Validation of a LC-MS/MS Method for Quantifying Urinary Nicotine, Six Nicotine Metabolites and the Minor Tobacco Alkaloids—Anatabine and Anabasine—in Smokers' Urine

Abstract

Tobacco use is a major contributor to premature morbidity and mortality. The measurement of nicotine and its metabolites in urine is a valuable tool for evaluating nicotine exposure and for nicotine metabolic profiling—i.e., metabolite ratios. In addition, the minor tobacco alkaloids—anabasine and anatabine—can be useful for monitoring compliance in smoking cessation programs that use nicotine replacement therapy. Because of an increasing demand for the measurement of urinary nicotine metabolites, we developed a rapid, low-cost method that uses isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) for simultaneously quantifying nicotine, six nicotine metabolites, and two minor tobacco alkaloids in smokers' urine. This method enzymatically hydrolyzes conjugated nicotine (primarily glucuronides) and its metabolites. We then use acetone pretreatment to precipitate matrix components (endogenous proteins, salts, phospholipids, and exogenous enzyme) that may interfere with LC-MS/MS analysis. Subsequently, analytes (nicotine, cotinine, hydroxycotinine, norcotinine, nornicotine, cotinine N-oxide, nicotine 1′-N-oxide, anatabine, and anabasine) are chromatographically resolved within a cycle time of 13.5 minutes. The optimized assay produces linear responses across the analyte concentrations typically found in urine collected from daily smokers. Because matrix ion suppression may influence accuracy, we include a discussion of conventions employed in this procedure to minimize matrix interferences. Simplicity, low cost, low maintenance combined with high mean metabolite recovery (76–99%), specificity, accuracy (0–10% bias) and reproducibility (2–9% C.V.) make this method ideal for large high through-put studies.

A high-throughput robotic sample preparation system and HPLC-MS/MS for measuring urinary anatabine, anabasine, nicotine and major nicotine metabolites

“…NHANES and PATH). We applied this system further to determine ANAT, ANAB, NIC and its seven major metabolites in human urine using a modified method based on the study by McGuffey et al [17]. We conducted the modifications aimed to increase the throughput using the robotic sample preparation system, mainly in 2 aspects: Liquid chromatography (column, buffer and gradient) and enzymatic hydrolysis.…”

“…Typical pre-treatment techniques include liquid–liquid extraction (LLE) [9,10], solid phase extraction (SPE) [11–13], soxhlet extraction [14], supercritical fluid extraction (SFE) [15], microwave-assisted extraction (MAE) and pressurized solvent extraction (PSE) [16]. The robotic sample preparation procedure described here uses a validated acetone precipitation step to deplete endogenous proteins, salts and phospholipids and to remove the exogenous enzyme that is added to the urine samples to hydrolyze conjugates [17]. …”

“…Tobacco use leads to concurrent exposure to NIC, ANAT, and ANAB, as well as the presence of measurable levels of biomarkers of exposure to these alkaloids [27]. Not surprisingly, ANAT and ANAB are variably glucuronidated [17] and excreted in the urine with elimination half-lives similar to those for nicotine and its metabolites. Thus the presence of biomarkers of exposure to NIC, ANAT, and ANAB has been validated as proving tobacco product use [27].…”

“…Conversely, nicotine replacement therapy uses pharmaceutical-grade nicotine that is essentially free of minor tobacco alkaloids; therefore subjects abstaining from tobacco use and undergoing nicotine replacement therapy have urinary ANAT and ANAB levels <2 ng/ml. [27] Based on these findings ANAT and ANAB are validated biomarkers for identifying non-compliance for participants using NIC replacement therapy (NRT) [17,27]. Our specific aim in this study was to develop and validate a robotic sample preparation method and a HPLC-MS/MS analytical method to simultaneously determine the urinary concentrations of NIC and seven major NIC metabolites in samples from NHANES and PATH studies including both “free” (non-conjugated) and “conjugated” (mostly glucuronide) forms.…”

Using Monte Carlo simulation to assess variability and uncertainty of tobacco consumption in a city by sewage epidemiology

“…Recognising this, the number of cigarettes consumed per smoker (n C ) was calculated using equation 4. ingestion and transdermal administration. [30][31][32][33][34][35][36][37][38] The analysis of compiled data suggests that the urinary excretion profile is quite similar when NIC is either administered through smoking or transdermal patches; however, the oral ingestion has higher extraction rates of OH-COT and COT (see online supplementary table S2). The main elements of the excretory profile of NIC are the NIC metabolites COT and OH-COT (and their conjugated forms), and NIC itself.…”