HPLC Assay with Ultraviolet Detection for Therapeutic Drug Monitoring of Sirolimus

Sirolimus (Rapamune®; rapamycin) is a macrocyclic lactone produced by Streptomyces hygroscopicus (1)(2). Studies in a variety of animal transplant models (3)(4)(5) and human clinical trials (6)(7)(8)(9) have shown that sirolimus is a potent immunosuppressive agent. Sirolimus is metabolized in humans by hepatic and intestinal cytochrome P450 3A4, primarily leading to demethylated and hydroxylated metabolites (10). When sirolimus is given in the presence of cytochrome P450 3A4 inducers or inhibitors or to patients with hepatic insufficiency, sirolimus blood concentrations may be affected and dose adjustments may be required. Therefore, therapeutic drug monitoring (TDM) of sirolimus concentrations plays an important role in the selection of the optimum dose of sirolimus; in Europe, the license for this drug stipulates that concentrations must be monitored.Sirolimus binds to the immunophilin FK506 binding protein and is sequestered in red blood cells, leading to whole-blood/plasma ratios of ∼38 (11)(12). Because of low sirolimus concentrations in plasma and limited stability in that matrix, whole blood is the matrix of choice for determining sirolimus concentrations (13)(14). An excellent correlation has been reported between sirolimus 24-h whole-blood trough concentrations ( c min) and sirolimus area under the concentration-time curve (12)(15). A strong correlation between sirolimus c min values and the evidence of efficacy and adverse events has also been reported (15). Therefore, the TDM of sirolimus c min values provides a useful prediction of inadequate immunosuppression or potential adverse events. When sirolimus is given with full-dose cyclosporine, the recommended therapeutic range for sirolimus whole-blood trough concentrations is 5–15 μg/L (15). For an analytical method to be suitable for the TDM of sirolimus, it should be simple, sensitive (lower limit of quantification, <5 μg/L), and rapid (turn-around time, <24 h).Several …