>84.10
0.05-5
0.9908
0.9
Arg
175.10>70.10
0.01-100
0.9956
0.5
*@ 0.5nmol/mL : except for Gly, 5nmol/mL : for Gly
The analysis of 20amino acids in food samples
The analysis of the amino acids contained in sports beverage on the market was carried out. In the case of sports beverage,
all amino acids written in the package were detected.
Sports Beverage
Pro
Thr
Lys
Gly
Phe
Trp
Leu Ile
Met
2.0
Ser
Ara
3.0
4.3
Tyr
Val
4.0
Thr
4.4
4.5
4.6
4.7
4.8
4.9
5.0
Asp
5.1
min
His
Arg
Glu
5.0
6.0
7.0
8.0
9.0
min
Figure 4 Mass Chromatograms of Sports Beverage (100 fold dilution with 0.1N HCl)
5
Furthermore, Japanese Sake, Beer and sweet cooking rice
wine (Mirin) were analyzed using this method. Japanese
Sake and Beer were diluted with 0.1N HCl. Sweet cooking
rice wine was diluted in the same way after a deproteinizing
Pro
preparation. These were filtered through a 0.2um filter and
then analyzed. MRM chromatograms of each food samples
are shown in Figure 5,6,7. Amino acids of each sample were
detected with high sensitivity.
Japanese Sake
Ala
Phe
Arg
Gly Ser
Thr
4.4
Leu
Trp Ile
Met
2.0
4.5
4.6
4.7
Tyr
Val
Ala
3.0
4.9
5.0
5.1
5.2min
His
Gln
Asn
Glu
4.0
4.8
Lys
(Cys)2
5.0
6.0
7.0
8.0
9.0
min
Figure 5 Mass Chromatograms of Japanese Sake (100 fold dilution with 0.1N HCl)
Beer
Pro
Asn
Asp
Ala
Thr
Trp
Phe
4.5
4.6
4.7
4.8
4.9
5.0
5.1
min
Tyr
Leu
Ile
Met
2.0
Gly Ser
Glu Gln
Val
3.0
Arg
His
4.0
Lys
(Cys)2
5.0
6.0
7.0
8.0
9.0
min
Figure 6 Mass Chromatograms of Beer (10 fold dilution with 0.1N HCl)
Trp
Sweet Cooking Rice Wine
Asn
Thr
Phe
Pro
4.5
Met
2.0
3.0
Gly
4.6
4.7
4.8
4.9
5.0
5.1
min
Gln
Tyr
Leu Ile
Asp
Ser
Ala
Glu
Val
(Cys)2
4.0
5.0
6.0
Arg
His
7.0
Lys
8.0
9.0
min
Figure7 Mass Chromatograms of Sweet Cooking Rice Wine (100 fold dilution with 0.1N HCl)
6
Conclusions
• 20 amino acids could be separated without derivatization using a typical volatile mobile phase suitable for LC/MS analysis
and detected with high sensitivity.
• This methods was able to be applied to the analysis of amino acids in various food samples.
Environment
• Page 170
Rapid screening and confirmation
analysis of polycyclic aromatic
hydrocarbons (PAHs) with DART mass
spectrometry
• Page 176
Fast and highly sensitive analysis of
multiple drugs in ground-, surface- and
wastewater
• Page 182
Multi-residue analysis of pyrethroids in soil
and sediment using QuEChERS by LC/MS/MS
PO-CON1455E
Rapid Screening and confirmation
analysis of polycyclic aromatic
hydrocarbons (PAHs) with DART
mass spectrometry
ASMS 2014
MP 551
Yu Takabayashi1, Jun Watanabe2, Motoshi Sakakura3,
Teruhisa Shiota3
1 SHIMADZU TECHNO-RESEARCH, INC., Tokyo, Japan;
2 Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan;
3 AMR Inc., Meguro-ku, Tokyo, Japan
Rapid Screening and confirmation analysis of polycyclic
aromatic hydrocarbons (PAHs) with DART mass spectrometry
Introduction
Recently, the regulation of the content of the polycyclic
aromatic hydrocarbons (PAHs) in goods which may put into
a mouth or may contact is advanced and the technologies
of measuring PAHs quickly are being developed.
The ionizing principle of DART (Direct Analysis in Real Time)
using the excitation helium gas is able to widely ionize the
wide-range compounds and it may also be able to ionize
the compounds which are not ionized by ESI. Since PAHs is
ionizable by DART, PAHs can be quickly screened by
holding up a sample directly to DART. In this research, the
technique detected by DART-MS was developed coupling
with LC and DART analysis after carrying out LC separation
was performed.
Commercial PAHs were used for the sample. The samples
were applied to DART MS with the solution formed in
suitable concentration or the powder formed. Small
amount of the samples were picked up and held in the
DART ionization gas stream using glass capillaries. In
LC-DART MS analysis, the mixed-solution of PAHs standard
was prepared and applied to HPLC. After carrying out
chromatogram separation using a reverse phased column,
LC-DART MS analysis was conducted by loading an eluate
to a DART ionization area continuously.
DART OS ion source and single/triple quadrupole type mass
spectrometer were used for this experiment. PAHs
measured in the detection mode which performs a full
scan mode with positive and negative simultaneous
ionization.
MS condition (LCMS-2020; Shimadzu Corporation)
Ionization
: DART (Direct Analysis in Real Time)
Heater Temperature (DART) : 300°C to 500°C
Measuring mode (MS)
: Positive/Negative scanning simultaneously
Ufswitching
Ufscanning
High-Speed Scanning 15,000u/sec
Figure 1 DART-OS ion source & LCMS-2020
2
Result
First, in order to verify whether PAHs ionizes in DART, PAH standard reagents were
analyzed in DART-MS. Benzo[a]anthracene, acenaphthene, anthracene, etc. were used as
typical PAHs. When benzo[a]anthracene was analyzed, in the positive spectrum, the signal
at m/z 229 which is equivalent to [M+H]+ was detected. Moreover, in the negative
spectrum, the signal at m/z 243 which is equivalent to [M+O-H]- was detected. Similarly,
acenaphthene and anthracene could also be ionized by DART-MS and were able to be
assigned as molecular related ion. Additionally pyrene and fluoranthene were also
examined. As each of these is structural isomers mutually in structural-formula C16H10, in
the negative spectrum, the signal of [M+O-H]- is detected by m/z 217 in each other, and
either was not able to identify whether the detected signal is pyrene or fluoranthene in
analysis by DART-MS without chromatogram separation.
12500000
1:BPC(+)
Positive TIC m/z 100-300
10000000
7500000
A
5000000
2500000
0
2:BPC(-)
750000
Negative TIC m/z 100-300
B
500000
250000
0
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
min
Inten. (x1,000,000)
7.0
6.0
229.3
Positive
[M+H]+
Benzo[a]anthracene
5.0
4.0
M+
C
3.0
2.0
245.2
1.0
0.0
100.0
125.0
Inten. (x100,000)
4.5
4.0
261.3
150.0
175.0
200.0
225.0
250.0
243.2
Negative
275.0
m/z
C18H12
Fw 228
[M+O-H]-
3.5
3.0
2.5
2.0
D
1.5
1.0
259.3
0.5
0.0
100.0
125.0
125.0
179.3
150.0
175.0
220.6
200.0
225.0
250.0
275.1
277.1
275.0
291.2
m/z
Figure 2 DART mass chromatogram and mass spectrum of Benzo[a]anthracene
A: positive mass chromatogram, B: negative mass chromatogram (The area with the orange dashed line is the time when sample was held in DART.)
C: positive mass spectrum, D: negative mass spectrum
3
Inten.(x100,000)
9.0
8.0
M+154.2
7.0
155.2
Positive
[M+H]+
6.0
5.0
Acenaphthene
C12H10
Fw 154
4.0
3.0
2.0
1.0
171.2
102.3
130.2 142.3
125.0
150.0
0.0
100.0
202.3
187.3
175.0
220.2
200.0
253.3
225.0
250.0
268.9
275.0
m/z
Inten.(x1,000,000)
3.0
179.2
2.5
Positive
[M+H]+
M+
2.0
Anthracene
C14H10
Fw 178
1.5
1.0
195.2
0.5
211.2
158.3
0.0
100.0
125.0
150.0
175.0
225.1
200.0
225.0
250.0
275.0
m/z
Inten.(x10,000,000)
1.00
Positive
204.2
0.75
0.50
Pyrene
C16H10
Fw 202
0.25
193.1
0.00
100.0
125.0
150.0
175.0
218.2
225.0
200.0
250.0
275.0
m/z
Inten.(x100,000)
1.75
217.2
[M+O-H]-
1.50
Negative
1.25
1.00
0.75
190.3
0.50
0.25
101.1
0.00
100.0
233.3
179.2
115.5
165.2
137.1
125.0
150.0
175.0
253.3
255.6
226.3
205.2
200.0
225.0
250.0
298.1
269.3
287.3
275.0
m/z
Inten.(x1,000,000)
208.3
2.5
2.0
fluoranthene
C16H10
Fw 202
122.3
1.5
1.0
220.3
169.2
0.5
0.0
100.0
222.3
183.2
136.3
108.2
4.0
Positive
194.2
236.3
125.0
150.0
175.0
200.0
225.0
250.0
275.0
m/z
Inten.(x100,000)
217.1
3.5
[M+O-H]-
Negative
3.0
2.5
2.0
1.5
167.1
1.0
0.5
0.0
100.0
165.8
125.0
150.0
181.1
175.0
194.2
208.3
200.0
233.3
222.7
225.0
247.0
256.2
250.0
270.9 283.0
275.0
m/z
Figure 3 DART mass spectra of acenaphthene (positive), anthracene (positive), pyrene (positive/negative) and fluoranthene (positive/negative)
4
Then, it examined the sample applied to DART
separating with LC in order to perform chromatogram
separation. As the suitable flow rate for DART ionization
was thought to be approximately 10uL/min, the splitter
located between column and DART ionization stage.
Furthermore, the closed interface was adopted for
sensitivity improvement.
Analytical Condition
Column
Mobile phase
Flow rate
DART heater temperature
Ionization
:
:
:
:
:
Unison UK-C8 (2.0mmI.D. x 100mm, 3um, Imtakt Corporation, Kyoto, Japan)
1mM Ammonium formate / Acetonitrile=75/25
0.2mL/min (to DART: 0.01mL/min)
500°C
Positive/Negative SIM mode
Injector
Pump
Column
splitter
Mobile phase
Figure 4 DART devices integrated with HPLC (AMR Inc.)
5
60000
2:325.00(+)
(a)
50000
40000
SIM 325(+) Quinine
30000
20000
10000
0
0.0
2.5
5.0
7.5
10.0
12.5
min
3:202.00(+)
50000
(b)
25000
0
SIM 202(+) pyrene
4:217.00(-)
7000
6000
SIM 217(-) pyrene
5000
4000
12500 3:228.00(+)
SIM 228(+)
benzo[a]anthracene
10000
7500
5000
5000 3:154.00(+)
SIM 154(+) acenaphthene
4000
3000
25000 3:178.00(+)
20000
SIM 178(+) anthracene
15000
10000
5000
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
min
Figure 4 LC-DART mass chromatogram
(a) Typical compound for DART; Quinine
(b) PAH mixture (4 compounds)
As a result, by measurement of each PAHs standard
reagent, each retention time was able to be confirmed
and also each PAH was able to be detected in each
retention time in the measurement using a PAH mixed
sample. The conclusion of this examination was
understood that DART MS is effective in quick screening,
and also LC-DART MS is effective in the confirmation
analysis of detected PAHs in analysis of PAHs.
Conclusions
DART mass spectrometer coupled with HPLC was valuable for confirmation analysis of polycyclic aromatic
hydrocarbons (PAHs)
PO-CON1448E
Fast and highly sensitive analysis
of multiple drugs in ground-,
surface- and wastewater
ASMS 2014
TP 583
Klaus Bollig1; Sven Vedder2, Anja Grüning2
1
2
Shimadzu Deutschland GmbH, Duisburg, Germany;
Shimadzu Europe GmbH, Duisburg, Germany
of multiple drugs in ground-, surface- and wastewater
Introduction
Many pharmaceuticals from medical treatments are
metabolized or partially degraded in the body. An even
larger amount of these compounds is excreted intact and
pollutes the aquatic environment. Relevant classes of drugs
are human or veterinary antibiotics, antiepileptics,
analgetics and lipid lowering drugs or radio-opaque
substances. The extent of damage caused to the
environment and the resulting health risk for humans or
animals should not be underestimated, even though it is
not understood in detail so far. The requirement for
universal, reliable and fast methods for drug determination
in water is steadily increasing. Highly sensitive
triple-quad-MS systems are suitable tools for the analysis of
residues in ground-, surface- and wastewater, but
development of a simple, rapid and robust method for
simultaneous measurement of trace levels of various
different classes of analytes in complex matrices is a
challenge.
Figure 1. LCMS-8050 triple quadrupole mass spectrometer
Method
This study describes a fast LC-MS/MS method for the
determination of trace levels of different classes of drugs in
water. With online SPE no further sample pretreatment is
necessary. The quaternary system with low pressure
gradient eluent (LPGE) and solvent blending functionality
renders addition of a third LC-Pump unnecessary. The
solvent blending function was further used for method
development. High speed values for MRM recording and
the fastest polarity switching time of 5 ms are essential
physical parameters for a maximum of data points on
various classes of analytes in different polarities during one
single analysis.
LC-MS/MS Method Optimization
One of the first steps during this automated process is the
precursor ion selection, followed by m/z adjustment of the
precursor. The collision energy is optimized for the most
abundant fragments and finally the fragment m/z is
adjusted. Six optimization steps were performed via flow
injection analysis, each taking 30 seconds (Figure 2). The
result of these automated steps was the automatic
generation of a final MRM method (Table 1).
