A Comparison of Direct and Indirect Analytical Approaches to Measuring Total Nicotine Equivalents in Urine

Abstract

Total nicotine equivalents (TNE), the sum of nicotine and metabolites in urine, is a valuable tool for evaluating nicotine exposure. Most methods for measuring TNE involve two-step enzymatic hydrolysis for indirect quantification of glucuronide metabolites. Here, we describe a rapid, low-cost direct LC/MS assay. In 139 smokers' urine samples, Bland-Altman, correlation, and regression analyses were used to investigate differences in quantification of nicotine and metabolites, TNE, and nicotine metabolite ratio (NMR) between direct and indirect LC/MS methods. DNA from a subset ( = 97 smokers) was genotyped for and, and the known impact of these variants was evaluated using urinary ratios determined by the direct versus indirect method. The direct method showed high accuracy (0%-9% bias) and precision (3%-14% coefficient of variation) with similar distribution of nicotine metabolites to literary estimates and good agreement between the direct and indirect methods for nicotine, cotinine, and 3-hydroxycotinine (ratios 0.99-1.07), but less agreement for their respective glucuronides (ratios 1.16-4.17). The direct method identified urinary 3HC+3HC-GLUC/COT as having the highest concordance with plasma NMR and provided substantially better estimations of the established genetic impact of glucuronidation variants compared with the indirect method. Direct quantification of nicotine and metabolites is less time-consuming and less costly, and provides accurate estimates of nicotine intake, metabolism rate, and the impact of genetic variation in smokers. Lower cost and maintenance combined with high accuracy and reproducibility make the direct method ideal for smoking biomarker, NMR, and pharmacogenomics studies. .

Cigarette consumption and biomarkers of nicotine exposure during pregnancy and postpartum

“…Spot urine samples (5 ml) were collected, frozen, and sent to University of Toronto for nicotine metabolite assessment. TNE was analyzed in urine using liquid chromatography tandem mass spectrometry (LC-MS/MS) as described previously (35). Urinary creatinine concentrations were determined using a colorimetric assay (Creatinine Assay Kit MAK080) from Sigma (St. Louis, MO) with a SynergyMX Analyzer (BioTek, Winooski, VT, USA) and were adjusted for the effect of pregnancy on creatinine clearance (i.e.…”

The Role of Pharmacogenetics in Smoking

“…Consumption or quantity measures include self‐reported cigarettes per day (CPD), which is subject to social desirability or recall bias. Consumption measures also include biomarkers of nicotine intake, typically metabolites of nicotine (from saliva, blood, or urine), 7 which include cotinine (nicotine’s major metabolite), cotinine plus 3′‐hydroxycotinine, or the sum of 90% of nicotine’s metabolites (total nicotine equivalents) 7 ; the latter 2 measurements are less subject to individual enzyme variability. Neuroimaging using functional magnetic resonance imaging and positron emission tomography allows for examination of reward/withdrawal neural activity and receptor/neurotransmitter availability.…”

“…The 1, 2, and 3 flavin‐containing monooxygenase (FMO) subtypes metabolize nicotine to its nicotine N′‐oxide metabolite, typically accounting for 4–7% of nicotine’s clearance 7 . FMOs are expressed both in the liver and in the brain 23 .…”

Pregnancy-Induced Increases in the Nicotine Metabolite Ratio: Examining Changes During Antepartum and Postpartum

“…Urinary concentrations of nicotine and metabolites were directly analyzed using liquid chromatography-tandem mass spectrometry as described by Taghavi et al (in press). 18 In brief, urine samples were diluted and prepared using solid-phase extraction adapted from a previously established method. 19 The limit of quantification was 1 ng/mL for all compounds.…”