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.
Sallustio
Benedetta C
BC
Clinical Pharmacology Laboratory, The Queen Elizabeth Hospital, Adelaide, South Australia, Australia; Discipline of Pharmacology, The University of Adelaide, Adelaide, South Australia, Australia.
Noll
Benjamin D
BD
Morris
Raymond G
RG
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