2
(x1,000,000)
6.25 1:Sulfamethazin 278.70(+)
1:Sulfamethazin 278.80(+)
5.25
(x1,000,000)
5.00
4.75
4.50
4.25
Inten. (x100,000)
186.1
3.5
186.2
3.0
5.00
4.00
4.75
3.75
4.50
3.50
4.25
3.25
4.00
2.5
124.2
124.2
92.2
3.75
3.00
2.0
3.50
2.75
92.2
3.25
2.50
1.5
2.75
2.00
92.2
92.2
2.50
1.75
2.25
1.50
1.75
1.00
1.50
0.75
1.25
0.50
1.00
0.25
0.75
65.2
53.2
0.0
50.0
0.25
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
min
0.00
0.05
1st Step: m/z Precursor adjustment
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.65
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
75.0
186.2
108.2
108.2
124.2
124.2
107.2
124.3
108.2
108.1
100.0
148.9
156.1
124.2
124.2
186.1
213.3
213.2
213.2
205.5
201.2
204.1
190.8
197.4
149.4
156.0
168.2
143.3
125.0
150.0
175.0
200.0
225.0
250.0
3rd Step: Product Ion / CE selection
(x1,000,000)
1.7 1:Sulfamethazin 279.10>92.20(+) CE: -35.0
1:Sulfamethazin 279.10>92.20(+) CE: -34.0
(x1,000,000)
1.0 2:Sulfamethazin
279.10>186.10(+) CE: -15.0
0.70
92.2
80.2
80.1 92.3
80.0
92.3
186.1
124.2
min
2nd Step: Setting Q1 Prerod Bias
(x1,000,000)
92.2
65.2
65.2
65.1
0.5
0.50
0.00
-0.25
156.1
156.1
108.2
1.0
2.00
1.25
124.2
108.2
108.2
3.00
2.25
0.00
4.0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.05
0.00
0.0
0.0
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
min
4th Step: m/z Product Ion adjustment
0.000
0.025
0.050
0.075
0.100
0.125
0.150
0.175
0.200
0.225
0.250
0.275
0.300
0.325
min
0.000
0.025
5th Step: Setting Q3 Prerod Bias
0.050
0.075
0.100
0.125
0.150
0.175
0.200
0.225
0.250
0.275
0.300
0.325
min
6th Step: CE fine tuning
Figure 2. Automated MRM Optimization of the drug Sulfamethazin
3
m
Table 1. Optimized MRM transitions of 9 drugs
Compound
Mode
MRM transitions
Collision energy (kV)
Sulfamethazin
ESI positive
279,10>186,10 / 279,10>92,20
-17 / -31
Sulfamethoxazol
ESI positive
253,90>92,20 / 253,90>156,15
-26 / -15
Bezafibrat
ESI positive
362,10>139,15 / 362,10>316,25
-25 / -15
Carbamazepine
ESI positive
237,10>194,20 / 237,10>179,20
-19 / -34
Diclofenac
ESI positive
296,00>214,15 / 296,00>215,15
-34 / -19
Clofibric acid
ESI negative
213,00>127,00 / 213,00>85,00
15 / 15
Ibuprofen
ESI negative
205,10>161,30
7
Iopamidol
ESI negative
775,80>126,95
22
Iopromid
ESI negative
790,00>127,00
26
Solvent Blending
The solvent blending functionality entails automated
mobile phase preparation on a LPGE (low pressure
gradient) unit which is integrated in the binary LC pumps.
The blending function eliminates the need of mobile phase
pre-mixing, as necessary with ordinary binary pumps.
Mobile phase composition can simply be changed in the
method without physically changing the solvents.
Therefore solvent blending is a powerful tool for easy and
efficient elucidation of the SPE, the gradient and the
starting conditions. During this study the solvent blending
function was used for optimization of the SPE conditions.
A second LPGE unit was used for the analytical gradient.
Traditional method
Step 1
Step 2
Step 3
1: prepare 5 mmol/L
Ammonium formate (pH 8.5)
200 mL
800 mL
200 mL
800 mL
2: prepare H2O
3: prepare MeOH
4: prepare 0,0025%NH4OH
5: prepare mobile phase A
(SPE loading condition);
different conditions tested !
6: prepare mobile phase B1 and B2
(analytical condition and gradient)
Set these to system
Mobile phase blending function
LPGE Unit:
1: prepare 5 mmol/L
Ammonium formate (pH 8.5)
Mobile phase composition for SPE loading, solvent
blending allows to change conditions automatically
Only step 1!
2: prepare H2O
3: prepare MeOH
2nd LPGE Unit:
Set these to system
Gradient for SPE release and separation
4: prepare 0,0025%NH4OH
Figure 3. Solvent blending functionality
4
HPLC/MS Workflow
A
Pump 1
SPE-Column
Analytical-Column
+ LCMS 8050
Autosampler
B
Pump 1
SPE-Column
Autosampler
Pump 2
Waste
Analytical-Column
+ LCMS 8050
Pump 2
Waste
Figure 4. Scheme of online-SPE extraction (A) and analytical separation (B)
Final method
SPE Conditions
Injection volume
SPE-column
SPE-flow rate
SPE-loading buffer
: 250 µl
: Strata-X , 25 µm , 20*2 mm
: 1 ml/min
: 1 mmol/L ammonium formate (LPGE Pump B)
Analytical Conditions (LPGE Pump A)
Column
Flow rate
Solvent A
Solvent B
Gradient
: 0 min
: 1 min
: 1.5 min
: 4.5 min
: 4.51 min
: 6 min
: Kinetex C8, 2.6 µm, 100*2.1 mm
: 0.5 ml/min
: 0.0025% NH4OH
: MeOH 1 min – 2.5 min analytical separation
: 30% B
: 30% B
: 95% B
: 95% B
: 30% B
: 30% B (Stop)
LCMS Conditions
Interface
Nebulizing Gas Flow
Heating Gas Flow
Desolvation Line Temperature
Heat Block Temperature
Drying Gas Flow
Polaritiy Switching Time
: ESI
: 2.2 L/min
: 12 L/min
: 400 ºC
: 150 ºC
: 400 ºC
: 6 L/min
: 5 ms
5
Results
In this study we developed a fast method for direct online
SPE LC-MS/MS analysis of 9 different drugs in water with a
minimal LC configuration of two binary pumps equipped
with LPGE units. The solvent blending function was used
for method development of the SPE extraction. The second
LPGE unit was used for SPE release and analytical gradient
separation. Each compound was quantified in a
concentration range from 0.05 ng/ml up to 2 ng/ml.
Measurements were performed on Shimadzu’s LCMS-8050
Triple Quad MS System. The calibration curves and lowest
calibration point is shown in figure 5.
Figure 5. Calibration curve and lowest calibration point at 0.05 ng/ml of each compound
PO-CON1444E
Multi-residue analysis of pyrethroids
in soil and sediment using QuEChERS
by LC/MS/MS
ASMS 2014
TP 560
Yuka Fujito1, Kiyomi Arakawa1, Yoshihiro Hayakawa1
1 Shimadzu Corporation. 1, Nishinokyo-Kuwabaracho
Nakagyo-ku, Kyoto 604–8511, Japan
Multi-residue analysis of pyrethroids in soil and sediment
using QuEChERS by LC/MS/MS
Introduction
Pytrethroids are one of the most widely used commercial
household insecticides in agricultural or non-agricultural
application sites. Synthetic pyrethroids are poorly
water-soluble, but are strongly adsorbed to soil, therefore
these compounds are increasingly being found in soil or
sediments. Recently, soil and sediment contamination by
pyrethroids has been detected in both urban and
agricultural area, and it’s becoming a global concern due
to the influence on the insects and aquatic invertebrates.
Therefore, quick, high-sensitive and universal analysis
methods are required. The analysis of pyrethroids is
typically performed by GC or GC/MS because of their
hydrophobicity. In this study, we report the development
of a simultaneous analysis technique for trace amounts of
pyrethroids by LC/MS/MS.
Materials
Sample
Sampling point
Soil
Residential garden (Kyoto, Japan)
Sediment
Lake Biwa (Shiga, Japan)
Pyrethrin
I : R=CH3
II : R=CO2CH3
Cyhalothrin
Permethrin
Tefluthrin
Esfenvalerate
Figure 1 Chemical structure of pyrethroids
Sample preparation
Sample preparation was carried out by the use of the
QuEChERS method. In case of the soil samples, hydration
of sample with water before acetonitrile extraction is
required to improve the recovery and operability. Result of
several different extraction methods that changed the
amount of the soil and water added, we finally adopted a
combination of 5 g soil (or 10 g sediment) and 5 mL water,
and the following procedures were based on the original
QuEChERS method.
2
Step 1 : Acetonitrile extraction
Step 2 : Clean-up
Weigh 5 g soil / 10 g sediment
(Add STDs solution)
Transfer 6mL Extract 1 into dSPE tube*2
• 900 mg MgSO4
• 150 mg PSA
• 45 mg GCB
Add 5mL water
Shake vigorously by hand 1min.
Add 10mL acetonitrile
Centrifuge for 5min.
Add salt mixture*1
• 4g MgSO4
• 1g NaCl
• 1g Trisodium citrate dehydrate
• 0.5g Disodium hydrogencitrate sesquihydrate
Transfer the supernatant into a vial
Shake vigorously by hand 1min.
Filtration using disposable filter
Centrifuge for 10min. (Extract 1)
LC/MS/MS analysis
*1 : Q-sep QuEChERS Extraction Salts (RESTEK)
*2 : Q-sep QuEChERS dSPE Tubes (RESTEK)
LC/MS/MS analsis
HPLC conditions (Nexera UHPLC system, Shimadzu)
Column
Mobile phase
Gradient program
Flow rate
Column temperature
Injection volume
: Phenomenex Kinetex 2.6 µm PFP 100Å (100 mm x 2.1 mm I.D.)
: A 5mM ammonium acetate - water
: B Methanol
: 40 % B (0 min.) → 100 % B (10 -12 min.) → 40 % B (12.01-15 min.)
: 0.2 mL / min.
: 40 ºC
: 1 μL
MS conditions (LCMS-8050, Shimadzu)
Ionization
Interface temperature
DL temperature
Heat block temperature
Nebulizing gas
Drying gas
Heating gas
: ESI (positive / negative)
: 100 ºC
: 100 ºC
: 400 ºC
: 3.0 L / min.
: 15.0 L / min.
: 3.0 L / min.
3
Table 1 MRM transitions of pyrethroids
Compounds
Polarity
Quantitative ion (m/z)
Confirmation ion (m/z)
pyrethrin-I
+
329.20>161.20
329.20>105.20
pyrethrin-II
+
373.20>161.20
373.20>105.20
fenpropathrin
+
367.20>125.20
367.20>181.20
cycloprothrin
+
498.90>181.10
498.90>229.20
deltamethrin
+
522.80>280.90
522.80>181.10
esfenvalrate
+
437.10>167.30
437.10>125.30
cypermethrin
+
433.10>191.10
433.10>181.20
cyfluthrin
+
450.90>191.00
450.90>206.10
ethofenprox
+
394.20>177.30
394.20>107.20
permethrin
+
408.10>183.30
408.10>355.20
cyhalothrin
+
467.10>225.10
467.10>141.10
bifenthrin
+
440.00>181.20
440.00>166.10
acrinathrin
+
559.00>208.20
559.00>181.10
acrinathrin
-
540.10>372.20
540.10>345.30
silafluofen
+
426.20>287.10
426.20>168.20
Ultra Fast Scanning
- 30,000 u / sec.
Ultra Fast Polarity Switching
- 5 msec.
Ultra Fast MRM
- Max. 555 transitions / sec
Result
MRM of pyrethroid standards
In this study, we selected and evaluated 15 pyrethroids
(pyrethrin, fenpropathrin, cycloprothrin, deltamethrin,
esfenvarelate, cypermethrin, cyfluthrin, ethofenprox,
permethrin, cyhalothrin, bifenthrin, acrinathrin, tefluthrin,
silafruofen) which are the most widely used for household or
agrocultural insecticides worldwide.
Except for tefluthrin, which was not ionized by LC/MS under
conditions tested, all other 14 compounds were successfully
detected in ESI positive mode or in both positive and
negative mode.
4
Table 2 Calibration curves
1500000
1400000
compounds
min. conc.
max. conc.
r2
pyrethrin I
0.5
500
0.9996
1300000
pyrethrin II
0.5
500
0.9997
1200000
fenpropathrin
0.02
100
0.9993
1100000
cycloprothrin
0.5
100
0.9991
pyrethrin-II
1000000
pyrethrin-I
900000
fentropathrin
cycloprothrin
deltamethrin
esfenvalrate
cypermethrin
cyfluthrin
ethofenprox
800000
700000
600000
500000
400000
permethrin
300000
cyhalothrin
200000
bifenthrin
100000
acrinathrin
silafluofen
0
7.0
8.0
9.0
10.0
deltamethrin
0.05
100
0.9992
esfenvalerate
0.5
100
0.9990
cypermethrin
0.05
100
0.9986
cyfluthrin
0.5
100
0.9976
ethofenprox
0.01
100
0.9993
trans-permethrin
0.02
100
0.9996
cis-permethrin
0.02
100
0.9994
cyhalothrin
0.1
100
0.9993
bifenthrin
0.02
100
0.9995
acrinathrin (+)
0.1
100
0.9987
acrinathrin (-)
0.5
500
0.9993
silafluofen
0.01
100
0.9999
(ppb)
min
Figure 3 MRM chromatograms
fenpropathrin
0.02 ppb
1.25
permethrin
0.02 ppb
(x1,000)
(x1,000)
0.75
0.50
2.50
2.25
cis-
1.50
1.25
(x1,000)
(x1,000)
1.75
1.00
silafluofen
0.01 ppb
bifenthrin
0.02 ppb
2.25
2.00
2.00
1.75
trans-
1.75
1.50
1.00
1.25
0.75
1.00
1.50
1.25
1.00
0.75
0.50
0.25
0.25
0.00
9.5
0.50
0.25
0.25
0.00
0.00
9.0
0.75
0.50
9.5
10.0
0.00
9.5
10.0
10.5
10.0
10.5
11.0
Figure 4 MRM chromatograms of the LOQs of typical pyrethroids
5
Recovery from soil and sediment matrices
All target compounds showed good recoveries from soil and
sediment matrices in the range 70-120% by the QuEChERS
method. Neither matrix effect (Ion suppression or
enhancement) nor sample preparation losses were observed.
sediment (lake)
140
120
120
100
100
80
80
60
60
40
40
20
20
0
0
Py
Py
STDs spiked after prep
STDs spiked before prep
re
P thr
Fe yre innp th 2
r
Cy rop in-1
cl at
o h
D pro rin
el
ta th
Es m rin
fe et
Cy nv hri
pe alr n
rm at
e e
Cy th
fl r
tr Eth ut in
an o h
s- fen rin
P
ci erm pr
o
sPe et x
rm hr
e
Cy t in
ha hr
lo in
Bi th
fe rin
A nt
cr h
in ri
Si at n
la hr
flu in
of
en
140
re
Py thr
Fe ret in-2
np hr
i
Cy rop n-1
cl at
op hr
D ro in
el
ta th
Es me rin
fe th
Cy nv rin
pe alr
rm ate
e
Cy thr
flu in
E
tr th t
an o hr
s- fen in
P
ci erm pro
sPe et x
rm hr
Cy et in
ha hri
lo n
Bi thr
f
i
A ent n
cr h
in ri
n
Si ath
la ri
flu n
of
en
Recovery (%)
soil (residential garden)
Figure 5 Recovery of 14 pyrethroids from soil and sediment matrices (10 ppb spiked)
Quantitative analysis of soil and sediment
The quantitative analysis of the soil and sediment sample
was performed. Ethofenprox and permethrin was detected
ethofenprox
from the soil sample at approximately 0.02 and 0.06 μg /
kg, respectively.
