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

Background: 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.
Methods: 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 (n = 97 smokers) was genotyped for UGT2B10*2 and UGT2B17*2, and the known impact of these variants was evaluated using urinary ratios determined by the direct versus indirect method.
Results: 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.Conclusions: 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.Impact: Lower cost and maintenance combined with high accuracy and reproducibility make the direct method ideal for smoking biomarker, NMR, and pharmacogenomics studies. Cancer Epidemiol Biomarkers Prev; 27(8); 882-91. ©2018 AACR. ©2018 American Association for Cancer Research.