Determination of Cotinine, 3′-Hydroxycotinine, and Their Glucuronides in Urine by Ultra-high Performance Liquid Chromatography

The measurement of the primary nicotine metabolites, cotinine and trans-3 '-hydroxycotinine, is a useful biomarker of nicotine exposure and metabolism genetics for smoking cessation research. Herein is described an ultra-high performance liquid chromatography-tandem mass spectrometry method for the determination of these primary nicotine metabolites in urine. Urine samples were diluted one hundred-fold with water and introduced into an ultra-high performance liquid chromatography triple quadrupole mass spectrometer using positive ion electrospray ionization with multiple reaction monitoring. Levels of urinary nicotine metabolites: cotinine, trans-3 '-hydroxycotinine, and their respective glucuronides were determined directly using deuterated internal standards and compared with indirect determination by enzymatic hydrolysis. The assay was applied to a community sample of smokers' urine (n=280). The assay demonstrated satisfactory performance (relative standard deviation of 1.6-6.5 percent at the 1000 nanograms per milliliter level and >98 percent recovery) suitable for application to smoking studies with a run time less than five minutes. The mean (min-max) levels of cotinine and cotinine-glucuronide were 968 (31-3831) and 976 (9-5607) nanograms per milliliter. The mean (min-max) levels of trans-3 '-hydroxycotinine and trans-3 '-hydroxycotinine-glucuronide were 3529 (13-21337) and 722 (0-4633) nanograms per milliliter. Direct determination of glucuronide metabolites was superior to indirect measurement using enzymatic hydrolysis, where there was evidence of loss of metabolites during sample preparation. A sensitive and selective ultra-high performance liquid chromatography-tandem mass spectrometry assay was successfully developed for the determination of cotinine, trans-3 '-hydroxycotinine, and their glucuronides in urine. The rapid and simple sample preparation makes this assay suitable for high throughput studies involving nicotine metabolism phenotype for both cytochrome P450 2A6 and uridine 5 '-diphospho-glucuronosyltransferase, smoking prevalence, and cessation studies.