Table 3 Result of quantitative analysis in the soil and sediment
permethrin
soil blank
solvent blank
Figure 6 Chromatograms of prethroids in the soil
soil
sediment
pyrethrin-I
n.d.
n.d.
pyrethrin-II
n.d.
n.d.
fenpropathrin
n.d.
n.d.
cycloprothrin
n.d.
n.d.
deltamethrin
n.d.
n.d.
esfenvalrate
n.d.
n.d.
cypermethrin
n.d.
n.d.
cyfluthrin
n.d.
n.d.
ethofenprox
0.01 ppb*
n.d.
permethrin
0.03 ppb
n.d.
cyhalothrin
n.d.
n.d.
bifenthrin
n.d.
n.d.
acrinathrin
n.d.
n.d.
silafluofen
n.d.
n.d.
n.d. : not detected
* : <LOQ
6
Conclusions
• A method for quantification of 14 pyrethroids in soil and sediment at ppt-level concentrations was developed by
LC/MS/MS.
• In this study, neither matrix effect nor sample preparation losses were observed in the recovery test, demonstrating the
applicability of QuEChERS method to sample preparation of soil and sediment.
Metabolism
• Page 197
High sensitivity analysis of metabolites in serum
using simultaneous SIM and MRM modes in a
triple quadrupole GC/MS/MS
• Page 202
Analysis of D- and L-amino acids using automated pre-column derivatization and liquid
chromatography-electrospray ionization
mass spectrometry
• Page 208
Characterization of metabolites in microsomal
metabolism of aconitine by high-performance
liquid chromatography/quadrupole ion trap/
time-of-flight mass spectrometry
• Page 213
Simultaneous analysis of primary metabolites
by triple quadrupole LC/MS/MS using pentafluorophenylpropyl column
PO-CON1443E
High Sensitivity Analysis of
Metabolites in Serum Using
Simultaneous SIM and MRM Modes
in a Triple Quadrupole GC/MS/MS
ASMS 2014
ThP 641
Shuichi Kawana1, Yukihiko Kudo2, Kenichi Obayashi2,
Laura Chambers3, Haruhiko Miyagawa2
1 Shimadzu, Osaka, Japan, 2 Shimadzu, Kyoto, Japan,
3 Shimadzu Scientific Instruments, Columbia, MD
High Sensitivity Analysis of Metabolites in Serum Using
Simultaneous SIM and MRM Modes in a Triple Quadrupole GC/MS/MS
Introduction
Gas chromatography / mass spectrometry (GC–MS) and a gas chromatography-tandem mass spectrometry (GC-MS/MS)
are highly suitable techniques for metabolomics because of the chromatographic separation, reproducible retention times
and sensitive mass detection.
MRM measurement mode
Some compounds with low CID efficiency produce insufficient product ions for MRM transitions, and the MRM mode is
consequently less sensitive than SIM for these compounds.
Our suggestion
SIM, MRM, and simultaneous SIM/MRM modes are evaluated for analysis of metabolites in human serum.
Materials and Method
Sample and Sample preparation
Sample
• Human serum
Sample Preparation1)
50uL serum
Supernatant 250 µL
Add 250 µL water / methanol / chloroform (1 / 2.5 / 1)
Add internal standard (2-Isopropylmalic acid)
Stir, then centrifuge
Extraction solution 225 µL
Add 200 µL Milli-Q water
Stir, then centrifuge
Freeze-dry
Residue
Add 40 µL methoxyamine solution (20 mg/mL, pyridine)
Heat at 30 ºC for 90 min
Add 20 µL MSTFA
Heat at 37 ºC for 30 min
Sample
1) Nishiumi S et. al. Metabolomics. 2010 Nov;6(4):518-528
Instrumentation
GC-MS
Data analysis
Database
Column
:
:
:
:
GCMS-TQ8040 (SHIMADZU)
GCMSsolution Ver.4.2
GC/MS Metabolite Database Ver.2 (SHIMADZU)
30m x 0.25mm I.D., df=1.00µm (5%-Phenyl)-methylpolysiloxane
2
Simultaneous SIM and MRM modes in GC/MS/MS
Figure 1 shows the theory of Simultaneous SIM and MRM modes. This analysis mode can measure SIM and MRM data in a
single analysis.
Q1
Collision Cell
Q3
SIM
CID
SIM
SIM
SIM
MRM
MRM
Figure 1 The concept of simultaneous SIM and MRM analysis mode.
Precursor ion (or SIM)
Product ion
%
100
100
75
%
361
73
50
25
0
103
147
100
169
75
50
217
271
191 243 319
200
CID
300
437
400
25
73
0
103
243
100
200
361
300
Figure 2 Mass Spectrum of Precursor (or SIM) and Product ion
Poor sensitivity of MRM in some compounds because of low CID efficiency
Method Creation using Database and SmartMRM
Figure 3 shows the GC/MS Metabolites Database Ver.2. This database involves conditions of SIM and MRM in 186
metabolites and a method creation function we call SmartMRM. SmartMRM creates MRM, SIM, SIM/MRM methods
from Database automatically.
Figure 3 GC/MS Metabolites Database Ver.2
• Select the MRM, SIM and SIM/MRM conditions of 186 TMS derivatization metabolites from GC/MS Metabolites
Database Ver.2.
• Select the two transitions (or ions) each metabolite.
3
Results
Comparison of the chromatogram between SIM and MRM in human serum
a) Glucuronic acid-meto-5TMS(2)
SIM
(x100,000)
333.10
3.5 160.10
MRM
(x10,000)
333.10>143.10
1.75 333.10>171.10
3.0
1.50
2.5
1.25
2.0
1.00
1.5
0.75
1.0
0.50
0.5
0.25
21.00
21.25
21.00
21.25
Detected the peak in MRM because of high selectivity
b) S-Benzyl-Cysteine-4TMS
SIM
(x100,000)
2.00 238.10
218.10
1.75
MRM
(x10,000)
218.10>73.00
7.5 238.10>91.00
1.50
5.0
1.25
1.00
0.75
(x100)
1.75 238.10>91.00
1.50
1.25
1.00
0.75
0.50
0.25
21.00
21.25
21.50
2.5
0.50
0.25
21.25
21.00
21.50
21.25
21.50
Peak was not detected in MRM because of low CID efficiency.
A number of Identification metabolites in serum
Table 1 shows the identification results of metabolites in human serum using SIM, MRM and simultaneous SIM/MRM
analysis modes in GC/MS/MS. In SIM/MRM, the metabolites, which were insufficient sensitivity in MRM, were measured by
SIM and the other metabolites were measured by MRM.
Table 1 The number of identified metabolites each analysis mode
Modes
A
B
C
Total
SIM
57
51
8
116
MRM
131
14
1
146
SIM/MRM
133
22
1
156
note) A:Target and Confirmation ions were detected.; B: Either Target or Confirmation ion was detected.
Another one was overlapped by contaminants.; C: Either Target or Confirmation ion was detected.
4
Fig.4 shows a number of metabolites in each mode can be measured. In metabolites with low CID efficiency, SIM are
superior to MRM if there are no interfering substances to the target metabolites.
MRM
SIM
40
106
10
Metabolites with low CID
efficiency in MRM
Metabolites with
interference in SIM
Figure 4 Detected metabolites in human serum each analysis mode.
The reproducibility(n=6) in MRM and SIM/MRM
Table 2 Comparison of the reproducibility results from MRM and SIM/MRM analysis.
A number of detected metabolites involves A, B and C in Table 1.
%RSD
MRM
SIM/MRM
Improvement
- 4.99%
73
76
+3
5 - 9.99%
26
30
+4
10 - 14.99%
8
10
+2
15 - 19.99%
9
10
+1
> 20%
30
30
0
146
156
+10
Conclusions
• Analytical results from the SIM and MRM modes identified 116 and 146 metabolites, respectively.
• In metabolites with poor CID efficiency, the sensitivity of SIM is more than 10 times higher than MRM.
• Simultaneous SIM and MRM modes in a single analysis (SIM/MRM) improves the sensitivity and reproducibility for
analysis of metabolites in human serum compared to MRM alone.
• A novel SIM/MRM expands the utility of a triple quadrupole GC/MS/MS
PO-CON1451E
Analysis of D- and L-amino acids using
automated pre-column derivatization
and liquid chromatography-electrospray
ionization mass spectrometry
ASMS 2014
MP739
Kenichiro Tanaka1; Hidetoshi Terada2; Yoshiko Hirao2;
Kiyomi Arakawa2; Yoshihiro Hayakawa2
1. Shimadzu Scientific Instruments, Inc., Columbia, MD;
2. Shimadzu Corporation, Kyoto, Japan
Analysis of D- and L-amino acids using automated pre-column
derivatization and liquid chromatography-electrospray ionization
mass spectrometry
Introduction
Recently, several species of D- amino acids have been
found in mammals including humans and their
physiological functions have been elucidated. Quantitating
each enantiomer of amino acids is indispensable for such
studies. In order to diagnose diseases, it is desirable that Dand L-amino acid can be separately quantitated and
applied to metabolic analysis.
Pre-column derivatization with o-phthalaldehyde (OPA) and
N-acetyl-L-cysteine(NAC) is widely utilized for the analysis
of D- and L- amino acids since the method can be
performed with a rapid reversed phase separation on a
relatively simple hardware (U)HPLC configuration with
good reliability. One of the drawbacks of pre-column
derivatization is less reproducibility due to the tedious
manual procedure and human errors. We have launched
an autosampler for a UHPLC system equipped with an
automated pretreatment function that allows overlapping
injections in which the next derivatization proceeds during
the current analysis for saving total analytical time. We
have applied this autosampler and its function to fully
automate pre-column derivatization for the determination
of amino acids. In this study, we developed a methodology
which enabled the automated procedure of pre-column
chiral derivatization of D- and L- amino acids.
Experimental
Instruments
The system used was a SHIMADZU UHPLC Nexera
pre-column Amino Acids (AAs) system consisting of
LC-30AD solvent delivery pump, DGU-20A5R degassing
unit, SIL-30AC autosampler, CTO-30A column oven, and
SHIMADZU triple quadrupole mass spectrometer
LCMS-8040. The software is integrated in the LC/MS/MS
workstation (LabSolutions, Shimadzu Corporation, Japan)
so that selected conditions can be seamlessly translated
into method files and registered to a batch queue, ready
for instant analysis. A 1.9um YMC-Triart C8 column (2.0
mm x 150 mm L.) was used for the analysis.
Derivatization Method
Derivatizing solutions:
0.1 mol/L boric acid buffer was prepared by dissolving 6.18
g of boric acid and 2.00 g of sodium hydroxide in 1 L of
water.
10 mmol/L NAC solution was prepared by dissolving 16.3
mg of N-acetyl-L-cysteine in 10 mL of the 0.1 mol/L boric
acid buffer.
10 mmol/L OPA solution was prepared by dissolving 6.7
mg of o-phthalaldehyde in 0.3 mL of ethanol, adding 0.7
mL of the 0.1 mol/L boric acid buffer and 4 mL of water.
Fig.1 shows the schematic procedure for amino acids
derivatization with the SIL-30AC.
Samples, including the derivatized amino acids, were
injected onto the UHPLC and separated under the
conditions shown in Table 1.
2
mass spectrometry
(1)
(3)
(2)
Supply 20 μL of
NAC solution to the
vial for mixing
Take 20 μL of 10 mmol/L
NAC solution
(6)
(7)
Supply 1 μL of
sample solution
to the vial for mixing
Supply 20 μL of
10 mmol/L OPA solution
to the vial for mixing
Take 20μL of
10 mmol/L OPA solution
(8)
Mix the sample solution
and derivatizing solutions
(5)
(4)
(9)
Wait for 3min until
the derivatization ends
Take 1 μL of
sample solution
(10)
Take 1μL of the mixed
solution
Inject 1μL of the mixed
solution to the column
Fig.1 Schematic procedure of automated pre-column derivatization
Table 1 UHPLC and MS analytical conditions
Mobile Phase
: A : 10 mmol/L Ammonium Bicarbonate solution
B : Acetonitrile/Methanol = 1/1(v/v)
Initial B Conc.
