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.

An immunoassay for the measurement of sirolimus.

OBJECTIVE: This study assessed the performance characteristics of a new microparticle enzyme immunoassay (MEIA) for the determination of sirolimus in whole blood. BACKGROUND: In clinical investigatory studies, dose adjustments of the immunosuppressive drug sirolimus have been carried out using either high-performance liquid chromatography (HPLC) or, more recently, this investigational immunoassay kit based on the MEIA technique. METHODS: Calibration was made over the linear range 0 to 30 ng/mL. Inaccuracy and imprecision were assessed by means of 3 control samples supplied with the kit (5, 11, and 22 ng/mL) and dilution of an above-quantitation-limit sample (154 ng/mL). Specificity was determined by the addition of 2 sirolimus metabolites to sirolimus-free human whole blood or to I of the control samples supplied with the kit. In addition, whole-blood samples from patients receiving either cyclosporine or tacrolimus (N = 24) were analyzed for sirolimus. A comparison of the MEIA and a validated HPLC/MS/MS assay analyzed both pooled samples from patients receiving sirolimus and spiked samples (sirolimus 2-60 ng/mL). In a more extensive comparison of patient samples measured by the MEIA assay, a validated HPLC assay with UV detection (HPLC-UV) was used (HPLC-UV sirolimus 7-64 ng/mL). RESULTS: Inaccuracy (between-run) was < or =16.2% at all 4 concentrations (N = 5). Within-assay imprecision (repeatability) was <6% (N = 5), and between-assay imprecision (reproducibility) for the same samples was < 11% (N = 5). Recovery, assessed by means of 3 in-house control samples prepared in both fresh and previously frozen sirolimus-free human whole blood, ranged from 93.9% to 109.5%. The limit of detection, determined by dilution of the lowest nonzero calibrator (3 ng/mL), was set at 1 ng/mL, at which repeatability was 20.5% (N = 5). Five ng/mL of hydroxysirolimus cross-reacted with the assay by a mean of between 44% and 50% (N = 4); 5 ng/mL of 41-O-demethylsirolimus cross-reacted with the assay by a mean of between 86% and 127% (N = 4). Assay specificity was further challenged by ethylenediamine-tetraacetic acid (EDTA)-whole-blood samples from transplant patients not receiving sirolimus. These samples had tacrolimus and cyclosporine concentrations of 7.8 to 15.9 ng/mL and 38 to 485 microg/L, respectively. The median result was 0 ng/mL (third quartile, 0.7 ng/mL; maximum, 1.4 ng/mL); no value was above the lowest nonzero calibrator. The results of the comparison between the MEIA and the HPLC/MS/MS assay showed mean positive biases of 21% and 8% for the MEIA in measuring sirolimus in pooled patient samples and spiked samples, respectively. The results of the comparison of the MEIA and HPLC-UV median sirolimus concentrations were 18.2 and 20.1. Whole-blood samples anticoagulated with EDTA and containing sirolimus were stable for analysis by MEIA for 3 freeze-thaw cycles when stored at -20 degrees C and for 10 days when stored at 4 degrees C or at ambient temperature. A decline in sirolimus concentration occurred when samples were stored at 37 degrees C. CONCLUSION: The MEIA showed suitable precision across a clinically relevant concentration range. In terms of patient management, the practical significance of cross-reactivity with sirolimus metabolites remains to be assessed.