An isotope dilution LC-MS/MS based candidate reference method for the quantification of cyclosporine A, tacrolimus, sirolimus and everolimus in human whole blood

• Published in: Clinical biochemistry Vol. 82; pp. 73 - 84
• Main Authors: Taibon, Judith, van Rooij, Milou, Schmid, Rupert, Singh, Neeraj, Albrecht, Eva, Anne Wright, Jo, Geletneky, Christian, Schuster, Carina, Mörlein, Sophie, Vogeser, Michael, Seger, Christoph, Pongratz, Stephan, Kobold, Uwe
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2020
• Subjects: Carbon Isotopes - chemistry; Chromatography, Liquid - methods; Chromatography, Liquid - standards; Cyclosporine - blood; Data Accuracy; Diagnostic Tests, Routine - methods; Diagnostic Tests, Routine - standards; Drug Monitoring - methods; Drug Monitoring - standards; Everolimus - blood; Humans; Immunosuppressive Agents - blood; Immunosuppressive drugs; Indicator Dilution Techniques; LC-MS/MS; Quantitative NMR; Reference measurement procedure; Reference Standards; Sensitivity and Specificity; Sirolimus - blood; Solid Phase Extraction - methods; Tacrolimus - blood; Tandem Mass Spectrometry - methods; Tandem Mass Spectrometry - standards; Whole blood; Whole blood; LC-MS/MS; Immunosuppressive drugs; Quantitative NMR; Reference measurement procedure
• ISSN: 0009-9120; 1873-2933
• DOI: 10.1016/j.clinbiochem.2019.11.006

•LC-MS/MS-based candidate reference method for the quantification of four immunosuppressant drugs in human whole blood.•Characterization of the primary reference materials by qNMR.•Uncertainty evaluation according to the GUM.•Method comparison study with an independent clinical laboratory measuring 237 native patient samples. An isotope dilution LC-MS/MS based candidate reference measurement procedure for the quantification of cyclosporine A, tacrolimus, sirolimus and everolimus in human whole blood is presented to be used for evaluation and standardization of routine assays applied for therapeutic drug monitoring. The assay allows baseline separation of the four immunosuppressive drugs within a total runtime of 9 minutes using a C4 reversed phase column. Sample preparation is based on protein precipitation with zinc sulphate followed by purification with solid phase extraction. Reference materials used in this reference measurement procedure were characterized by qNMR and an absolute content of analytes calculated to guarantee traceability to SI units. As internal standards the corresponding deuterated and 13C-labelled analytes were used. The method allows the measurement of cyclosporine A in the range of 5 ng/mL to 2100 ng/mL; tacrolimus, sirolimus and everolimus were analysed in the range of 0.25 ng/mL to 50 ng/mL. Imprecision for inter-day measurements were found to be ≤3.5% for cyclosporine A and ≤4.4% for tacrolimus, sirolimus and everolimus. Accuracy was found to be within 101% and 108% for cyclosporine A and between 95% and 104% for the macrolide compounds. The uncertainty was evaluated according to the GUM. Expanded measurement uncertainties were found to be ≤7.2% for cyclosporine A, ≤6.8% for tacrolimus, ≤9.0% for sirolimus and ≤8.9% for everolimus (k = 2).