MSACL 2019 EU Abstract
Self-Classified Topic Area(s): Small Molecules / Tox / TDM
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 High-throughput Quantification of Immunosuppressant Drugs in Human Blood by LC-MSMS for Clinical Research
Magnus Olin (1), Victoria Barclay (2), Anna Hansson (2), Sara Bildsten (2), Miguel Gambell-Barroso (2), Louise Gustafsson (2)
(1) Thermo Fisher Scientific, Hägersten, Sweden (2) Karolinska University Hospital, Huddinge Sweden
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Presenter Bio: Sales support Expert for Thermo Fisher Scientific. Experience from qualitative and quantitative mass spectrometry using both nominal mass and HRAM mass spectrometers. Expert in quantitation in biological fluids; Sample preparation, Validation, troubleshooting. 20 years experience of mass spectrometry from Pharma industry, both regulated and non regulated bionanalysis.
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Thermo Fisher Scientific |
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Abstract Introduction
The analytical validation of a clinical research method for the quantification of four immunosuppressant drugs (Cyclosporin A (CyA), Tacrolimus (TAC), Sirolimus (SIR) and Everolimus (EVE)) in whole blood is reported. The present method allows low level quantitation (CyA 10 ng/mL, TAC, SIR, EVE at 0.5 ng/mL) which makes it especially suitable for clinical research studies. At the same time the method was adapted for high throughput analysis using multi-channeling, delivering results in less than one minute per injection. Method performance was evaluated in terms of linearity of response within the calibration ranges, selectivity, accuracy, and intra- and inter-assay precision and carry-over for each analyte.
Methods
Whole blood was extracted by offline protein precipitation and internal standard addition into a 96 well plate by an automated procedure using a Hamilton™ robotic liquid handler. The plates were centrifuged and the supernatant injected onto a Thermo Scientific™ Transcend II™ system connected to a Thermo Scientific™ TSQ Quantis™ triple quadrupole mass spectrometer. Detection was performed by selected reaction monitoring (SRM) using four isotopically labeled internal standards. The LC column used was a Thermo Scientific™ Accucore RPMS 2.1x30 mm. The system was used in multi-channel mode and the cycle time was less than 2 min/channel providing results from one injection in less than one minute. Data was acquired and processed using Thermo Scientific™ TraceFinder™ 4.1 software. The method was evaluated using the MassTox® calibrators and Quality Control Samples from Chromsystems Instruments & Chemicals GmbH (Munich, Germany). An additional level for both calibrator and Quality Control sample at LLOQ was obtained by dilution of a low level Qiuality Control sample with blank whole blood, providing calibration samples at 7 levels and Quality Control samples at 5 levels. The calibrated range was 9.5 -1950 ng/mL (CyA), 0.5-42 ng/mL (EVE, TAC) and 0.5 – 47 ng/mL (SIR). Five batches of data was collected on two indentical LC-MS/MS systems.
Results
The method proved to be linear in the ranges covered by the calibrators. The data from both systems demonstrated acceptable accuracy and precision with a bias between nominal and average calculated concentration for the control samples within ±20% at LLOQ and ±10% at higher levels. The %CV for inter- and intra-assay precision was <20% at LLOQ and <10% at higher levels for all the analytes.
Conclusions
A liquid chromatography-tandem mass spectrometry method for clinical research for the quantification of four different immunosuppressant drugs in whole blood was implemented. The method offers high throughput and accurate results to a level suitable for clinical research studies. The described method meets research laboratory requirements in terms of sensitivity, linearity of response, accuracy and precision and throughput.
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= Discovery stage.
= Clinically available.
