Determination of rapamycin in whole blood by HPLC.

Sirolimus (rapamycin, RAPA) is a natural fermentation product (macrolide antibiotic) that has demonstrated potent immunosuppressive activity. A reverse-phase high-performance liquid chromatographic (HPLC) method is described for analysis of the drug in whole blood. The samples are purified by using liquid-liquid extraction with butyl chloride/diethyl ether combined with reversed-phase solid-phase extraction. RAPA and internal standard were traced by UV-detection at 278 nm. Linear calibration curves with correlation coefficients > 0.999 were obtained (range, 1-50 ng/ml). Minimum detectable concentration was approximately 0.4 ng/ml and recovery approximately 45% for both RAPA and internal standard. Coefficient of variation (day to day) was 9.8% at 5 ng/ml (n = 6) and 5.6% at 40 ng/ml (n = 6). The chromatography requires < 10 min per sample. The assay has proved to be free of interference peaks from cyclosporine or tacrolimus. The method has been used to determine the whole blood concentrations in samples from healthy volunteers and renal transplant recipients receiving single and multiple doses of oral rapamycin.