Evaluation of Two Methods for Analysis of Blood Sirolimus

Sirolimus, a potent immunosuppressive drug, is used for prevention of organ rejection in renal transplant recipients. Therapeutic monitoring of sirolimus blood level is recommended, by the intra-patient and inter-patient variability of absorption and metabolism together with the concentration-dependent inhibitory effeets and toxicity. Sirolimus is usually assayed in whole blood by high-performance liquid chromatography (HPLC). The study evaluated an in-house HPLC method with ultraviolet detection (HPLC-UV) and a recently developed commercial assay based on microparticle enzyme immunoassay (MEIA) technology applied to IMx analytical platform (Abbott Diagnostics). The aims of this study were evaluation and comparison of both methods for sirolimus assay. The sample preparation for HPLC-UV required addiction of 32-desmethoxysirolimus as internai standard and precipitation of blood matrix with zinc sulphate solution 5% and acetone. After sample alkalinization and centrifugation, a liquid-liquid extraction was performed with 1-chlorobutane. The dried extracts were reconstituted with the mobile phase (60% acetonitrile/water mixture) and purified by a second liquid-liquid extraction with hexane. The samples were analysed with a reverse-phase CI8 column, at 50°C under a flow rate of 0.5 mL/min for 15 minutes in the HPLC system. The retention times of sirolimus and internai standard were 10 and 12.5 minutes, respectively, and they were detected by ultraviolet absorption at 278 nm. Also the MEIA method required a manual pre-treatment step of the whole blood sample, by extraction with zinc sulphate solution in methanol and ethylene glycol and collection of supernatant after centrifugation. The assay was a competitive fluoroimmunoassay where monoclonal anti-sirolimus antibody coated microparticles are the capture reagent and sirolimus conjugated alkaline phosphatase is the competitive reagent. The method is applied to automated IMx instrument. The clinical blood specimens used for correlation study were obtained from renal transplant recipients receiving sirolimus therapy. The specimens were immediately chilled and stored at -20°C for analysis within 1 week after collection. The assay applied to HPLC showed linearity up to 100 ng/mL, recovery of 105.6±4.4% (mean±SD), intra- and inter-assay imprecision CVs of 3.0-3.4% and 3.0- 6.5% at control levels of 25 and 6 ng/mL, respectively. The MEIA method was investigated for recovery (91.3±4.9) and intra- and inter-assay imprecision (CV 4.8 and 8.0%, respectively). Patient correlation study ( n = l l l ) with HPLC-UV and MEIA assays yielded a mean results (SD) of 9.2(6.3) ng/mL (range 3.2-40.3) and 8.3(6.2) ng/mL (range 2.5 - 42.0), respectively. The Passing-Bablok correlation function was as follows: HPLC-UV = 1.031 x MEIA + 0.74 ng/mL; the 95% confidence intervals were 0.929-1.139 for the slope and 0.08-1.34 ng/mL for the intercept. Thus MEIA gave lower sirolimus results than HPLC-UV method and that was confirmed by Bland-Altman analysis too. Some previous studies presented different results about this comparison: the disagreement may be clarifìed through a better selection and characterisation of study populations. The study showed that both HPLC-UV and MEIA methods have good analytical performance. However, sirolimus blood monitoring by MEIA offers the advantages of shorter turnaround time and automation.