Sample clean-up and high-performance liquid chromatographic techniques for measurement of whole blood rapamycin concentrations.

Determination of rapamycin (RAPA), a potential adjunct to cyclosporin immunosuppressive therapy in solid organ transplantation, presents a challenge because of the agent's unusual chemical properties. RAPA is not readily extracted from biological matrices, is light-sensitive, and exists in numerous isomeric forms. Using liquid-liquid extraction techniques with tert.-butyl methyl ether followed by ethanol, one achieves 96% recovery of RAPA and 70% recovery of the internal standard, beta-estradiol-3-methyl ether, from human whole blood. Subsequent high-performance liquid chromatography (HPLC) utilizing two heated reversed-phase C18 columns in tandem and an 85% methanol-water mobile phase with ultraviolet detection at 276 nm provides measurement of RAPA from 1-ml samples of human whole blood. The chromatography requires less than 40 min per sample. The assay exhibits < or = 10% standard error of the mean and < or = 12% coefficient of variation over the concentration range 2-100 ng/ml. The method has been tested using pharmacokinetic profiles from renal transplant recipients receiving bolus intravenous RAPA infusions.