Simple, fast and sensitive LC-MS/MS analysis for the simultaneous quantification of nicotine and 10 of its major metabolites

Urinary determination of nicotine metabolites provides an ideal tool for the quantitative assessment of the tobacco use-related nicotine dose, provided that the considered metabolites comprise a large share of the amount taken up. A method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the sensitive, fast and robust analysis of nicotine and 10 major nicotine metabolites (“Nic+10”), including cotinine, trans-3′-hydroxy-cotinine, nicotine-N-glucuronide, cotinine-N-glucuronide, trans-3′-hydroxy-cotinine-O-glucuronide, nornicotine, norcotinine, nicotine-N′-oxide, cotinine-N′-oxide and 4-hydroxy-(3-pyridyl)-butanoic acid. Corresponding deuterated internal standards were spiked prior to a simple and straightforward solid phase extraction (SPE) procedure. Liquid chromatography was performed on a reversed phase C8 column and mass-specific detection was conducted in scheduled-MRM mode. The method was validated according to FDA Guidelines, showing excellent selectivity, precision, accuracy and robustness. The limits of quantification were in the range 0.2-2.3 ng/ml for all analytes. The novel method was applied to human urine samples derived from 25 smoking subjects. Quantitative results were correlated against a previously used LC-MS/MS method and compared to reports from the literature. The relative molar profile of nicotine and its 10 major metabolites was in good agreement with the literature. In addition, correlation amongst the two methods was excellent for almost all analytes, whereas the accordance between both methods was moderate for hydroxy-cotinine-O-glucuronide and norcotinine. These deviations, however, could be explained. The current method allows the simultaneous determination of nicotine and its 10 major metabolites (metabolite coverage about 95% of the absorbed dose) from a small sample volume and within a reasonable amount of time. Due to its wide dynamic range, high sensitivity and high throughput capabilities, this method could serve as a powerful tool for quantifying the nicotine dose of smokers, passive smokers as well as novel tobacco and nicotine product users in clinical and epidemiological studies.