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Abstract INTRODUCTION:
TSNAs, or tobacco-specific nitrosamines, are carcinogens found in tobacco products, including e-cigarettes and smokeless tobacco. NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol), and NNN (n-nitrosonornicotine) are the most analyzed TSNAs. In laboratory animal studies, NNK has been identified as a cause of lung cancer, a finding also associated with its metabolite, NNAL. Furthermore, NNN has been observed to induce esophageal cancer in these same animal models.
TSNAs can be difficult to accurately detect, as, like nicotine, there can be false positives for secondhand and third-hand contamination. Contamination due to secondhand and thirdhand smoke causes non-smokers to test positive for TSNAs if the detection limits for the method are low. Additionally, due to the ubiquitous nature of nitrosamines, contamination could originate from test tubes, extraction media, pipette tips, or any number of items used throughout the sample extraction and analysis processes.
OBJECTIVES:
This study demonstrates an optimized and streamlined extraction workflow for TSNAs in urine with high analyte recoveries, low matrix effect, and excellent reproducibility.
METHODS:
TSNAs (NNAL, NNK, and NNN) were spiked at 200pg/mL and extracted from non-hydrolyzed, non-smoker human donor urine. Sample extractions were investigated using ISOLUTE® SLE+ (Supported Liquid Extraction) plates comparing extraction performance with different elution solvents. Extraction performance was evaluated by assessing recovery, matrix effect, and reproducibility. Calibration curves were constructed using concentrations ranging from 0.1 to 200 pg/mL of synthetic urine (UriSub, pH 7.4) to avoid signal interferences. Limit of quantifications (LoQs) were determined from the calibration curves for each analyte by applying linear regression model. LC-MS/MS analysis was performed using a Shimadzu Nexera X2 UHPLC system coupled to a SCIEX 5500 QTrap MS system.
RESULTS:
Extraction recovery for TSNAs with ethyl acetate as elution solvent were 91.8%, 92.1%, and 80.7% for NNAL, NNK, and NNN respectively, which shows robustness of the method. Matrix effects for TSNAs were 0.82, 0.92, and 0.87 for NNAL, NNK, and NNN respectively indicating relatively lower matrix suppression for the analytes representing clean sample extract. Other elution solvents such as hexane, dichloromethane,10% isopropanol in dichloromethane showed consistent matrix effect (>0.80) but gave a wide range of extraction recovery (30-104%). Calibration curves for TSNAs from ethyl acetate elution showed good linearity with R2 values > 0.99 for each analyte. LoQs obtained from linear regression were 16.0, 9.0, and 3.0 pg/mL for NNAL, NNK, and NNN respectively.
CONCLUSIONS:
The load-wait-elute extraction procedure utilizing ISOLUTE® SLE+ offers a simple, fast, and automation compatible solution to extract TSNAs for LC-MS/MS analysis with high recovery and less matrix interference. The ability to quantify TSNAs at low pg/mL levels would allow the accurate monitoring of second-hand tobacco exposure and greatly facilitate toxicological studies.
REFERENCES:
1. Smith, C. J.; Hansch, C. The Relative Toxicology of Compounds in Mainstream Cigarette Smoke Condensate. Food Chem. Toxicol. 2000, 38, 637–646.
2. Xia, B., Xia, Y., Wong, J., Nicodemus, K.J., Xu, M., Lee, J., Guillot, T. and Li, J. (2014), Quantitative analysis of five tobacco-specific N-nitrosamines in urine by liquid chromatography–atmospheric pressure ionization tandem mass spectrometry†. Biomed. Chromatogr., 28: 375-384.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
