A Simple and High-Throughput LC-MS-MS Method for Simultaneous Measurement of Nicotine, Cotinine, 3-OH Cotinine, Nornicotine and Anabasine in Urine and Its Application in the General Korean Population

Measuring nicotine metabolites is the most objective method for identifying smoke exposure. Liquid chromatography - tandem mass spectrometry (LC-MS-MS) can measure multiple metabolites and is sensitive enough to detect low concentrations of metabolites. Therefore, we developed a simple and high-throughput method for measuring nicotine, cotinine, trans-3-hydroxycotinine (3-OH cotinine), nornicotine and anabasine for population-based studies using LC-MS-MS. Each 30 μL of urine sample was diluted with 90 μL of acetonitrile containing five deuterated internal standards. Chromatographic separation used a C18 column, and LC-MS-MS analysis was performed with a multiple reaction monitoring mode. The chromatographic run time for each sample was 6.5 min. The method was validated by evaluating selectivity, interference, limit of detection, lower limit of quantification, precision, accuracy, linearity, extraction recovery, matrix effect and carryover according to guidelines. Our methods required a short preparation time (∼20 min) while simultaneously measuring five markers for smoking status. No endogenous or exogenous interference was found. Our method showed excellent precision and accuracy: within-run coefficient of variation (CV) 2.9-9.4%, between-run CV 4.8-8.7% and bias -10.1 to 5.3%. Linear dynamic ranges were 1-10,000 ng/mL for nicotine, nornicotine and anabasine; 2-5,000 ng/mL for cotinine and 5-15,000 ng/mL for 3-OH cotinine. Extraction recovery was consistent (87-109%) across concentrations. No significant matrix effect or carryover was observed. The validated method was applied to 849 urine samples. In samples from the 125 current smokers, nicotine, cotinine, 3-OH cotinine, nornicotine and anabasine were detected in 97.6, 99.2, 98.4, 96.8 and 87.2%, respectively. No markers were detected in 93.9% of 609 nonsmokers. The overlapping detection of multiple markers made it possible to identify the smoking status even in current smokers with a low concentration of cotinine. Our LC-MS-MS method using a simple sample preparation technique is sensitive and effective for screening of smoking status in the general population.