Measurement of sirolimus in whole blood using high-performance liquid chromatography with ultraviolet detection

Abstract

Background Sirolimus, a potent immunosuppressive drug, exhibits intrapatient and interpatient variability of absorption and metabolism. Thus, therapeutic drug monitoring is important. Objective This paper describes a reverse-phase high-performance liquid chromatography (HPLC) method, using ultraviolet (UV) absorption for detection, for measuring sirolimus levels in human whole-blood samples. Methods The stability of sirolimus in whole blood was assessed under conditions likely to be encountered during transport of study samples to a central laboratory. The performance of the HPLC-UV assay in measuring sirolimus was compared with that of 3 established, validated HPLC assays with tandem mass-spectrometric (MS/MS) detection. Results of the HPLC-UV assay also were compared with results produced by a prototype microparticle enzyme immunoassay (MEIA). Results Inaccuracy for 3 in-house control samples was ≤4%, whereas within-assay repeatability (coefficient of variation [CV]) was ≤5% and between-assay reproducibility was ≤6.6%. Mean recovery of sirolimus from blood was 81.5%±4.3%. The lower limit of quantification was set at 6.5 ng/mL, and the repeatability CV at this concentration was 4.2% (n = 6). Sirolimus-containing whole-blood samples were stable for 3 freeze/thaw cycles when stored at −20 °C and for ≥2 days when stored at ambient temperature. The sample extract was shown to be stable for up to 54 hours at ambient temperature (~22 °C) after extraction. Results of the HPLC-UV assay were consistent with those of the HPLC/ MS/MS assays but lower than those produced by MEIA. Conclusion This HPLC-UV method is considered suitable for therapeutic drug monitoring of sirolimus.