High-performance liquid chromatographic assay for N-glucuronidation of nicotine and cotinine in human liver microsomes.
A method for the determination of N-glucuronidation of nicotine and cotinine in human liver microsomes by high-performance liquid chromatography was developed. Nicotine or cotinine was incubated with human liver microsomes and UDP-glucuronic acid in a 200-microl incubation mixture. The nicotine N-glucuronide (Nic-glu) and cotinine N-glucuronide (Cot-glu) formed were analyzed by ion-pair chromatography with a C-18 column. The sensitivity of quantification at 260 nm absorption was improved by using a noise-base clean Uni-3, and the limit of quantification was 10 pmol/200 microl mixture for both Nic-glu and Cot-glu. Linear standard curves were obtained within the concentration ranges 25-1000 pmol/200 microl mixture for Nic-glu and 100-5000 pmol/200 microl mixture for Cot-glu. The intraassay precision and accuracy were < or =11.1% coefficient of variation (CV) and 97.5-106.6% for Nic-glu and < or =4.6% CV and 96.7-100.4% for Cot-glu. The interassay precision and accuracy were < or =7.2% CV and 98.2-106.1% for Nic-glu and < or =4.6% CV and 96.8-99.3% for Cot-glu. This is the first report of the in vitro determination of Nic-glu and Cot-glu in human liver microsomes. Furthermore, this highly sensitive HPLC method can be used for the determination of Nic-glu and Cot-glu in biological specimens in vivo.
Nakajima
Miki
M
Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Takara-machi 13-1, Kanazawa, 920-0934, Japan. nmiki@kenroku.kanazawa-u.ac.jp
Kwon
Jun-Tack
JT
Tanaka
Eriko
E
Yokoi
Tsuyoshi
T
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003367:Cotinine / Q000737:chemistry* / Q000378:metabolism; D020719:Glucuronides / Q000737:chemistry*; D014453:Glucuronosyltransferase / Q000378:metabolism; D006801:Humans; D008862:Microsomes, Liver / Q000737:chemistry; D009538:Nicotine / Q000737:chemistry* / Q000378:metabolism
D020719:Glucuronides; D009538:Nicotine; D014453:Glucuronosyltransferase; D003367:Cotinine