Benedetta C Sallustio1, Benjamin D Noll, Raymond G Morris. 1. Clinical Pharmacology Laboratory, The Queen Elizabeth Hospital, Adelaide, South Australia, Australia; Discipline of Pharmacology, The University of Adelaide, Adelaide, South Australia, Australia.
Abstract
OBJECTIVES: 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. DESIGN AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. Crown
OBJECTIVES: 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. DESIGN AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. Crown
Authors: Nicholas A Kolaitis; Daniel R Calabrese; Patrick Ahearn; Aida Venado; Rebecca Florez; Huey-Ling Lei; Karolina Isaak; Erik Henricksen; Emily Martinez; Tiffany Chong; Rupal J Shah; Lorriana E Leard; Mary Ellen Kleinhenz; Jeffrey Golden; Teresa De Marco; John R Greenland; Jasleen Kukreja; Steven R Hays; Paul D Blanc; Jonathan P Singer Journal: Am J Health Syst Pharm Date: 2019-12-02 Impact factor: 2.637