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


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.
Clinical Pharmacology Laboratory, The Queen Elizabeth Hospital, Adelaide, South Australia, Australia; Discipline of Pharmacology, The University of Adelaide, Adelaide, South Australia, Australia.
Benedetta C
BC
Sallustio
Benjamin D
BD
Noll
Raymond G
RG
Morris
D002851:Chromatography, High Pressure Liquid; D016903:Drug Monitoring; D000068338:Everolimus; D006801:Humans; D007118:Immunoassay; D007166:Immunosuppressive Agents / Q000097:blood; D020123:Sirolimus; D016559:Tacrolimus / Q000097:blood; D053719:Tandem Mass Spectrometry
D007166:Immunosuppressive Agents; D000068338:Everolimus; D020123:Sirolimus; D016559:Tacrolimus