: 0%
Flow Rate
: 0.4 mL/min
Column Temperature
: 40 ºC
Injection Volume
: 1 μL
LC Time Program
: 0 -> 5%(0.01min), 5%(0.01-1.00min), 5 ->20%(1.00 - 15.00min),
20 - 25%(15.00 - 24.00min), 25 – 90%(24.00 - 24.50min),
90%(24.50 - 27.50min), 90 - 0% (27.50 – 28.50min)
Ionization Mode
: ESI
Nebulizing Gas Flow Rate
: 3 L/min
Drying Gas Flow Rate
: 15 L/min
DL Temperature
: 300 ºC
Heating Block Temperature : 450 ºC
Result
Analysis of Standard Solution
A standard solution containing 27 amino acids was
prepared at 1 mmol/L concentration each in 0.1 mol/L HCl
solution. The MS conditions such as ESI positive and
negative ionization modes were optimized in parallel with
the column separation, and compound dependent
parameters such as CID and pre-bias voltage were adjusted
using the function for automatic MRM optimization. The
transition that provided the highest intensity was used for
quantification.
Table 2 shows the MRM transition of each derivatized
amino acid.
The MRM chromatogram is illustrated in Fig.2.
3
mass spectrometry
Table 2 Compounds, Ionization polarity and MRM transition
Compound
Polarity
Precursor m/z
Product m/z
Aspartic acid
+
395.00
130.00
Glutamic acid
+
409.10
130.05
Serine
+
367.00
130.00
Glutamine
+
408.20
130.05
Glycine
+
337.00
130.00
Histidine
+
417.10
244.05
Threonine
+
381.20
130.05
Arginine
+
436.10
263.10
Tyrosine
+
443.00
130.05
Valine
+
379.10
250.05
Tryptophan
+
466.20
337.10
Isoleucine
+
393.00
264.05
Phenylalanine
+
427.20
298.05
250000
9
1
225000
2
6
200000
175000
3 4
5
7
150000
125000
11 15
16
17
14
8
10
20
13
12
100000
18
75000
19
24
21
22
50000
27
23
26
25
25000
0
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
min
■Peaks
1. D-Aspartic acid, 2. L-Aspartic acid, 3. L-Glutamic acid, 4. D-Glutamic acid, 5. D-Serine, 6. L-Serine, 7. L-Glutamine
8. D-Glutamine, 9. Glycine, 10. L-Histidine, 11. D-Histidine ,12. D-Threonine, 13. L-Threonine, 14. L-Arginine
15. D-Arginine, 16. D-Alanine, 17. L-Alanine, 18. D-tyrosine, 19. L-Tyrosine, 20. L-Valine, 21. D-Valine
22. L-Tryptophan, 23. D-Tryptophan, 24. L-Isoleucine, 25. D-Phenylalanine, 26. L-Phenylalanine, 27.D-Isoleucine
Fig. 2 Chromatogram of a 27 amino acid standard solution
4
mass spectrometry
Method Validation
Reproducibility and linearity in this analysis were evaluated
with a plasma spiked standard solution. As a result, less
than 5% relative standard deviation of peak areas were
obtained. Table 3 shows the reproducibility of repeated
analysis of spiked sample (n=6). Five different levels of
spiked sample concentration from 1 to 100 μmol/L
standard solution were used for the linearity evaluation.
The coefficients of determination (r2) were approximately
0.999. Table 4 shows the summary for the linearity results.
Table 3 Reproducibility
Compound
Repeatability (%RSD)
5 μmol/L
25 μmol/L
D-Aspartic acid
3.5
2.5
D-Glutamic acid
3.7
3.1
D-Serine
4.8
3.0
D-Glutamine
4.1
3.4
D-Histidine
4.3
1.8
D-Threonine
3.8
2.6
D-Arginine
3.4
1.7
D-Alanine
4.0
2.3
D-Tyrosine
3.2
2.9
D-Valine
3.3
2.2
D-Tryptophan
3.9
3.2
D-Isoleucine
3.1
2.9
D-Phenylalanine
3.5
1.8
Table 4 Linearity
Compound
Cali.F
r2
D-Asparic acid
Y = (44661.8)X + (1829.61)
0.998
D-Glutamic acid
Y = (12191.8)X + (10390.7)
0.999
D-Serine
Y = (22319.5)X + (-2869.30)
0.999
D-Glutamine
Y = (3458.60)X + (1521.83)
0.999
D-Histidine
Y = (5778.33)X + (-341.182)
0.998
D-Threonine
Y = (10800.6)X + (-1874.07)
0.999
D-Arginine
Y = (10535.7)X + (-1298.12)
0.998
D-Alanine
Y = (15349.1)X + (-4719.98)
0.999
D-Tyrosine
Y = (17098.7)X + (-1812.69)
0.999
D-Valine
Y = (23707.7)X + (772.548)
0.999
D-Tryptophan
Y = (18089.1)X + (-3620.41)
0.998
D-Isoleucine
Y = (44017.1)X + (67903.1)
0.999
D-Phenylalanine
Y = (22426.0)X + (-736.090)
0.999
5
mass spectrometry
Table 5 Recovery
Compound
Recovery (100%)
5 μmol/L
25 μmol/L
D-Asparic acid
100.3
107.1
D-Glutamic acid
92.8
97.8
D-Serine
97.9
100.6
D-Glutamine
103.2
104.3
D-Histidine
104.8
100.4
D-Threonine
101.1
98.8
D-Arginine
102.4
99.6
D-Alanine
93.5
99.5
D-Tyrosine
98.1
101.0
D-Valine
101.0
99.2
D-Tryptophan
97.8
100.4
D-Isoleucine
98.8
102.4
D-Phenylalanine
104.5
100.9
Considering the frequency of amino acids analysis in physiological samples, the recovery of spiked samples were
confirmed. In addition, the results indicated that the recovery ratio of most amino acids are around 100%.
Table 5 shows the summarized results for the recovery of each amino acid.
Conclusions
• The combination of Shimadzu triple quadrupole mass spectrometer and Nexera UHPLC provides reliable pre-column
derivatized AAs analysis with enhanced productivity.
• An established method was successfully applied to the separation of D- and L- amino acids with excellent reliability.
PO-CON1476E
Characterization of metabolites in
microsomal metabolism of aconitine
by high-performance liquid
chromatography/quadrupole ion
trap/time-of-flight mass spectrometry
ASMS 2014
WP 739
Cuiping Yang1, Changkun Li2, Tianhong Zhang1,
Qian Sun2, Yueqi Li2, Guixiang Yang2, Taohong Huang2,
Shin-ichi Kawano2, Yuki Hashi2, Zhenqing Zhang1,*
1
Beijing Institute of Pharmacology & Toxicology,
2
Shimadzu Global COE, Shimadzu (China) Co., Ltd., China
Characterization of metabolites in microsomal metabolism
of aconitine by high-performance liquid chromatography/quadrupole
ion trap/time-of-flight mass spectrometry
Introduction
Aconitine (AC) is a bioactive alkaloid from plants of the
genus Aconitum, some of which have been widely used as
medicinal herbs for thousands of years. AC is also well
known for its high toxicity that induces severe arrhythmias
leading to death. Although numerous studies have raised
on its pharmacology and toxicity, data on the identification
metabolites of AC in liver microsomes are limited. The
study of metabolic pathways is very important for efficacy
of therapy and evaluation of toxicity for those with narrow
therapy window.
The aim of our work was to obtain the metabolic pathways
of AC by the human liver microsomes.
Sample Preparation
The typical reaction mixture incubation contained 10 μ
mol/L aconitine and was preincubated at 37 ºC for 3 min.
Reactions were initiated by adding 50 μL of NADPH (20
mmol/L), then incubated at 37 ºC in a waterbath shaker for
Instrument
60 min. The reactions were terminated by adding 3-volume
of ice-cold acetonitrile, then vortexed and centrifuged to
remove precipitated protein.
: LCMS-IT-TOF (Shimadzu Corporation, Japan);
UFLCXR system (Shimadzu Corporation, Japan);
: Shim-pack XR-ODS II (2.0 mmI.D. x 75 mmL.,2.2 μm)
: A: water (0.1% formic acid+5 mmol ammonium formate),
B: acetonitrile
: 30%B (0-4 min)-80%B (8 min)-80%B (8-11 min)-30%B (11.01-17 min)
: 0.3 mL/min
Column
Mobile phase
Gradient program
Flow rate
Results
(x1,000,000)
1:TIC (1.00)
7.5
A
5.0
2.5
0.0
0.0
7.5
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
32.5
35.0
(x1,000,000)
B
5.0
M10
M3
M2
2.5
M1
M5
M12
M6
M4
M7
M9
M11 M13
M8
M14
M15
M16
M0
0.0
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
32.5
Fig.1 TIC chromatogram (A) and mass chromatograms of the metabolites of AC in the microsomal incubation mixture of human (B)
2
OH
O
OH
OH+
OO
O
N
OH
O
O
O
O
O
OH
+
OH
OH
+
O
O
OH
C34H48NO11+
Exact Mass: 646.3227
O
C32H44NO9+
OH
OH
+
C31H40NO8+
OH
O
OHH+
OH
OH
+
OH
OH
O
HN
H
O
O
O
N
H
C25H36NO9+
O
O
HN
O
O
OH
O
OHH+
O
N
H
O
O
O
O
O
N
OH
H
C29H36NO8+
H
O
O
O
HN
OH
OH
O
O
O
N
H
O
O
OH
+
O
O
H
HO
C22H26NO4+
Exact Mass 368.1862
H
C25H34NO8+
C21H25NO4+
Exact Mass 354.1705
Fig. 2 Proposed fragmentation pathway of AC
OH
OH
OH
O
O
HOH2C
N
O
N
OH
HO
O
O
O
O
H
O
M8
O
O
O
O
N
OH
OH
H
OH
O
O
O
O
O
N
OH
O
O
H
O
O
H
O
OH
O
O
O
O
M13
N
OH
O
O
OH
OH
O
H
O
O
M7
O
O
O
O
O
OH
HO
OH
OH
O
O
O
N
HO
O
O
O
O
O
H
M10
O
N
OH
HO
O
O
O
O
H
O
N
M14
O
O
H
O
O
O
HO
O
OO
O
OH
O
HN
H
O
N
M12
O
OH
O
O
H
M1
O
N
OH
O
OO
O
O
O
H
OH
O
M5
H
O
OH
O
O
O
O
M3
OH
O
N
OH
OH
OH
OH
OH
M15
OO
O
OH
O
H
OH
OO
OH
OH
HO
O
OH
N
O
N
OH
OH
M0
O
O
M4
O
O
O
H
O
OH
HOH2C
M6
OH
O
M11
O
O
O
OH
OH
OH
O
O
O
O
N
OH
H
OH
HO
OH
O
O
O
HO
O
N
OH
O
OO
HOH2C
O
H
M9
O
OH
HO
OH
HO
M2
OH
O
O
N
O
O
O
OH
O
OO
OH
O
O
OH
OH
O
O
H
M16
Fig. 3 Proposed metabolic profile of AC in the human liver microsomes
3
Table1 Mass data for characterization of metabolites in of AC in the microsomal
incubation mixture of human
No.
RT
(min)
Meas.MW
(m/z)
Pred.MW
(m/z)
M0
22.3
646.3230
646.3222
0.8
M1
10.5
618.2922
618.2909
M2
11.2
616.2754
M3
11.3
M4
mDa ppm
error error
MS2 data
Formula
Biotransformation
1.26
586.3000, 554.2752, 526.2785, 494.2536,
476.2431, 404.2432, 368.1847, 354.1687
C34H47NO11
Parent
1.3
2.10
558.2710, 498.2469, 480.2378, 436.2093,
354.1725
C32H43NO11
deethylation
616.2752
0.2
0.26
556.2510, 554.2335, 494.2106, 478.2321,
434.1908, 402.1682
C32H41NO11
bidemethylation+
dehydrogenation
604.3140
604.3116
2.4
3.94
554.2744, 522.2398, 434.1898
C32H45NO10
deacetylation
11.8
630.2930
630.2909
2.1
3.35
570.2686, 552.2576, 510.2457, 492.2381
C33H43NO11
demethylation+
dehydrogenation
M5
12.2
586.3005
586.3011
0.6
0.96
568.2938, 554.2705, 522.2537, 466.2168,
434.1922
C32H43NO9
deacetylation+
dehydration
M6
13.3
616.2769
616.2752
2.3
3.68
584.2477, 524.2316, 434.1941
C32H41NO11
bidemethylation+
dehydrogenation
M7
13.5
632.3035
632.3065
3.0
4.81
572.2866, 512.2638, 494.2468, 480.2283,
462.2214, 290.2236, 354.1652, 340.1871
C33H45NO11
demethylation
M8
13.7
648.3016
648.3015
0.1
0.23
588.2702, 570.2654, 528.2566, 510.2434,
406.2161
C33H45NO12
oxidation+
demethylation
M9
13.8
618.2935
618.2909
3.0
4.88
558.2714, 494.2109, 476.2400, 340.1548
C32H43NO11
bidemethylation
M10
14.1
618.2890
618.2909
1.5
2.43
558.2722, 494.2127, 476.2009, 354.1635
C32H43NO11
bidemethylation
M11
15.0
662.3179
662.3171
0.8
1.21
602.2964, 570.2654, 542.2750, 510.2434,
420.2416
C34H47NO12
oxidation
M12
15.1
602.2948
602.2960
1.6
2.66
584.2533, 524.2249, 510.2179, 406.1582
C32H43NO10
deacetylation+
dehydrogenation
M13
16.0
632.3054
632.3065
1.1
1.80
572.2853, 512.2661, 480.2368, 476.2445,
436.2082, 368.1812
C33H45NO11
demethylation
M14
17.3
662.3209
662.3171
3.8
5.74
602.2947, 570.2654, 542.2766, 510.2434,
478.2187
C34H47NO12
oxidation
M15
17.6
632.3068
632.3065
0.3
0.42
586.2973, 526.2738, 508.2273, 494.2490
C33H45NO11
demethylation
M16
17.9
584.2826
584.2854
2.8
4.82
552.2669, 492.2111, 460.2063
C32H41NO9
deacetylation+dehydration+
dehydrogenation
4
Conclusions
In this study, totaling 16 metabolites were found and characterized in the humam liver microsomes incubation mixture,
including O-demethylation, oxidation, bidemethylation, dehydrogenation, N-deethylation, deacetylation, dehydration and
besides M1, M3, M4, M9, M13 and M15, all the left ten of them were first identified and reported. Collectively, these
data provide a foundation for the clinical use of AC and contributes to a wider understanding of xenobiotic metabolism
and toxicity evaluation.
