Abstract
A high performance liquid chromatographic (HPLC) method was developed for the quantitation of rapamycin, an immunosuppressive agent, in biological specimens. The method employs a 15 cm Supelco LC-18 column (5 μm) interconnected to a 25 cm Supelco LC-18 column (5 μm). The mobile phase is a methanol/water gradient system. The flow rate is 0.51 ml/min and detection is by ultraviolet (UV) absorption at 276 nm. The method was validated for its specificity, precision, linearity and sensitivity in rat serum. Endogenous compounds in rat serum did not interfere with the detection of rapamycin or the internal standard (β-estradiol-3-benzoate). Based on a 1.0 ml serum sample, the assay was linear from 5 to 500 ng/ml. The intra-day coefficients of variation were below 10% and independent of concentration. Inter-day precision values ranged from 5.5 to 13.6%, the difference being independent of concentration. The specificity, linearity and sensitivity of the method was also demonstrated in cynomolgus monkey serum, rat plasma and hemolyzed rat whole blood. In each case, the method was specific, with no endogenous interferences. Furthermore, the method showed no interference from two newly reported rapamycin degradation products.