SIMPLIFIED HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY ASSAY FOR MEASUREMENT OF TACROLIMUS AND ITS METABOLITES AND CROSS-VALIDATION WITH MICROPARTICLE ENZYME-IMMUNOASSAY

In this study, a modified, specific assay for measurement of tacrolimus and its metabolites in blood and urine from transplant patients using high-performance liquid chromatography (HPLC) linked to mass spectrometry (MS) is described. Samples were prepared for HPLC-MS by modified solid-liquid extraction. The original two-step washing procedure was replaced by a single washing step, and samples were eluted with acetonitrile/water instead of dichloromethane, thus avoiding an evaporation step. Samples were injected automatically every 3 min into the HPLC-MS system. Time-consuming gradient elution was replaced by isocratic elution. This procedure resulted in a lower limit of quantitation of 0.2 mu g/L. The interassay variability was 14.5% for 5 mu g/L and 15.8% for 25 mu g/L. The intraassay variability was 11.2% for 5 mu g/L and 4% for 25 mu g/L. The recovery for tacrolimus in blood was 90.4% for I mu g/L, 78.9% for 10 mu g/L, and 81.3% for 25 mu g/L. Measurement of tacrolimus and its metabolites in samples from various transplant patients showed that the main metabolites found in blood and urine are demethyl-tacrolimus, didemethyl-tacrolimus and demethyl-hydroxy-tacrolimus. Cross validation of the modified HPLC-MS assay with a microparticle enzyme immunoassay showed a significant correlation between the two assays, with r = 0.915.