PO-CON1447E
Simultaneous analysis of primary
metabolites by triple quadrupole LC/MS/MS
using pentafluorophenylpropyl column
ASMS 2014
WP 613
Tsuyoshi Nakanishi1, Takako Hishiki2, Makoto Suematsu2,3
1 Shimadzu Corporation, Kyoto, Japan,
2 Department of Biochemistry, School of Medicine,
Keio University, Tokyo, Japan,
3 Japan Science and Technology Agency,
Exploratory Research for Advanced Technology,
Suematsu Gas Biology Project, Tokyo, Japan
Simultaneous analysis of primary metabolites by triple
quadrupole LC/MS/MS using pentafluorophenylpropyl column
Introduction
Various metabolic pathways are controlled to keep a
biological function in the cell and to monitor the rapid
and slight changes of these metabolism, a simple
simultaneous analysis is required for quantification of
primary metabolites. A typical LC/MS system with an
ODS column is not effective to measure primary
metabolites because of low affinity of ODS column to
hydrophilic metabolites. Here we report the
simultaneous measurement of 97 metabolites by triple
quadrupole LC/MS/MS using pentafluorophenylpropyl
column. In this experiment, MRM transitions of these
metabolites were optimized and this method was
applied to biological samples. Furthermore, to evaluate
the accuracy of developed method for quantification,
simultaneous analysis by PFPP column was compared to
measurement of ion-paring chromatography.
Methods and materials
Commercially available compounds were used as
standards to optimize MRM transition and LC condition
for separation. Mixed standard solutions were diluted to
a range of 10 nM~10000 nM for a calibration curve and
an aliquot of 3 µL was subjected to LC/MS/MS
measurement.
Mice were sacrificed under anesthesia and the isolated
heart/liver tissues were rapidly frozen in liquid nitrogen.
Frozen liver or heart tissues (>50 mg) from mice were
homogenized in 0.5 mL methanol including
L-methionine sulfone and 2-morpholinoethanesulfonic
acid (MES) as internal standards. After a general
chloroform/methanol extraction, upper aqueous layer
filtered through 5-kDa cutoff filter. The filtrate was dried
up and dissolved in 0.1 mL purified water. Further, the
solution was diluted to 20-100 folds in purified water.
An aliquot of 3 µL was analyzed to measure primary
metabolites by LC/MS instrument, Nexera UHPLC system
and LCMS-8030/LCMS-8040 triple quadrupole mass
spectrometer. The following is detailed conditions of
LC/MS mesurement.
2
UHPLC conditions (Nexera system using a PFPP column)
Column
Mobile phase A
B
Flow rate
Time program
Injection vol.
Column temperature
: Discovery HS F5 150 mm×2.1 mm, 3.0 µm
: 0.1% Formate/water
: 0.1% Formate/acetonitrile
: 0.25 mL/min
: B conc.0%(0-2.0 min) - 25%(5.0 min) - 35%(11.0 min)
- 95%(15.0.-20.0 min) - 0%(20.1-25.0 min)
: 3 µL
: 40°C
MS conditions (LCMS-8030/LCMS-8040)
Ionization
DL Temp.
HB Temp
Drying Gas
Nebulizing Gas
: Positive/Negative, MRM mode
: 250°C
: 400°C
: 10 L/min
: 2.0 L/min
Result
Optimization of MRM transition
The MRM transitions for 97 standard compounds were
optimized on both positive and negative mode by flow
injection analysis (FIA). The MRM transitions of the 97
metabolites were determined as described in Table 1.
Subsequently, LC condition was investigated to separate
the 97 metabolites with a good resolution. As a
consequence, the 97 metabolites were eluted from a PFPP
column with a gradient of acetonitrile for <15 min in the
condition described in Figure 1. The linearity of this
method was also confirmed by the simultaneous analysis of
a serial of diluted calibration curve.
Figure 1 shows the MRM chromatogram of 97 metabolites
at a concentration of 5 µM. In this figure, we can see the
peak from all metabolites with a good separation.
3
Table 1 MRM transition of 97 metabolites
No.
Name
Product ion
No.
Name
Product ion
1
2-Aminobutyrate
104.10
58.05
+
0.99
51
Inosine
269.10
137.05
+
0.99
2
Acetylcarnitine
204.10
85.05
+
0.99
52
Kynurenine
209.10
192.05
+
0.99
3
Acetylcholine
147.10
87.05
+
0.99
53
Leu
132.10
86.05
+
0.99
4
Adenine
136.00
119.05
+
0.98
54
L-Norepinephrine
170.10
152.15
+
0.99
5
Adenosine
268.10
136.05
+
0.99
55
Lys
147.10
84.10
+
0.99
6
Adenylsuccinate
464.10
252.10
+
0.99
56
Met
149.90
56.10
+
0.99
7
ADMA
203.10
70.10
+
0.99
57
Methionine-sulfoxide
166.00
74.10
+
0.99
8
Ala
89.90
44.10
+
0.99
58
Nicotinamide
123.10
80.05
+
0.99
9
AMP
348.00
136.05
+
0.99
59
Nicotinic acid
124.05
80.05
+
0.99
10
Arg
175.10
70.10
+
0.99
60
Ophthalmic acid
290.10
58.10
+
0.99
11
Argininosuccinate
291.00
70.10
+
0.99
61
Ornitine
133.10
70.10
+
0.99
12
Asn
133.10
87.15
+
0.99
62
Pantothenate
220.10
90.15
+
0.99
13
Asp
134.00
74.05
+
0.99*
63
Phe
166.10
120.10
+
0.99
14
cAMP
330.00
136.05
+
0.99
64
Pro
115.90
70.10
+
0.99
15
Carnitine
162.10
103.05
+
0.99
65
SAH
385.10
134.00
+
0.98
16
Carnosine
227.10
110.05
+
0.99*
66
SAM
399.10
250.05
+
0.99*
17
cCMP
306.00
112.10
+
0.99
67
SDMA
203.10
70.15
+
0.99
18
cGMP
346.00
152.05
+
0.99
68
Ser
105.90
60.10
+
0.99*
19
Choline
104.10
60.05
+
0.99
69
Serotonin
177.10
160.10
+
0.99
20
Citicoline
489.10
184.10
+
0.99*
70
Thr/Homoserine
120.10
74.15
+
0.99
21
Citrulline
176.10
70.05
+
0.99
71
Thymidine
243.10
127.10
+
0.99
22
CMP
324.00
112.05
+
0.99
72
Thymine
127.10
54.05
+
0.99*
23
Creatine
132.10
44.05
+
0.99
73
TMP
322.90
81.10
+
0.99*
24
Creatinine
114.10
44.05
+
0.99
74
Trp
205.10
188.15
+
0.99
25
Cys
122.00
76.05
+
0.99*
75
Tyr
182.10
136.10
+
0.99
26
Cystathionine
223.00
88.05
+
0.99
76
Uracil
113.00
70.00
+
0.99*
27
Cysteamine
78.10
61.05
+
0.98*
77
Uridine
245.00
113.05
+
0.99
28
Cystine
241.00
151.95
+
0.99
78
Val
118.10
72.15
+
0.99
29
Cytidine
244.10
112.05
+
0.99
79
2-Oxoglutarate
144.90
101.10
-
0.98*
30
Cytosine
112.00
95.10
+
0.99
80
Allantoin
157.00
97.10
-
0.98*
31
Dimethylglycine
104.10
58.05
+
0.99
81
Cholate
407.20
343.15
-
0.99**
32
DOPA
198.10
152.10
+
0.99*
82
cis-Aconitate
172.90
85.05
-
0.99
33
Dopamine
154.10
91.05
+
0.99*
83
Citrate
191.20
111.10
-
0.99*
34
Epinephrine
184.10
166.10
+
0.99
84
FMN
455.00
97.00
-
0.99
35
FAD
786.15
136.10
+
0.99*
85
Fumarate
115.10
71.00
-
0.99**
36
GABA
104.10
87.05
+
0.99
86
GSSG
611.10
306.00
-
0.99*
37
gamma-Glu-Cys
251.10
84.10
+
0.99*
87
Guanine
150.00
133.00
-
0.99*
38
Gln
147.10
84.15
+
0.99
88
Isocitrate
191.20
111.10
-
0.99*
39
Glu
147.90
84.10
+
0.99*
89
Lactate
89.30
89.05
-
0.97*
40
Gly
75.90
30.15
+
0.99*
90
Malate
133.10
114.95
-
0.99*
41
GMP
364.00
152.05
+
0.99
91
NAD
663.10
541.05
-
0.99*
42
GSH
308.00
179.10
+
0.99*
92
Orotic acid
155.00
111.10
-
0.99
43
Guanosine
284.00
152.00
+
0.99
93
Pyruvate
86.90
87.05
-
0.99*
44
His
155.90
110.10
+
0.99
94
Succinate
117.30
73.00
-
0.99*
45
Histamine
112.10
95.05
+
0.99*
95
Taurocholate
514.20
107.10
-
0.99*
46
Homocysteine
136.00
90.10
+
0.99*
96
Uric acid
167.10
123.95
-
0.99*
47
Homocystine
269.00
136.05
+
0.99
97
Xanthine
151.00
108.00
-
0.99*
48
Hydroxyproline
132.10
86.05
+
0.99
49
Hypoxanthine
137.00
55.05
+
0.98*
50
Ile
132.10
86.20
+
0.99
Precursor ion Polarity Linearity (R2)
Precursor ion Polarity Linearity (R2)
Calibration curve was obtained at a range of concentration from 10 nM to 10000 nM.
* Calibration curve was obtained at a range of concentration from 100 nM to 10000 nM.
** Calibration curve was obtained at a range of concentration from 1000 nM to 10000 nM.
4
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
Figure 1 MRM chromatogram of 97 compounds
Application to tissue extracts as biological samples
Simultaneous analysis of 99 compounds was performed for
heart / liver tissue extracts as biological samples. Figure 2
shows MRM chromatograms of 99 compounds from tissue
extracts (liver/heart). In this measurement, 83/97
metabolites were detected from liver tissue extracts and
88/97 metabolites were confirmed from heart tissue
extracts. These results show this method is also effective to
simultaneous analysis of biological samples. As shown in
the resulting MRM chromatogram, some major peaks were
derived from the metabolites which were known to be
characteristic to each tissue. Furthermore, this
characteristic difference in each tissue was also confirmed
in some faint peaks (e.g., cholate, cystine and
homocysteine).
5
10000000
GSH
9000000
Liver Tissue
8000000
7000000
Guanosine
6000000
5000000
Ophtalmic acid
4000000
AMP
3000000
GSSG
2000000
1000000
0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
30000000
Creatine
Heart Tissue
25000000
S-Adenosylhomocysteine
20000000
Acetylcarnitine
15000000
10000000
5000000
0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
Figure 2 MRM chromatogram of liver/heart tissue extracts
Correlation between PFPP and ion pairing Methods
We have previously reported simultaneous analysis of 55
metabolites which were related to central carbon
metabolic pathway by using ion pairing chromatography at
ASMS conference 2013. To evaluate the accuracy of this
simultaneous method using PFPP column, we compared
the resulting peak area of 25 metabolites, which were
covered as targets in both methods. The 25 metabolites
are Lysine, Arginine, Histidine, Glycine, Serine, Asparagine,
Alanine, Glutamine, Threonine, Methionine, Tyrosine,
Glutamate, Aspartae, Phenylalanine, Tryptophan, Cysteine,
CMP, NAD, GMP, TMP, AMP, cGMP, cAMP, MES and
L-Methionine sulfone as internal standards. Heart tissue
extracts were prepared from mice (n=9) according to the
method described above and the aliquots were measured
by the simultaneous method using either ion pairing
chromatography or PFPP separation system. As a result, we
could see the similar trend of elevation/decrease of peak
area in metabolites of 20/25 between nine samples. The
peak areas between 9 samples of representative
metabolites are shown in Figure 3. This result shows that a
ratio of areas between 9 samples is kept in both methods.
The four metabolites (TMP, cGMP, cAMP and Cysteine)
could be hardly detected on simultaneous analysis by
alternately ion-paring chromatography or PFPP column.
Tryptophan had a faint peak in this experiment and led to
the low similarity.
6
MES
L-Methionine sulfone
1.5E+06
PFPP
1.0E+06
5.0E+05
0.0E+00
2.0E+06
4.0E+05
4.0E+05
1.5E+06
3.0E+05
3.0E+05
2.0E+05
2.0E+05
1.0E+05
1.0E+05
0.0E+00
0.0E+00
1 2 3 4 5 6 7 8 9
MES
5.0E+05
0.0E+00
1 2 3 4 5 6 7 8 9
1 2 3 4 5 6 7 8 9
L-Methionine sulfone
Threonine
Serine
1.0E+06
4.0E+04
2.5E+04
8.0E+05
8.0E+05
3.0E+04
2.0E+04
6.0E+05
6.0E+05
4.0E+05
4.0E+05
2.0E+05
2.0E+05
1.0E+04
0.0E+00
0.0E+00
0.0E+00
4.0E+06
6.0E+06
3.0E+06
4.0E+06
2.0E+06
2.0E+06
1.0E+06
0.0E+00
Aspartate
4.0E+05
3.0E+04
3.0E+05
5.0E+06
0.0E+00
1.0E+04
1.0E+05
0.0E+00
0.0E+00
1 2 3 4 5 6 7 8 9
1 2 3 4 5 6 7 8 9
GMP
1 2 3 4 5 6 7 8 9
1 2 3 4 5 6 7 8 9
AMP
6.0E+05
5.0E+05
4.0E+05
3.0E+05
2.0E+05
1.0E+05
0.0E+00
2.0E+04
2.0E+05
1 2 3 4 5 6 7 8 9
3.0E+05
2.5E+05
2.0E+05
1.5E+05
1.0E+05
5.0E+04
0.0E+00
1.0E+07
Phenylalanine
4.0E+04
0.0E+00
AMP
1 2 3 4 5 6 7 8 9
5.0E+05
5.0E+03
1.5E+07
0.0E+00
1 2 3 4 5 6 7 8 9
1.0E+04
1 2 3 4 5 6 7 8 9
Phenylalanine
8.0E+06
1.5E+04
2.0E+04
1 2 3 4 5 6 7 8 9
Aspartate
Ion pairing
1.0E+06
1.0E+06
1 2 3 4 5 6 7 8 9
PFPP
Serine
5.0E+05
1 2 3 4 5 6 7 8 9
Ion pairing
Threonine
5.0E+05
GMP
6.0E+04
5.0E+04
4.0E+04
3.0E+04
2.0E+04
1.0E+04
0.0E+00
1 2 3 4 5 6 7 8 9
1 2 3 4 5 6 7 8 9
Figure 3 Correlation of peak areas between PFPP and ion-pairing method
Conclusions
• The 97 metabolites were separated by PFPP column with high resolution and this method was applied to biological
samples.
