Topic: Tox / TDM / Endocrine
Podium Presentation in Room 1 on Wednesday at 16:15 (Chair: Mark Marzinke / Ganesh Moorthy)
Authors: Natalicia de Jesus Antunes1,2,3*, Gilberto De Nucci1, Karin Kipper3,4, Lewis Couchman3,5, David W Holt3 and Atholl Johnston2,3
Introduction. Therapeutic drug monitoring (TDM) of immunosuppressive drugs (IMS) is a necessary tool for minimizing drug toxicity and the prevention of organ rejection and graft loss [1, 2]. The most commonly prescribed IMS are cyclosporin A (CYC), everolimus (EVE), sirolimus (SIR), tacrolimus (TAC), and mycophenolic acid (MYC) . The aim of the study was to develop and validate an ultra-high performance liquid chromatography assay with tandem mass spectrometry (UHPLC-MS/MS) to quantify CYC, EVE, SIR and TAC in human blood for use in the assay of patient samples as part of a routine therapeutic drug monitoring (TDM) service.
Methods. CYC, EVE, SIR and TAC were extracted from 50 μL human whole blood by protein precipitation. The internal standard (IS) solution consisted of cyclosponin-D12, ascomycin and 32-desmethoxyrapamycin in methanol/1,2-ethanediol/de-ionised water (100/50/50, v/v/v). A 1.0 M ZnSO4 solution (50 µL) was used to perform haemolysis followed by protein precipitation with 150 μL acetonitrile. Analytes were quantified by UHPLC-MS/MS. The separation of all drugs was performed on a Waters UPLC BEH C18 column kept at 50 ºC. Analytes were eluted with mobile phase A consisting of 2 mM ammonium acetate with 0.1% formic acid (v/v) in de-ionised water, and mobile phase B consisting of 2 mM ammonium acetate with 0.1% formic acid (v/v) in methanol at a flow rate of 300 μL/min. The applied gradient program consisted of 10% B for 0.5 min, thereafter the percentage of B was increased to 50% over 0.5 min, and followed by an increase to 99% B over 1.5 min. Mobile Phase B was then kept at 99% for 2.7 min. At 4.2 min, the mobile phase reverted to 10% B. The total run time was 5 min. Assay performance was evaluated by analysing external proficiency testing samples. The method was used to evaluate the drug concentrations in blood for TDM patients (n = 30).
Results. The method was linear from 1.23 to 41.6 µg/mL, 1.29 to 47.0 µg/mL, 1.28 to 42.4 µg/mL, and 23.75-1094.0 µg/mL for EVE, SIR, TAC, and CYC, respectively. The within- and between-assay reproducibility were 0.6 and 10.7% for EVE, 1.1 and 0.6% for SIR, 2.1 and 9.1% for TAC, and 1.0 and 5.1% for CYC. The matrix interference was minimal. All drugs demonstrated stability in whole blood for at least 7 days at room temperature and at +4 ⁰C, 5 days in the auto-sampler at +4 ⁰C, 1 month at -20 °C and after 3 freeze/thaw cycles, with accuracy ranging from 88% to 111%. Results from proficiency testing and patient samples quantification were comparable to our previous method using a liquid-liquid extraction.
Conclusion. This method showed good analytical performances for the determination of everolimus, sirolimus, tacrolimus, and cyclosporin in whole blood over their respective calibration ranges.
 Zhang Y, Zhang R. Drug Test Anal. 2018;10(1):81-94.
 Freudenberger K, Hilbig U, Gauglitz G. Trac‐Trend Anal Chem. 2016;79:257‐268.
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