Identification of 5-Hydroxycotinine in the Plasma of Nicotine-Treated Mice: Implications for Cotinine Metabolism and Disposition in Vivo

Abstract

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 an e-cigarette (e-cig) containing 2.4% nicotine. Liquid chromatography-mass spectrometry 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 minutes postinjection, and CNO was readily detectable. In e-cig-exposed mice with plasma nicotine levels resembling that of human smokers, plasma 5-HC and CNO, as well as 3-HC, were readily quantifiable at the end of the 4-hour exposure time. In nicotine-injected mice, the combined concentration of 3-HC plus 5-HC plus CNO, all formed from cotinine by CYP2A5, was higher (P < 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 < 0.001) than males (N = 8 per 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 liquid chromatography with tandem mass spectrometry in mice and is controversial in human smokers. SIGNIFICANCE STATEMENT: Nicotine is the active ingredient of tobacco, but its elimination route through its biomarker cotinine is not fully understood. By liquid chromatography-mass spectrometry, this study has identified and quantified for the first time 5-hydroxycotinine and cotinine N-oxide, 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.