Document 6528966

A Novel Concept for Sample Collection and Sample Preparation
Stefan König, Orhan Yildiz, Norbert Hermann, Andreas Steurer, Maharajah Singrasa, Werner Döbelin, INOVALAB AG, Kägenstrasse 17, 4153 Reinach, Switzerland
Calibration curve for Bosentan
(1.00 to 4096 ng/mL)
Complete instrument setup with autosampler, HPLC and mass spectrometer
OVERVIEW
HPLC Conditions:
This poster demonstrates a novel concept for sample collection and sample preparation based on a
prototype instrument and newly designed consumables. A prototype instrument of a modified CTC PAL
autosampler instrument (designed and constructed by Prolab Instruments GmbH) was used for the
analysis of whole blood samples.
HPLC: Shimadzu 10ADvp pumps for the high pressure gradient on the analytical column and Merck 6200
and 6000 pumps for injecting / loading, trapping and transferring the samples.
Mobile Phase A: H2O + HCOOH + NH3(aq.), (100 + 1 + 0.05)
Mobile Phase B: MeCN + MeOH + HCOOH + NH3(aq.), (50 + 50 + 1 + 0.05)
HPLC column 1: 2.0 x 25 mm, LiChrospher ADS (RAM), RP-18, 25 um, Merck
HPLC column 2: 2.0 x 20 mm, C18, 5 um, Clipeus, Higgins Analytical
HPLC column 3: 2.0 x 50 mm, C6-Phenyl, 3um, Phenomenex
A.rdb (Ro47-0203): "Linear" Regression ("1 / (x * x)" weighting): y = 0.0008 x + 0.000651 (r = 0.9979)
INTRODUCTION
Calibration curve for Hydroxy Bosentan
(2.00 to 512 ng/mL)
B.rdb (Ro48-5033): "Linear" Regression ("1 / (x * x)" weighting): y = 0.00241 x + 0.00037 (r = 0.9977)
3.3
1.25
3.2
1.20
1.15
3.0
1.10
2.8
1.05
1.00
2.6
0.95
2.4
0.90
0.85
2.2
0.80
2.0
0.75
0.70
1.8
0.65
1.6
0.60
0.55
1.4
0.50
1.2
0.45
0.40
1.0
0.35
0.8
0.30
0.25
0.6
0.20
0.4
0.15
0.10
0.2
0.05
0.0
During the last few years several new approaches for sample preparation, either on-line or off-line,
were developed due to a constant need for smaller sample volumes, easier handling and easier
logistics. Many of these approaches offer some advantages compared to the traditional process of
blood collection in vacutainers, centrifugation and plasma processing, but none of these offers a
completely integrated solution.
0.00
0
WORKFLOW
200
400
600
800
1000
1200
1400
1600
1800 2000 2200 2400
Analyte Conc. / IS Conc.
2600
2800
3000
3200
3400
3600
3800
4000
Calibration curve for Hydroxy Desmethyl Bosentan
(2.00 to 512 ng/mL)
A modified electronic pipette (RAININ EDP 3) which is capable of multiple aspiration steps is used for the
following procedure:
B.rdb (Ro64-1056): "Linear" Regression ("1 / (x * x)" weighting): y = 0.0021 x + 0.00841 (r = 0.9978)
Stock solutions at 1.00 mg/mL: all compounds were dissolved in acetonitrile and DMSO (9 + 1) and
stored at – 80ºC. Working solutions (stored at 6ºC) were prepared in a mixture of methanol and water
(1 + 1) and spiked to whole blood. Calibration ranges of 1.00 ng/mL to 4096 ng/mL for Bosentan and
2.00 ng/mL to 512 ng/mL for the three measured metabolites were used. Bosentan and the three
measured metabolites were obtained from Toronto Research Chemicals.
100
120
140 160
180 200
220 240 260 280 300
Analyte Conc. / IS Conc.
320 340
360
380 400
420
440 460
480 500
Calibration curve for Desmethyl Bosentan
(2.00 to 512 ng/mL)
0.32
0.30
0.28
0.26
0.75
0.24
0.70
0.65
0.22
0.60
0.20
0.55
0.18
0.50
0.16
0.45
0.14
0.40
0.12
0.35
0.10
0.30
0.25
0.08
0.20
0.06
0.15
0.04
0.10
0.02
0.05
Autosampler rack with whole blood
samples ready to analyze
0.00
HPLC flow scheme
0
20
40
60
80
100
120
140 160
180 200
220 240 260 280 300
Analyte Conc. / IS Conc.
320 340
360
380 400
420
440 460
480 500
0
20
40
60
80
100
120
140 160
180 200
220 240 260 280 300
Analyte Conc. / IS Conc.
