With the U.S. Food and Drug Administration (FDA) having oversight for legal distribution and marketing of tobacco products, a rigorous approach to the analysis of tobacco-related compounds in biological samples for product assessment studies is prudent to ensure compliance with regulatory expectations. The measurement of total nicotine equivalents (i.e. nicotine plus its major metabolites) in urine is a direct and noninvasive method for assessment of human exposure to tobacco products. Selectivity for all of the analytical compounds in a method is paramount for accuracy and reproducibility. Although some methods may quantify nicotine and its metabolites from a single injection, the selectivity of such methods may not be sufficient for regulated bioanalysis (i.e. GLP-compliant).

A method for the determination of nicotine, cotinine, trans-3’-hydroxycotinine, and their N-, N-, and O-glucuronides (respectively) from 0.250 mL of urine has been validated with lower limits of quantification (LLOQs) of 10/10/10/10/20/50 ng/mL, respectively. Liquid-liquid and solid-phase extraction were combined with reversed-phase gradient chromatography and detection by multiple reaction monitoring (MRM) of ions on an AB Sciex API 4000™ triple quadrupole mass spectrometer for accurate and precise analysis with complete, FDA-compliant selectivity against trace urinary interferences. Development of a complementary method for analysis of nornicotine, norcotinine, nicotine oxide, and cotinine oxide, from 0.100 mL of urine with target LLOQs of 2/1/5/2 ng/mL, respectively, is in progress and nearing completion.

Concentrations of the analytes (or lack thereof) in smokers’ and nonsmokers’ urine, along with sample chromatography, are presented to demonstrate the accuracy, precision, sensitivity, and selectivity of the comprehensive methodology.