Validation of an Assay for Routine Monitoring of Sirolimus Using HPLC with Mass Spectrometric Detection

[摘要] Sirolimus (rapamycin, Rapamune®; Wyeth-Ayerst Research) is a potent immunosuppressive drug with a molecular mass of 913.6 Da (1). When licensed in the United States, measurement of circulating sirolimus concentrations was recommended for pediatric patients, for those with hepatic impairment, and for patients in whom potent inducers or inhibitors of the enzyme CYP3A4 are co-administered or in whom concomitantly administered cyclosporine dosing is markedly reduced or discontinued. Because 95% of the drug is sequestered within erythrocytes, whole blood has been recommended as the sample matrix (2).In a series of concentration-controlled clinical trials, sirolimus was measured by HPLC with mass spectrometric (MS) detection (3) or HPLC with ultraviolet (UV) detection as a guide to dose adjustment (4)(5). For the double-blind pivotal phase III clinical studies, the drug was measured by an investigational microparticle enzyme immunoassay (Abbott Diagnostics) on the IMx® clinical analyzer (6). The immunoassay is not commercially available, and there is an acute need for an accurate, rapid, and simple chromatographic assay to manage patient care. Current clinical trials target predose (trough) whole-blood sirolimus concentrations within the approximate range 5–30 μg/L.HPLC-UV assays for sirolimus have throughputs of only ∼20–30 samples per day, and current sirolimus dosage regimes tend to achieve sirolimus trough concentrations close to the reported lower limits of quantification (LLOQ) of these assays, ∼1–5 μg/L, despite the use of blood sample volumes in the range 1–2 mL (7)(8)(9). For most laboratories, the only feasible alternative to HPLC-UV currently available is HPLC-MS (10)(11), which has a shorter chromatographic run time (leading to a higher sample throughput), smaller sample volume, increased selectivity for sirolimus, and a lower LLOQ. With sample volumes of 0.5 or 1 mL, the LLOQ is 0.2 or 0.25 μg/L, respectively (10)(11 …

[学科分类] 过敏症与临床免疫学