• The utility of this simultaneous analysis using PFPP column was confirmed by comparing between PFPP and ion paring
chromatography.
Life Science
• Page 222
Surface analysis of permanent wave processing
hair using DART-MS
• Page 229
Analysis of allergens found in cosmetics using
MDGC-GCMS (Multi-Dimensional Gas Chromatograph Mass Spectrometer)
PO-CON1454E
Surface analysis of permanent wave
processing hair using DART-MS
ASMS 2014
MP 476
Shoji Takigami1, Erika Ikeda1, Yuta Takagi1,
Jun Watanabe2, Teruhisa Shiota3
1 Gunma University, Kiryu, Japan;
Surface analysis of permanent wave processing hair
using DART-MS
Introduction
Permanent wave processing of hair is carried out at two
processes as follows;
(A) Reducing agent (permanent wave 1 agent) makes the
bridge construction between the keratin protein molecular
chains of hair, especially disulfide (S-S) bond of cystine
residue cleaved to thiol (-SH) group and hear results a wave
and curl.
(B) Oxidizing agent (permanent wave 2 agent) makes -SH
group oxidized to be reproduced S-S bond. As reducing
agents used for permanent wave 1 agent, the thing of
cosmetics approval, such as cysteamine hydrochloride and
a butyrolactone thiol (brand name Spiera, other than quasi
drugs, such as ammonium thioglycolate, acetyl cystein, and
thiolactic acid, are used.
After hair is applying permanent wave processing and
coloring repeatedly, the chemical structure of a keratin
molecule and fine structure in the hair have been damaged
and it resulted as damage hair. It is thought that hair
becomes dryness and twining if the cuticle which covers
hair is damaged, so it is important to investigate the
surface structure of hair and its chemical structure
changing.
DART (Direct Analysis in Real Time), a direct atmospheric
pressure ionization source, is capable of analyzing samples
directly with little or no sample preparation. Here, analysis
of the ingredient which has deposited on the permanent
wave processing hair surface was tried using this DART
combined with a mass spectrometer.
Ufswitching
Ufscanning
Figure 1 DART-OS ion source & LCMS-2020
TGA
(thioglycolate)
CA
(cysteamine hydrochloride)
BLT
(butyrolactone thiol)
O
O
HCl
SH
H2N
SH
HO
Fw 92
Wave efficiency is good in a
weak alkaline (pH 8 - 9.5)
Fw 113
weak alkaline (pH 8 - 9.5)
O
SH
Fw 118
weak acid (pH 6)
The chemical state and property were investigated in the surface of the hair which repeated permanent wave processing
with these reducing agents.
2
using DART-MS
The Chinese virgin hair purchased from the market was
washed with the 0.5% non-ionic surfactant containing
saturated EDTA solution, and then it was considered as
untreated hair sample. Permanent wave processing of hair
was prepared as following; the 0.6M TGA solution and
0.6M CA solution which were adjusted to pH8.5 with
aqueous ammonia and the 0.6M BLT solution adjusted to
pH6.0 with arginine water, which were used as a reducing
agent. After hair sample was reduced for 15 minutes at
35°C using each solvent, it was carried out oxidation
treatment at 35°C by being immersed in 8% sodium
bromate solution (pH7.2) for 15 minutes.
LCMS-2020 (Shimadzu) was coupled with DART-OS ion
source (IonSense) and hear samples were held onto DART
gas flow directly, then their surface analyzed.
Ionization
Heater Temperature (DART) : 350°C
Measuring mode (MS)
Chinese Virgin Hair
0.5% Laureth - 9 solution - EDTA saturated 35°C 1h
Water washing and air drying
Untreated
Permanent wave processing
by agent 1 & 2 at 0.6M each
permanent wave 1 agent :
TGA or CA (pH 8.5; aqueous ammonium)
BLT (pH 6; arginine) 35°C 15min
Repeat
6 times
Water washing
permanent wave 2 agent :
8% NaBrO3 solution (pH 7.2) 35°C 15min
Water washing
Britton - Robinson buffer (pH 4.6) 35°C 15min
Water washing
Air drying
Analyzed by DART-MS
3
using DART-MS
Result
After repeating operation of permanent wave processing
1-6 times using TGA (thioglycollic acid), CA (cysteamine),
and BLT (Butyrolactonethiol), hair was immersed for 15
minutes at 35°C and with a flush and air-drying, then
permanent wave processing hair was prepared. In order to
investigate the ingredient which has deposited on the
permanent wave processing hair surface, DART-MS analysis
#1
75000000
#2
was performed.
DART-MS analysis was conducted in order of #1 Untreated
(woman hair), #2 control; ammonia treatment (pH 8.5), #3
0.6M thioglycolic acid (TGA) processing, #4 0.6M
butyrolactone thiol (BLT) processing, #5 0.6M cysteamine
hydrochloride (CA) processing and #6 control; arginine
processing (pH 6).
#3
#4
2:TIC(+)
#5
#6
50000000
25000000
0
15000000
4:TIC(-)
10000000
5000000
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0 min
Figure 2 TIC chromatogram of each sample analyzing with DART
In the DART mass spectra of #1 untreated and #6 control,
many signals considered as triglyceride and diglyceride
were detected in both positive and negative spectra
obtained by DART-MS. In #3 0.6M thioglycolic acid (TGA)
processing spectra, the signal in particular of TGA origin
was not detected.
In #4 BLT processing spectra (Figure 3), the signals
considered to be oxidized BLT (3, 3'-dithiobis
(tetrahydrofuran2-one), molecular weight 234) were
detected at m/z 235 and 252 in the positive mode. The
signal m/z 235 is equivalent to [M+H]+ and m/z 252,
[M+NH4]+. In the negative mode, the signals, m/z 115,
231 were detected. They were considered the signal
equivalent to [M-H]- and [2M-H]- of BLT oxide compound
(C4H4O2S, molecular weight 116) in which two hydrogen
atoms were removed from BLT. Carrying out permanent
wave processing by BLT, it was found that the dimer of BLT
accumulated on the cuticle surface.
In #5 CA processing spectrum (Figure 5), the signal
considered to be the dimer (Fw152) origin in which CA
carried out S-S bond in the positive mode was detected at
m/z 153.
This is equivalent to [M+H]+.
4
using DART-MS
Inten. (x10,000,000)
Positive
252
1.50
[M+NH4]+
1.25
[M+H]+
235
1.00
0.75
0.50
0.25
282
0.00
5.0
4.0
3.0
100
200
368 424
300
486
516
400
500
600
700
800
900
1000
1100
m/z
Inten. (x100,000)
179
Negative
[M-H]115
[2M-H]231
2.0
1.0
0.0
321
347
100
200
300
411
501
400
500
579
600
700
800
900
1000
1100
m/z
Figure 3 DART-MS spectra of #4 BLT processing
The BLT-related signals were detected from the positive and the negative spectra.
Inten. (x1,000,000)
1.50
1.25
Positive
282
124
[2M+H]+
391
1.00
252
0.75
153
0.50
0.25
0.00
102
100
468
424
184
200
300
400
563
600 644
500
600
691
700
769
851
800
922
900
1000
1100
m/z
Figure 4 DART-MS spectra of #5 CA processing
The CA-related signal was detected from the positive spectrum
5
using DART-MS
100000
4:325.15(-)
#2
#1
#3
#4
#5
#6
Negative XIC m/z 325
50000
0
10000000 2:234.70(+)
Positive XIC m/z 235
5000000
0
2:251.75(+)
10000000
5000000
0
4:114.95(-)
250000
0
4:230.90(-)
500000
0
1500000 2:123.85(+)
1000000
500000
0
1000000 2:152.85(+)
500000
0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0 min
Figure 5 XIC chromatorgam of each sample analyzing with DART
In order to indicate clearly the signals specifically
detected in each sample, the extraction chromatograms
(XIC) were shown (Figure 5). It turned out that
BLT-related signals were detected only in #4 and the
CA-related signal in #5.
Moreover, although the signal intensity was weak, the
signal at negative m/z 325 was detected from all
samples. Negative m/z 325 is equivalent to [M-H]- of 18
methyl eicosanoic acid (18MEA, molecular weight 326).
18MEA is one of lipid components which protect a
cuticle. There is no significant difference of this signal in
the hair between treated hair and untreated hair. We
would like to inquire so that intensity difference can be
found out by further verifying the detection technique in
the future.
6
using DART-MS
Conclusions
By direct analysis of the hair by DART-MS, the chemical structure change in the surfaces of hair, such as permanent wave
processing, was able to be observed.
PO-CON1469E
Analysis of allergens found in cosmetics
using MDGC-GCMS (Multi-Dimensional
Gas Chromatograph Mass Spectrometer)
ASMS 2014
TP761
Sanket Chiplunkar, Prashant Hase, Dheeraj Handique,
Ankush Bhone, Durvesh Sawant, Ajit Datar,
Jitendra Kelkar, Pratap Rasam
Analysis of allergens found in cosmetics using MDGC-GCMS
(Multi-Dimensional Gas Chromatograph Mass Spectrometer)
Introduction
Cosmetics, fragrances and toiletries (Figure 1) are used
safely by millions of people worldwide. Although many
people have no problems, irritant and allergic reactions
may occur. Irritant and allergic skin reactions are the types
of contact dermatitis. Essential oils present in fragrance
contain some natural and synthetic compounds, which
may cause allergic reactions to the end user after
application. There are 26 potential allergens listed by
European Directive (EU) 2003/15/EC and International
Fragrance Association (IFRA)[1] labeled on cosmetics.
Shimadzu MDGC-GCMS technology facilitates the
identification and quantification of these allergens to
comply with the threshold limits of 100 ppm for rinse-off
products.
Co-eluting peaks were resolved completely with the help
of MDGC-GCMS heart-cut technique.
Figure 1. Cosmetics, fragrances and toiletries
Method of Analysis
Extraction of allergens from shampoo sample
Shampoo samples were collected from local market.
Standard solutions of 23 allergens were procured from
ACCU Standard and dilutions were carried out in
Ethanol/Acetonitrile to yield 1000 ppm concentration.
Further dilutions were made in methanol.
MDGC-GCMS technique was effectively used to minimize
matrix effect. Co-eluting peaks were resolved with
heart-cut technique using two columns of different
polarities. In MDGC-GCMS, 1st instrument was GC-2010
Plus equipped with FID as a detector and 2nd instrument
was GCMS-QP2010 Ultra with MS as a detector. Columns
in both the instruments were connected with Deans
switch. Allergens in shampoo samples were determined by
using this technique. For sample preparation, following
methodology was adopted.
1) Blank Solution : 10 mL of methanol was transferred in 20 mL centrifuge tube and vortexed for 5 minutes. The mixture
was then centrifuged for 5 minutes at 3000 rpm. This solution was filtered through 0.2 µm nylon syringe filter. Initial 2
mL was discarded and remaining filtrate was collected.
2) Sample Solution : 1 g of shampoo sample was weighed in 10 mL volumetric flask and diluted up to the mark with
methanol. Above mixture was transferred in 20 mL centrifuge tube. Further processing was done as mentioned in
blank solution.
3) Spike Sample Solution : For recovery study, 1 g of sample was spiked with different volumes of standard stock
solution. The above procedure was repeated for preparing different concentration levels of allergens in samples. These
spiked samples were treated as mentioned in sample solution.
Part method validation was carried out by performing
system precision, sample precision, linearity and recovery
study. For validation, solutions of different concentrations
were prepared using 40 ppm (actual concentration)
standard stock solution mixture of allergens.
2
Table 1. Method validation parameters
Parameter
Concentration
System Precision
10 ppm
Sample Precision
10 % in Methanol
Linearity
2.5, 5, 7.5, 10, 15 (ppm)
Accuracy / Recovery
5, 10, 15 (ppm)
MDGC-GCMS Analytical Conditions
The instrument configuration used is shown in Figure 2. Samples were analyzed using Multi-Dimensional GC/GCMS as per
the conditions given below.
Figure 2. Multi-Dimensional GC/GCMS System by Shimadzu
Figure 3. Schematic diagram of multi-Deans switch in MDGC-GCMS
3
MDGC-GCMS analytical parameters
Chromatographic parameters (1st GC : GC-2010 Plus)
• Column
• Injection Mode
• Split Ratio
• Carrier Gas
• Column Flow
• Detector
• APC Pressure
• Column Oven Temp.
:
:
:
:
:
:
:
:
Stabilwax (30 m L x 0.25 mm I.D.; 0.25 μm)
Split
5.0
Helium
2.27 mL/min
FID
200 kPa (For switching)
Rate (ºC /min)
Temperature (ºC)
50.0
15.00
100.0
5.00
240.0
Hold time (min)
0.00
0.00
43.67
Chromatographic parameters (2nd GCMS : GCMS-QP2010 Ultra)
• Column
• Detector
• Interface Temp.
