High-Performance Liquid Chromatographic Assay for N-Glucuronidation of Nicotine and Cotinine in Human Liver Microsomes

✍ Scribed by Miki Nakajima; Jun-Tack Kwon; Eriko Tanaka; Tsuyoshi Yokoi

• Publisher: Elsevier Science
• Year: 2002
• Tongue: English
• Weight: 79 KB
• Volume: 302
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2001.5528

✦ Synopsis

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-l incubation mixture. The nicotine Nglucuronide (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 l mixture for both Nic-glu and Cot-glu. Linear standard curves were obtained within the concentration ranges 25-1000 pmol/200 l mixture for Nic-glu and 100 -5000 pmol/200 l mixture for Cotglu. The intraassay precision and accuracy were <11.1% coefficient of variation (CV) and 97.5-106.6% for Nic-glu and <4.6% CV and 96.7-100.4% for Cot-glu. The interassay precision and accuracy were <7.2% CV and 98.2-106.1% for Nic-glu and <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 Nicglu and Cot-glu in biological specimens in vivo.