High-performance liquid chromatographic assay for glucuronidation activityof 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), in human liver microsomes

A simple and sensitive assay for glucuronidation activity of 7-ethyl-10-hyd roxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11). in human liver microsomes by high-performance liquid chromatography (HPLC) wit h fluorescence detection is reported. The method was validated for the dete rmination of SN-38 glucuronide (SN-38G) with respect to specificity, linear ity, recovery, stability, precision, accuracy, and limits of detection and quantitation. There was no interference from matrix and non-enzymatic react ions. The calibration curve for SN-38G was linear from 5 to 500 nm. Average recoveries ranged from 98 to 100% in spiked human liver microsome samples, and the SN-38G was stable at 4 degreesC for at least 72 h. The newly devel oped method was found to be more sensitive and selective than previous meth ods using thin layer chromatography and HPI-C. The limit of quantitation fo r SN-38G was 5 nm (2.5 pmol/assay). The intra- and inter-day precision and accuracy were less than 7 and 4%. respectively. The intra- and inter-day pr ecision of enzyme assay for UDP-glucuronosyltransferase (UGT) activity towa rd SN-38 in human liver microsomes was less than 4%. With this improved sen sitivity, the kinetics of SN-38 glucuronidation in human liver microsomes c ould be determined more precisely. Therefore, this method is applicable to in vitro study on the side effects and drug interactions of CPT-11 using sm all amounts of biological sample. Copyright (C) 2001 John Wiley & Sons, Ltd .