• Ionization Mode
• Event Time
• Mode
:
:
:
:
:
:
:
:
Rxi-1ms (30 m L x 0.25 mm I.D.; 0.25 μm)
Mass spectrometer
200 ºC
240 ºC
EI
0.30 sec
SIM and SCAN
Rate (ºC /min)
Temperature (ºC)
80.0
3.00
180.0
10.00
260.0
: 75.00 min
Hold time (min)
13.00
0.00
20.67
Results
Sample analysis using MDGC-GCMS
MDGC-GCMS technique was used to avoid matrix interference from sample. Using multi-Deans switch and heart-cut
technique (Figure 3), co-eluted components from the 1st column were transferred to the 2nd column with different polarity.
4
uV (x100,000)
Chromatogram
10.0
uV (x10,000)
Chromatogram
Fernesol - 2
uV (x10,000)
Chromatogram
9.5
9.0
Fernesol - 2
0.1
5.0
7.5
10.0
12.5
15.0
Benzyl Alcohol
Hexyl cinnam aldehyde
Amyl cinnamal
4.5
3.0
2.0
Fernesol - 1
Isoeugenol
5.0
3.5
1.5
17.5
20.0
22.5
25.0
1.0
27.5
25.5
30.0
26.0
26.5
27.0
32.5
27.5
28.0
28.5
min
Benzyl Cinnamate
25.0
Benzyl salicylate
min
Sample - 6
17.0
Amyl cinnamal
Anisyl alcohol
Cinnamyl alcohol
16.0
Eugenol
15.0
Cinnamal
14.0
Hydroxy-citronellal
0.2
5.5
2.5
Geraniol
Benzyl Alcohol
Sample - 1
0.3
13.0
Citral - 2
Citronellol
0.4
Methyl heptine carbonate
Sample - 2
Sample -Citral
3 -1
Limonene
0.5
Linalool
0.6
12.0
6.0
4.0
0.5
0.7
6.5
Sample - 5
Benzyl benzoate
1.0
7.0
Amylcin namyl alcohol
0.8
7.5
Coumarin
1.5
8.0
Fernesol - 1
Fernesol -Isoeugenol
2
Sample - 2
2.0
Geraniol
0.9
2.5
8.5
Citral - 2
3.0
Sample - 3
Citral - 1
3.5
Citronellol
4.0
1.0
Anisyl alcohol
Cinnamyl alcohol
4.5
Fernesol - 2
5.0
1.1
35.0
37.5
40.0
42.5
45.0
47.5
min
Figure 4. Chromatogram of spiked sample solution before switching
3.25
(x100,000)
uV (x10,000)
164.00 (100.00)
149.00 (100.00)
6.0 103.00 (100.00)
138.00 (100.00)
109.00 (100.00)
5.0 137.00 (100.00)
92.00 (100.00)
115.00 (100.00)
4.0 134.00 (100.00)
Chromatogram
Target compound - Isoeugenol
28.105
3.50
3.00
2.75
2.50
2.25
2.00
3.0
1.75
2.0
26.491
1.00
26.256
1.50
1.25
Target compound - Isoeugenol
1.0
0.75
0.0
0.50
0.25
26.5
27.0
27.5
28.0
min
-1.0
27.0
27.5
28.0
28.5
29.0
29.5
Figure 6. SIM chromatogram with 2 column (MS)
Figure 5. Chromatogram with 1 column (FID)
nd
st
Summary of results
Table 2. Summary of results for precision on GC and GCMS
Sr. No.
Type of sample
Sample name
Concentration
Result
1
Standard
2
Cosmetic
23 Allergens mixture
10 ppm
% RSD for area (n=6) < 2.0
Shampoo
Unknown
% RSD for area (n=6) < 2.0
5
Table 3. Linearity by GC
Area (x10,000)
Sr. No.
Name of allergen
Linearity (R2)
1
Linalool
0.9945
2
0.9949
3
Citronellol
0.9965
5.0
4
Geraniol
0.9962
4.0
5
Hydroxy citronellal
0.9973
6
Cinnamal
0.9959
7
Amyl Cinnamal
0.9976
2.0
8
Coumarin
0.9971
1.0
9
Amylcin namyl alcohol
0.9983
0.0
10
Benzyl benzoate
0.9979
7.0
6.0
3.0
0.0
2.5
5.0
7.5
10.0
12.5
Conc.
Figure 7. Linearity graph for linalool
Table 4. Linearity by GCMS
Area(x10,000)
Sr. No.
Name of allergen
Linearity (R2)
1
Limonene
0.9945
2
Benzyl alcohol
0.9871
3
Citral - 1
0.9889
4
Citral - 2
0.9902
5
Eugenol
0.9894
6
Anisyl alcohol
0.9916
7
Cinnamyl alcohol
0.9937
8
Isoeugenol
0.9902
9
Farnesol - 1
0.9919
10
Farnesol - 2
0.9929
11
0.9932
12
Benzyl salicylate
0.9853
13
Benzyl cinnamate
0.9927
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
0.0
2.5
5.0
7.5
10.0
12.5
Conc.
Figure 8. Linearity graph for benzyl cinnamate
Quantitation of allergens in shampoo sample
For the quantitation studies, the shampoo sample was
spiked with allergens standard to achieve 5, 10 and 15
ppm concentrations. Recovery studies were performed on
13 allergens, having co-elution or matrix interference, using
heart-cut technique. The quantitation of these allergens
was carried out using 2nd detector (MS) in SIM mode.
In below recovery study, some allergens had recovery value
out side the acceptance limit (70-130 %). Optimization can
be done by means of change in sample clean up procedure
and filtration study.
6
Table 5. Quantitation of allergens – Recovery Study
(x1,000)
3.00
% Recovery
Sr. No.
Name of allergen
Level -1
5 ppm
Level -2
10 ppm
Level -3
15 ppm
2.75
m/z : 69.00
Farnesol-1
Farnesol-2
2.50
1
Limonene
127
126
129
2
Benzyl alcohol
114
114
123
2.25
3
Citral - 1
101
106
114
2.00
4
Citral - 2
97
103
112
1.75
5
Eugenol
96
105
116
1.50
6
Anisyl alcohol
94
105
116
7
Cinnamyl alcohol
98
106
115
8
Isoeugenol
103
108
118
1.25
1.00
Spiked
0.75
9
Farnesol - 1
83
95
107
10
Farnesol - 2
84
95
106
0.50
11
121
122
130
0.25
12
Benzyl salicylate
63
47
32
13
Benzyl cinnamate
66
61
56
Unspiked
25.0
27.5
30.0
32.5
min
Figure 9. Overlay SIM chromatogram
of unspiked and spiked sample
Conclusion
• MDGC-GCMS method was developed for quantitation of allergens present in cosmetics. Part method validation was
performed as per ICH guidelines.[2] Results obtained for reproducibility, linearity and recovery studies were well within
acceptable limits.
• Simultaneous SCAN/SIM and high-speed scan rate 20,000 u/sec are the characteristic features of GCMS-QP2010 Ultra,
which enables quantitation of allergens at very low concentration level.
• Matrix effect from cosmetics was selectively eliminated using MDGC-GCMS with multi-Deans switching unit and
heart-cut technique.
• MDGC-GCMS was found to be very useful technique for simultaneous identification and quantitation of components
from complex matrix.
Reference
[1] IFRA guidelines (International Fragrance Association), GC/MS Quantification of potential fragrance allergens, Version 2,
(2006), 6.
[2] ICH guidelines, Validation of Analytical Procedures: Text And Methodology Q2(R1), Version 4, (2005).
Technical
Applications
•Page238
Applicationsofdesorptioncoronabeam
ionization-massspectrometry
•Page243
Rapidanalysisofcarbonfiberreinforcedplastic
usingDART-MS
•Page249
Analysisofstyreneleachedfrompolystyrene
cupsusingGCMScoupledwithHeadspace(HS)
sampler
PO-CON1474E
Applications of Desorption Corona
Beam Ionization-Mass Spectrometry
ASMS 2014
WP 393
Yuki Hashi1, Shin-ichi Kawano1, Changkun Li1, Qian Sun1,
Taohong Huang1, Tomoomi Hoshi2, Wenjian Sun3
Shimadzu (China) Co., Ltd., Shanghai, China
2
Shimadzu Corporation, Kyoto, Japan
3
Shimadzu Research Laboratory (Shanghai) Co., Ltd.,
Shanghai, China
1
Applications of Desorption Corona Beam
Ionization-Mass Spectrometry
Introduction
Numerous ambient ionization mass spectrometric
techniques have been developed for high throughput
analysis of various compounds with minimum sample
pretreatment.(1) Desorption corona beam ionization (DCBI)
is a more recent technique.(2) In DCBI, helium is used as
discharge gas and heating of the gas is required for sample
desorption. A visible thin corona beam is formed by using
hollow needle/ring electrode structure. This feature
facilitates localizing sampling areas and obtaining good
reproducibility of data. Details of DCBI hardware are
shown in Figs. 1 and 2. In this study, DCBI was applied for
analysis of various samples.
Helium flow
HVDC
-
Heated thin
wall tubing
+
LVDC
Discharge
needle
Counter
electrode
Sampling
capillary
MS inlet
Sample and stage
Figure 1 Schematic diagram of DCBI
DCBI probe
Corona beam
MS Inlet
Manual liquid
sampler
Figure 2 DCBI interface
2
Method
Sample Preparation
Samples (melamine, saturated hydrocarbon mixture, polyaromatic hydrocarbon mixture, testosterone, pirimicarb, and
methomyl) were dissolved in methanol or acetonitrile.
DCBI-MS Analysis
Samples were analyzed using a DCBI system coupled to a LCMS-2020 quadrupole mass spectrometer (Shimadzu
Corporation, Japan). The system was operated with the DCBI control software and LabSolutions for LCMS version 5.42.
DCBI
Flow rate
HV discharge
He gas temperature
Sample volume
:
:
:
:
0.6 L/min
+2.0-3.0 kV
350 ºC
1, or 2 µL
MS (LCMS-2020 quadrupole mass spectrometer)
Polarity
DL temperature
BH temperature
Mass range
:
:
:
:
Positive
250 ºC
400 ºC
m/z 100-500
In this experiment, all compounds with variety of polarity
from non- to high-polar gave protonated molecules (Figs.
3-8). Methomyl gave also fragment ions (m/z 106) by
cleavage at methylcarbamoyl group, while fragment ions
with significant intensity were not observed for other
compounds. Analysis time was less than 1 minute.
Inten. (x1,000)
127.1
2.25
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
136.0
100.0
105.0
110.0
115.0
120.0
125.0
130.0
135.0
148.6
140.0
145.0
m/z
Figure 3 Mass spectrum of melamine (0.5 mg/mL)
3
Inten. (x100,000)
1.50
213.2
1.25
241.3
255.3
269.3
199.2
1.00
283.3
297.3
185.2
0.75
311.3
0.50
171.2
325.3
0.25
339.3
157.2
115.1 143.2
0.00
100
367.4
150
200
250
300
350
Compound
C 10H 22
C 11H 24
C 12H 26
C 13H 28
C 14H 30
C 15H 32
C 16H 34
C 17H 36
C 18H 38
C 19H 40
C 20H 42
C 21H 44
C 22H 46
C 23H 48
C 24H 50
C 25H 52
MW
142
156
170
184
198
212
226
240
254
268
282
296
310
324
338
352
Compound
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Anthracene
Phenanthrene
Pyrene
Fluoranthene
Chrysene
Benzo[a]anthracene
MW
128
152
154
166
178
178
202
202
228
228
m/z
Figure 4 Mass spectrum of saturated hydrocarbon mixture (1 mg/mL)
6.5
Inten. (x10,000)
153.1
6.0
5.5
155.2
5.0
4.5
179.1
4.0
3.5
3.0
2.5
167.2
2.0
1.5
1.0
0.5
0.0
100.0
209.1
195.1
129.1
141.2
115.1
125.0
150.0
203.1
235.1
175.0
200.0
225.0
276.2
250.0
275.0
m/z
Figure 5 Mass spectrum of polyaromatic hydrocarbon mixture (2 mg/mL)
Inten. (x10,000)
289.2
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
112.1
331.2
150
200
250
300
350
424.5 461.4
400
450
m/z
Figure 6 Mass spectrum of testosterone (1 mg/mL)
4
Inten. (x100,000)
Inten. (x100,000)
239.2
9.0
163.0
1.2
1.1
8.0
105.9
1.0
7.0
0.9
6.0
0.8
0.7
5.0
0.6
4.0
0.5
3.0
0.4
0.3
2.0
0.2
1.0
0.0
100
182.2
150
200
0.1
250
300
350
400
450
Figure 7 Mass spectrum of pirimicarb (0.5 mg/mL)
m/z
194.0
121.9
0.0
100
208.0
150
252.0
200
250
354.1 394.3
300
350
400
450
m/z
Figure 8 Mass spectrum of methomyl (0.5 mg/mL)
Conclusion
The DCBI system was successfully applied for analysis of samples with various polarity.
Mass spectra were quickly obtained after sample introduction to the DCBI probe.
The method is useful for fast identification of various compounds.
References
(1) Monge ME et al, Chem. Rev. 113 (2013), 2269-2308
(2) Hua W et al, Analyst 135 (2010), 688-695
PO-CON1456E
Rapid analysis of carbon fiber
reinforced plastic using DART-MS
ASMS 2014
TP 782
Hideaki Kusano1, Jun Watanabe1, Yuki Kudo2,
Teruhisa Shiota3
2 Bio Chromato, Inc., Fujisawa, Japan;
Rapid analysis of carbon fiber reinforced plastic
using DART-MS
Introduction
DART (Direct Analysis in Real Time) can ionize and analyze
samples directly under atmospheric pressure, independent
of the sample forms. Then it is also possible to measure in
form as it is, without sample preparation. Qualitative
analysis of target compounds can be conducted very fast
and easily by combining DART with LCMS-2020/8030
which have ultra high-speed scanning and ultra high-speed
polarity switching.
