Clinical, Forensic and Pharmaceutical Applications

Transcription

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 1a,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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© Shimadzu Corporation, 2014
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
Shimadzu France SAS, Le Luzard 2, Boulevard Salvador
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.
Materials and Methods
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.
Analytical Conditions
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
• Nitrogen gas flow
• 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
Liquid Chromatography (UHPLC) Nexera coupled with
LCMS-8040 triple quadrupole system (Shimadzu
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,
Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh
Chambers, Makwana Road, Marol, Andheri (E),
Mumbai-400059, Maharashtra, India.
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.

Clinical, Forensic and Pharmaceutical Applications

Transcription

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