320 340
360
380 400
420
440 460
480 500
CONCLUSIONS / OUTLOOK
RESULTS
After initial optimization for the HPLC parameters performance tests were carried out in order to verify
the performance of the complete system. Analytical runs containing 2 calibration curves (at the
beginning and at the end) and 6 replicates of 4 QC levels were analyzed. Data for intra- and inter-assay
precision and accuracy were within the requirements for bioanalytical method validations. Typical
chromatograms for a Cal 1 sample (ISTDs not shown), a Cal 6 sample and a Blank sample are shown
beneath.
Typical Cal 1 sample
XIC of +MRM (8 pairs): 552.3/202.1 amu from Sample 4 (A-Cal1) of Serie1.wiff (Turbo Spray)
Whole blood samples are transferred to
injection / clamp module
Typical Cal 6 sample
Max. 1937.5 cps.
2175
2100
XIC of +MRM (8 pairs): 552.3/202.1 amu from Sample 9 (A-Cal6) of Serie1.wiff (Turbo Spray)
XIC of +MRM (8 pairs): 552.3/202.1 amu from Sample 3 (A-Blank) of Series1.wiff (Turbo Spray)
Max. 75.0 cps.
275
Simultaneous determination of bosentan and its three major metabolites in various biological matrices and species using
narrow bore liquid chromatography with ion spray tandem mass spectrometric detection
B. Lausecker , B. Hess, G. Fischer, M. Mueller and G. Hopfgartner, Journal of Chromatography B, 2000, 67-83
260
6.5e5
2.72
240
6.0e5
1800
220
5.5e5
2.74
1600
200
5.0e5
1500
1400
180
4.5e5
1300
160
4.0e5
1200
1100
An alternative concept for sample collection, preparation, processing and analysis has been developed
and successfully implemented for the analysis of Bosentan and its metabolites in human whole blood.
Further experiments, for examples the use of the sample cartridges during tox studies are currently
ongoing. The prototype autosampler instrument has been further developed and is now marketed
under the brand name ScapTM (sample cartridge and prep system) by Prolab Instruments GmbH
(http://www.prolab.ch).
REFERENCES
Typical blank sample
Max. 5.2e5 cps.
7.0e5
2000
1900
1700
ISTDs for Bosentan and the three metabolites (d4-labeled)
556.3
202.1
80 msec
542.3
189.1
80 msec
572.3
202.1
80 msec
558.3
189.2
80 msec
80
0.38
0.85
0.80
Mass Spectrometry:
Mass spectrometer: MDS SCIEX API 4000
Positive electrospray ionization (needle voltage 5000 V, orifice 60 V and collision energy 40 V)
Curtain gas: 20, Gas 1: 40, Gas 2: 50, ion source temperature: 500ºC
Analyst software, version 1.4.1 (all HLPC components were controlled by contact closure)
The following SRM transitions were used for the experiments:
Bosentan and the three metabolites
Q1 mass
Q3 mass
Dwell time
552.3
202.1
80 msec
(Bosentan)
538.3
189.1
80 msec
(Desmethyl Bosentan)
568.3
202.1
80 msec
(Hydroxy Bosentan)
554.3
189.2
80 msec
(Hydroxy Desmethyl Bosentan)
60
0.36
0.90
Calibration and Quality Control Samples:
40
0.34
1.00
0.95
EXPERIMENTAL CONDITIONS
20
B.rdb (Ro47-8634): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000686 x + 0.00137 (r = 0.9980)
1.10
1.05
1) Take a 10 uL sample tip with the multiple aspiration pipette
2) Aspirate 1 uL of internals standard solution containing EDTA
3) Aspirate 5 uL of calibration or quality control sample
4) Aspirate 1 uL of internal standard solution containing EDTA
5) Close the sample tip with a cap and store in autosampler or freezer
0
140
3.5e5
1000
The sample cartridge acts as a disposable loop. During the
injection the sample is loaded onto the RAM column and
washed. Then the cleaned sample is transferred onto the
trapping column for preconcentration before it is transferred
to the analytical column and then eluted to the mass
spectrometer for analysis. The overall analysis time per
sample was 7 minutes.
120
3.0e5
900
800
100
2.5e5
ACKNOWLEDGEMENTS
700
80
2.0e5
600
500
2.73
60
1.5e5
400
300
40
1.0e5
200
20
5.0e4
1.00
100
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Time, min
4.0
4.5
5.0
5.5
6.0
6.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Time, min
4.0
4.5
5.0
5.5
6.0
6.5
0
0.5
1.0
2.78
1.5
2.0
2.5
3.0
3.5
Time, min
4.0
4.5
5.0
5.5
6.0
6.5
We gratefully acknowledge all members of Inovalab for their contributions and Dr. Jasper Dingemanse and Dr. Laurent Nicolas
from Actelion for their donation of the internal standards.