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High‐performance liquid chromatographic assay for glucuronidation activity of 7‐ethyl‐10‐hydroxycamptothecin (SN‐38), the active metabolite of irinotecan (CPT‐11), in human liver microsomes
Author(s) -
Hanioka Nobumitsu,
Jinno Hideto,
Nishimura Tetsuji,
Ando Masanori,
Ozawa Shogo,
Sawada Junichi
Publication year - 2001
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.76
Subject(s) - chemistry , chromatography , glucuronidation , sn 38 , microsome , metabolite , active metabolite , detection limit , high performance liquid chromatography , glucuronide , microsoma , enzyme , irinotecan , biochemistry , medicine , colorectal cancer , cancer
A simple and sensitive assay for glucuronidation activity of 7‐ethyl‐10‐hydroxycamptothecin (SN‐38), the active metabolite of irinotecan (CPT‐11), in human liver microsomes by high‐performance liquid chromatography (HPLC) with fluorescence detection is reported. The method was validated for the determination of SN‐38 glucuronide (SN‐38G) with respect to specificity, linearity, recovery, stability, precision, accuracy, and limits of detection and quantitation. There was no interference from matrix and non‐enzymatic reactions. The calibration curve for SN‐38G was linear from 5 to 500 n M . Average recoveries ranged from 98 to 100% in spiked human liver microsome samples, and the SN‐38G was stable at 4°C for at least 72 h. The newly developed method was found to be more sensitive and selective than previous methods using thin layer chromatography and HPLC. The limit of quantitation for SN‐38G was 5 n M (2.5 pmol/assay). The intra‐ and inter‐day precision and accuracy were less than 7 and 4%, respectively. The intra‐ and inter‐day precision of enzyme assay for UDP‐glucuronosyltransferase (UGT) activity toward SN‐38 in human liver microsomes was less than 4%. With this improved sensitivity, the kinetics of SN‐38 glucuronidation in human liver microsomes could be determined more precisely. Therefore, this method is applicable to in vitro study on the side effects and drug interactions of CPT‐11 using small amounts of biological sample. Copyright © 2001 John Wiley & Sons, Ltd. Abbreviations used: CPT‐11 7‐ethyl‐10‐[4‐(1‐piperidino)‐1‐piperidino]carbonyloxycamptothecineSN‐38 7‐ethyl‐10‐hydroxycamptothecinUGTs UDP‐glucuronosyltransferases.