TY - JOUR T1 - <strong>Identification of 5-hydroxycotinine in the plasma of nicotine-treated mice -</strong><strong>implications for cotinine metabolism and disposition in vivo</strong> JF - Drug Metabolism and Disposition JO - Drug Metab Dispos DO - 10.1124/dmd.122.001059 SP - DMD-AR-2022-001059 AU - Keiko Kanamori AU - Syed M Ahmad AU - Chang Sung Shin AU - Abdul Hamid AU - Kabirullar Lutfy Y1 - 2022/01/01 UR - http://dmd.aspetjournals.org/content/early/2022/10/02/dmd.122.001059.abstract N2 - Two oxidation products of cotinine, 5-hydroxycotinine (5-HC) and cotinine N-oxide (CNO), were identified for the first time in vivo in the plasma of C57BL/6 mice after injection of nicotine (1 mg/kg) or exposure to e-cigarette containing 2.4% nicotine. Liquid-chromatography mass-spectrometry (LCMS) was used to separate 3-hydroxycotinine (3-HC), 5-HC and CNO and to quantify each by the sensitive direct detection of their parent ion with m/z of 193.097. In nicotine-injected mice, 5-HC was as abundant as 3-HC 15 min post-injection, and CNO was readily detectable. In e-cig-exposed mice with plasma nicotine level resembling that of human smokers, plasma 5-HC and CNO, as well as 3-HC, were readily quantifiable at the end of the 4-h exposure time. In nicotine-injected mice, the combined concentration of 3-HC + 5-HC + CNO, all formed from cotinine by CYP2A5, was higher (P&lt;0.01) in females than in males, although the male-female difference in cotinine plasma level did not reach statistical significance. The result highlights the importance of considering these three oxidation products of cotinine in examining cotinine metabolism and disposition. Coumarin 7-hydroxylase activity, a specific marker of CYP2A5, measured in the hepatic microsomes of untreated mice showed that females have higher activity (P&lt;0.001) than males (N=8/sex). The abundance of plasma 5-hydroxycotinine in nicotine-treated mice raises intriguing questions about the site of its origin (hepatic or possibly kidney CYP2A5) and the routes of its disposition because urinary excretion of 5-HC has not been detected by LC-MS/MS in mice, and is controversial in human smokers. Significance Statement Nicotine is accounted for the addictive property of tobacco, but its elimination route through its biomarker cotinine is not fully understood. By liquid-chromatography mass-spectrometry, we have identified and quantified for the first time 5-hydroxycotinine (5-HC) and cotinine N-oxide (CNO), which are oxidation products of cotinine, in the plasma of mice treated with nicotine or exposed to e-cigarettes. The results raise intriguing questions about nicotine disposition in vivo in this well-established preclinical model of human smokers. ER -