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.