Carbon-fiber-reinforced plastics, CFRP is the
fiber-reinforced plastic which used carbon fiber for the
reinforced material, which is only called carbon resin or
carbon in many cases. An epoxy resin is mainly used for a
base material in CFRP. While CFRP is widely used taking
advantage of strength and lightness, most approaches
which measure CFRP with analytical instruments were not
tried, triggered by the difficulty of the preparation.
DART (Direct Analysis in Real Time), a direct atmospheric
pressure ionization source, is capable of analyzing samples
with little or no sample preparation. Here, rapid analysis of
carbon fiber reinforced plastic was carried out using DART
combined with a mass spectrometer.
Figure 1 CFRP：carbon-fiber-reinforced plastic
Thermosetting polyimide (carbon-fiber-reinforced plastics)
and thermoplastic polyimide (control sample) were
privately manufactured. After cutting a sample in a suitable
size, it applied DART-MS analysis. They were introduced to
the DART gas using tweezers. The DART-OS ion source
(IonSense, MA, USA) was interfaced onto the single
quadrupole mass spectrometer LCMS-8030 (Shimadzu,
Kyoto Japan). Ultra-fast polarity switching was utilized on
the mass spectrometer to collect full scan data.
LCMS-8030 can achieve the polarity switching time of
15msec and the scanning speed of up to 15,000u/sec,
therefore the loop time can be set at less than 1 second
despite the relatively large scanning range of 50-1,000u.
Ionization
2
using DART-MS
UFswitching
UFscanning
Figure 2 DART-OS ion source (IonSense) & triple quadrupole LCMS (Shimadzu)
Result
3 CFRP samples were analyzed by DART-MS. Mass chromatograms of each sample were shown in Figure 3 and mass
spectra in Figure 4.
Sample
#1 thermoplastic polyimide (control)
#2 thermosetting polyimide (molded; dried)
#3 thermosetting polyimide (immediately after molded; wet state with solvent)
Heater Temperature (DART) : 300ºC
Measuring mode (MS)
1:MIC1(+)
50000000
25000000
0
6000000
5000000
2:MIC1(-)
4000000
3000000
2000000
1000000
#1
0
7.5
8.0
8.5
9.0
#2
9.5
10.0
10.5
#3
11.0
11.5
12.0
min
Figure 3 TIC chromatogram of CFRP samples #1, #2, #3
3
using DART-MS
Inten.
7.5
(x1,000,000)
Positive, m/z 50-300
#1
5.0
2.5
0.0
100.1
50
Inten.
7.5
172.1
100
282.2
228.3
200
250
m/z
(x1,000,000)
#2
2.5
N-methyl pyrrolidone
C5H9NO
Mw 99
5.0
[M+H]+
[2M+H]+
199.1
100.1
0.0
199.1
150
172.2
50
100
150
282.3
200
250
m/z
Inten. (x1,000,000)
7.5
100.1
199.1
#3
5.0
2.5
0.0
50
100
150
200
250
m/z
Figure 4 DART-MS spectra of each sample
Since the thermosetting polyimide used for this
measurement was molded using the organic solvent
(N-methyl pyrrolidone, C5H9NO, molecular weight 99),
molecular related ions of N-methyl pyrrolidone, [M+H]+
(m/z 100) and [2M+H]+ (m/z 199), were detected very
strongly in the mass spectrum of #1. The mass spectrum
of #2 also showed the same ions that intensity was
intentionally detected strongly compared with #3
although intensity was weak compared with #1. Even if
it raised the heating gas temperature of DART to high
temperature (up to 500°C), MS signal considered to
originate in the structural information of CFRP was not
able to be obtained.
Then, the optional heating mechanism, ionRocket (Bio
Chromato, Inc.; Figure 5), in which a sample could be
heated directly was developed to the sample stage of
DART, and analysis of CFRP was verified by heating the
sample directly up to 600°C.
Sample
#4 thermosetting polyimide (molded; dried)
#5 thermoplastic polyimide (control)
Heater Temperature (DART)
: 400°C
Temperature control (ionRocket) : 0-1min room temp., 4min 600°C
Measuring mode (MS)
: Positive scanning
4
using DART-MS
600°C
r.t.
1
4
time[min]
evaporated ingredient
excitation helium
MS
spectrometer
DART ion source
sample pot
small heating furnace
heater
Figure 5 DART-MS system integrated with ionRocket
When heating temperature was set to 600ºC, the rudder
shape signals of 28u (C2H4) interval was appeared
around m/z 900. This signal was more notably detected
with the thermosetting polyimide sample than the
thermoplastic sample. Since the sample was heated at
high temperature, it was considered that the thermal
decomposition of resin started, the thermal
decomposition ingredient of polyimide clustered, and
possibly the structures of the rudder signals of equal
interval were generated.
5
using DART-MS
#4
Zoom
#5
#4
thermosetting polyimide
#5
thermoplastic polyimide
Figure 6 DART-MS with ionRocket spectra of each sample
Conclusions
The result of having analyzed the carbon fiber plastic CFRP (thermosetting polyimide and thermoplastic polyimide)
using DART-MS,
a. residue of the solvent used in fabrication was able to be checked by direct analysis of CFRP by DART.
b. analyzing CFRP by DART and the heating option ionRocket, the difference between thermosetting polyimide and
thermoplastic polyimide was able to be found out.
Acknowledgment
We are deeply grateful to Mr. Yuichi Ishida, Japan Aerospace Exploration Agency (JAXA), offered the CFRP sample
used for this experiment.
PO-CON1464E
Analysis of styrene leached from
polystyrene cups using GCMS coupled
with Headspace (HS) sampler
ASMS 2014
TP763
Ankush Bhone(1), Dheeraj Handique(1), Prashant Hase(1),
Sanket Chiplunkar(1), Durvesh Sawant(1), Ajit Datar(1),
Jitendra Kelkar(1), Pratap Rasam(1), Nivedita Subhedar(2)
(1) Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh
(2) Ramnarain Ruia College, L. Nappo Road,
Matunga (E), Mumbai-400019, Maharashtra, India.
Analysis of styrene leached from polystyrene cups
using GCMS coupled with Headspace (HS) sampler
Introduction
Worldwide studies have revealed the negative impacts of
household disposable polystyrene cups (Figure 1) on
human health and environment.
Molecular structure of styrene is shown in Figure 2. Styrene
is considered as a possible human carcinogen by the WHO
and International Agency for Research on Cancer (IARC).[1]
Migration of styrene from polystyrene cups containing
beverages has been observed.[2] Styrene enters into our
body through the food we take, mimics estrogens in the
body and can therefore disrupt normal hormonal
functions. This could also lead to breast and prostate
cancer.
The objective of this study is to develop a sensitive,
selective, accurate and reliable method for styrene
determination using low carryover headspace sampler,
HS-20 coupled with Ultra Fast Scan Speed 20,000 u/sec,
GCMS-QP2010 Ultra to assess the risk involved in using
polystyrene cups.
Figure 1. Polystyrene cup
Figure 2. Structure of styrene
Method of Analysis
Extraction of styrene from polystyrene cups
This study was carried out by extracting styrene from commercially available polystyrene cups and recoveries were
established by spiking polystyrene cups with standard solution of styrene. Solutions were prepared as follows,
1) Standard Stock Solution:
1000 ppm of styrene standard stock solution in DMF: Water-50:50 (v/v) was prepared. It was further diluted with
water to make 100 ppm and 1 ppm of standard styrene solutions.
2) Calibration Curve:
Calibration curve was plotted using standard styrene solutions in the concentration range of 1 to 50 ppb with water as
a diluent. 5 mL of each standard styrene solution was transferred in separate 20 mL headspace vials and crimped with
automated crimper.
3) Sample Preparation:
150 mL of boiling water (around 100 ºC)[1] was poured into polystyrene cups. The cup was covered with aluminium foil
and kept at room temperature for 1 hour. After an hour, 5 mL of sample from the cup was transferred into the 20 mL
headspace vial and crimped with automated crimper.
Method was partly validated to support the findings by performing reproducibility, linearity, LOD, LOQ and recovery
studies. For validation, solutions of different concentrations were prepared using standard stock solution of styrene (1000
ppm) as mentioned in Table 1.
2
Table 1. Method validation parameters
Parameter
Concentration (ppb)
Linearity
1, 2.5, 5, 10, 20, 50
Accuracy / Recovery
2.5, 10, 50
Precision at LOQ level
1
Reproducibility
50
HS-GCMS Analytical Conditions
Figure 3 shows the analytical instrument, HS-20 coupled with GCMS-QP2010 Ultra on which samples were analyzed with
following instrument parameter.
Figure 3. HS-20 coupled with GCMS-QP2010 Ultra by Shimadzu
HS-GCMS analytical parameters
Headspace parameters
• Sampling Mode
• Oven Temp.
• Sample Line Temp.
• Transfer Line Temp.
• Equilibrating Time
• Pressurizing Time
• Pressure Equilib. Time
• Load Time
• Load Equilib. Time
• Injection Time
• Needle Flush Time
• GC Cycle Time
:
:
:
:
:
:
:
:
:
:
:
:
Loop
80.0 ºC
130.0 ºC
140.0 ºC
20.00 min
0.50 min
0.10 min
0.50 min
0.10 min
1.00 min
10.00 min
23.00 min
3
Chromatographic parameters
• Column
• Injection Mode
• Split Ratio
• Carrier Gas
• Flow Control Mode
• Linear Velocity
• Pressure
• Column Flow
• Total Flow
:
:
:
:
:
:
:
:
:
:
:
Rxi-5Sil MS (30 m L x 0.25 mm I.D., 0.25 μm)
Split
10.0
Helium
Linear Velocity
36.3 cm/sec
53.5 kPa
1.00 mL/min
14.0 mL/min
12.42 min
Rate (ºC /min)
Temperature (ºC)
50.0
40.00
200.0
30.00
280.0
Hold time (min)
0.00
1.00
5.00
Mass Spectrometry parameters
• Interface Temp.
• Ionization Mode
• Event Time
• Mode
• m/z
• Start Time
• End Time
:
:
:
:
:
:
:
:
200 ºC
230 ºC
EI
0.20 sec
SIM
104,103 and 78
1.00 min
5.00 min
Results
Fragmentation of styrene
Mass spectrum of styrene is shown in Figure 4. From the
mass spectrum, base peak of m/z 104 was used for
quantitation where as m/z 103 and 78 were used as
reference ions.
SIM chromatogram of 50 ppb standard styrene solution
with m/z 104, 103 and 78 is shown in Figure 5.
Method validation data is summarized in Table 2. Figures 6
and 7 show overlay of SIM chromatograms for m/z 104 at
linearity levels and calibration curve respectively.
4
Inten.
104
100
75
50
103
78
25
51
44
52
63
58
0
45.0
50.0
55.0
60.0
74
65
65.0
70.0
75.0
85
80.0
85.0
89
90.0
98
95.0
100.0
105.0 m/z
Figure 4. Mass spectrum of styrene
(x1,000,000)
104.00 (10.00)
7.5 103.00 (10.00)
78.00 (10.00)
5.0
2.5
0.0
2.325
2.350
2.375
2.400
2.425
2.450
2.475
2.500
2.525
min
Figure 5. SIM chromatogram of 50 ppb standard styrene solution
Summary of validation results
Table 2. Validation summary
Sr. No.
Compound Name
Parameter
Concentration in ppb
Result
1
Reproducibility (% RSD)
50
% RSD : 1.74 (n=6)
2
Linearity* (R2)
1 – 50
R2 : 0.9996
3
4
5
LOD
Styrene
LOQ
Precision at LOQ
1 – 50
1
LOD : 0.2 ppb
LOQ : 1 ppb
S/N ratio : 38 (n=6)
% RSD : 3.2 (n=6)
* Linearity levels – 1, 2.5, 5, 10, 20 and 50 ppb.
5
(x1,000,000)
2.00
Area
m/z : 104.00
1.75
1250000
50 ppb
1.50
R2 = 0.9996
1000000
20 ppb
1.25
10 ppb
1.00
5 ppb
0.75
2.5 ppb
0.50
1 ppb
750000
500000
250000
0.25
0.00
0
2.2
2.3
2.4
2.5
2.6
0
min
10
Figure 6. Overlay of SIM chromatograms for m/z 104 at linearity levels
20
30
40
Conc.
Figure 7. Calibration curve for Styrene
Quantitation of styrene in polystyrene cup sample
Analysis of leachable styrene from polystyrene cups was
done as per method described earlier. Recovery studies
were carried out by spiking 2.5, 10 and 50 ppb of standard
styrene solutions in polystyrene cups. Figure 8 shows
overlay SIM chromatogram of spiked and unspiked
samples. Table 3 shows the summary of results.
(x100,000)
m/z : 104.00
7.5
5.0
Spiked
2.5
Unspiked
0.0
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
min
Figure 8. Overlay SIM chromatograms of spiked and unspiked samples
Table 3. Summary of results for sample analysis
Sr. No.
Sample Name
Parameter
1
Unspiked sample
Precision
2
Spiked polystyrene cups
Recovery
Observed
Concentration
in ppb
Spiked
Concentration
in ppb
% Recovery
9.8
NA
NA
12.0
2.5
88.0
18.5
10
87.0
55.9
50
92.2
6
Conclusion
• HS-GCMS method was developed for quantitation of styrene leached from polystyrene cup. Part method validation was
performed. Results obtained for reproducibility, linearity, LOQ and recovery studies were within acceptable criteria.
• With low carryover, the characteristic feature of HS-20 headspace, reproducibility even at very low concentration level
could be achieved easily.
• Ultra Fast Scan Speed 20,000 u/sec is the characteristic feature of GCMS-QP2010 Ultra mass spectrometer, useful for
quantitation of styrene at very low level (ppb level) with high sensitivity.
References
[1] Maqbool Ahmad, Ahmad S. Bajahlan, Journal of Environmental Sciences, Volume 19, (2007), 422, 424.
[2] M. S. Tawfika; A. Huyghebaerta, Journal of Food Additives and Contaminants, Volume 15, (1998), 595.

ASMS 2014 Poster Collection

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