Determination of UDP-glucuronosyltransferase UGT2B7 activity in human liver microsomes by ultra-performance liquid chromatography with MS detection.
A rapid and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) method was developed for the qualitative and quantitative determination of UGT2B7 activity using 3'-azido-3'-deoxythymidine (AZT) as probe substrate in human liver microsomes (HLMs). The method was validated for the determination of AZT glucuronidation (AZTG) with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. The chromatographic separation was achieved on a UPLC BEH C18 column (50 mm x 2.1mm i.d., 1.7 microm), with phase of acetonitrile-water (ratio 6:94). Selective ion reaction (SIR) monitor was specific for AZT, AZTG and I.S. The method was linear over the concentration range 0.5-500 microM for AZTG in spiked HLMs. Good precision and accuracy were obtained for concentrations over the standard curve range. AZTG was stable at 4 degrees C for at least 72 h in spiked liver microsomes samples. The method was successfully used to determine the kinetics of UGT activities toward AZT in HLMs. In addition, the method could determine the effects of fluconazole, a known UGT2B7 selective inhibitor, on AZTG in HLMs. Therefore, this method is suitable for in vitro studies using AZTG formation as an index reaction for UGT2B7 activity.
Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457, Zhong-shan Road, Dalian 116023, China.
Hui-Xin
HX
Liu
Yu-Qi
YQ
He
Ying
Y
Hu
Yong
Y
Liu
Jiang-Wei
JW
Zhang
Wei
W
Li
Zheng-Tao
ZT
Wang
Ling
L
Yang
D002853:Chromatography, Liquid / Q000379:methods*; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000737:chemistry / Q000201:enzymology*; D013058:Mass Spectrometry / Q000379:methods*; D008861:Microsomes / Q000737:chemistry / Q000201:enzymology*; D015215:Zidovudine / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism
D015215:Zidovudine; C491474:UGT2B7 protein, human; D014453:Glucuronosyltransferase