Therapeutic monitoring of sirolimus in human whole-blood samples by high-performance liquid chromatography.
BACKGROUND: Sirolimus is a macrolide antibiotic isolated from Streptomyces hygroscopicus that has demonstrated immunosuppressive activity. Human and animal studies have shown a good correlation of trough sirolimus concentrations with immunosuppressive efficacy. OBJECTIVE: This report describes a reverse-phase high-performance liquid chromatography (HPLC) method used for therapeutic drug monitoring of sirolimus. METHODS: A reverse-phase C18 column method was developed using an automated HPLC system and ultraviolet (UV) detection. Whole-blood samples collected in ethylenediamine-tetraacetic acid (EDTA) are first hemolyzed, and an internal standard (desmethoxysirolimus) is added to 1.0 mL of sample, which is then extracted with 1-chlorobutane and, after the organic layer is removed, evaporated to dryness. The residue is reconstituted in a 70% methanol/water mixture. Reconstituted extracts are analyzed by HPLC at a column temperature of 60 degrees C and a flow rate of 1.0 mL/min. Typically, chromatography requires 35 minutes between each sample injection. The UV detector is set at 278 nm with a response sensitivity of 0.010 AUFS (absorbance units full scale). Standards and controls prepared in hemolyzed EDTA-anticoagulated whole blood are extracted and run in parallel. Identification of peaks of interest is by retention time; quantification of sirolimus in controls and clinical samples uses a peak-height ratio (sirolimus/internal standard). RESULTS: The assay's precision (coefficients of variation, 5.7%-14.4%) and sensitivity (2.5 ng/mL) were found to be appropriate for therapeutic monitoring purposes. Analytical recovery of 88.0% to 106.3% was observed throughout the assay's linear range (2.5-150.0 ng/mL). Stability studies at 20 degrees C to 25 degrees C and 2 degrees C to 8 degrees C showed an estimated recovery of sirolimus ranging from 85% to 110% of target concentrations (10-90 ng/mL). In a study comparing the results of 194 samples from kidney transplant recipients assayed by the HPLC-UV assay and by a microparticle enzyme immunoassay, the HPLC-UV method provided approximately 10% lower values. CONCLUSION: The HPLC-UV assay is analytically capable of providing useful data for the clinical assessment of patients receiving sirolimus.