Comparison of blood sirolimus, tacrolimus and everolimus concentrations measured by LC-MS/MS, HPLC-UV and immunoassay methods

Abstract

<h4>Objectives</h4>An LC-MS/MS method was developed for simultaneous quantitation of tacrolimus, sirolimus and everolimus in whole blood, and compared to HPLC-UV and immunoassay methods.<h4>Design and methods</h4>Blood (0.1mL) was analysed following solid-phase extraction and chromatographic resolution using a C18 column (45°C) and mobile phase of methanol/40mM ammonium acetate/glacial acetic acid (83/17/0.1) at 200μL/min, with positive electrospray ionisation and multiple reaction monitoring.<h4>Results</h4>Intra- and inter-day imprecision and inaccuracy were ≤12.2% over a 1.5-40μg/L calibration range. An external quality assurance programme confirmed acceptable inaccuracy and imprecision of the LC-MS/MS method, but highlighted problems with immunoassay quantitation, particularly for everolimus, showing a >30% bias in FPIA everolimus concentrations measured in pooled patient samples versus spiked drug-free whole blood.<h4>Conclusions</h4>LC-MS/MS provides significant accuracy and precision advantages compared to HPLC and immunoassays. Discrepancies in everolimus concentrations measured by the Seradyn FPIA immunoassay require further investigation.