| Summary: | Introduction. Popularity of electronic cigarettes (e-cigarettes) has increased dramatically in recent years, especially among adolescents. The most recent data from the National Institute on Drug Abuse (NIDA) cites that >16% of 12th graders have tried e-cigarettes, and >30% of those individuals will start smoking within 6 months1. E-cigarettes are available in a variety of ‘strengths’ indicating the labeled nicotine concentration in the product. In this project, we sought to investigate the accuracy of nicotine labeling found in some commercially available e-liquids. As such, we developed and validated a liquid chromatography-mass spectrometry (LC-MS) assay to determine the nicotine concentration in these products.
Methods. A literature search revealed that the two most commonly used chromatographic approaches for nicotine are hydrophilic interaction liquid chromatography (HILIC) and reversed phase chromatography (RP). Both options were evaluated, and nicotine peak quality and reproducibility were assessed. Mass spectrometric conditions for positive electrospray (+ESI) ionization were optimized, including collision energy and ion accumulation time. The optimized method included a gradient separation using a UCT C18 column (2.1 x 100 mm; 1.8 micron) with acetonitrile as the organic phase and 0.1% formic acid in water as the aqueous phase. Nicotine stock solutions were prepared in 100% ethanol and diluted in acetonitrile to achieve calibration concentrations (5 – 75 micrograms/mL). Deuterium labeled nicotine (d4) was used as the internal standard at a concentration of 10 micrograms/mL. The method was evaluated for precision, reflected by percent relative standard deviation (%RSD) and accuracy, or percent error, at each concentration for three days. The method was applied to the assessment of nicotine concentration in samples of e-liquids labeled as 3 mg/mL nicotine.
Results. Reversed phase chromatography outperformed HILIC separation for nicotine under the conditions tested. The final RP-LC-MS/MS method involves direct monitoring of m/z 163.1219 for quantification of nicotine (167.1219 for d4-nicotine). The method exhibits < 15% RSD and < 15% error for all concentrations in the calibration range (< 20% at the lower limit of quantification). The developed method allows for rapid throughput, with a run time of 5 minutes. The lower limit of detection was determined to be 1 microgram/mL. Of the e-liquids evaluated, variations of up to 37% from the labeled amount of 3 mg/mL were detected. Additionally, a product labeled ‘zero nicotine’ contained no detectable nicotine.
Conclusions. A fast, accurate, and reproducible LC-MS/MS assay has been developed and validated for the determination of nicotine in e-cigarette liquids. This method was applied to the evaluation of e-liquids, which showed significant variability in nicotine content from the labeled amount.
1 https://www.drugabuse.gov/related-topics/trends-statistics/infographics/teens-e-cigarettes
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