A validated single-step saliva and serum sample extraction LC-MS/MS method for the analysis of nicotine, cotinine and 3'-hydroxycotinine for clinical vaping studies

Introduction: Quantifying low nicotine and metabolite concentrations in biofluids is challenging due environmental nicotine contamination. However, accurate quantification of low concentrations is crucial for studies on electronic nicotine delivery systems (ENDS) using e-liquids with varying nicotine content. Methods: We developed an LC-MS/MS method to quantify nicotine, cotinine, and 3'-hydroxycotinine (3-OH-cotinine) in serum and saliva for pharmacokinetic (PK) analyses and large studies. Results: For reliable chromatography and to limit bench work, C18 chromatography was used with single-step extraction using methanol and 0.1 M ZnSO4 (4:1, v/v) in serum and 80 % methanol in saliva. Environmental nicotine contamination was addressed through implementation of a C18 delay column, which separated the environmentally abundant nicotine present in the mobile phases from sample nicotine peaks. Total run-time was 6 min and lower limits of quantification were 0.5, 0.25 and 0.5 ng/ml for nicotine, cotinine and 3-OH-cotinine, respectively, in serum and 3, 1 and 2 ng/ml in saliva. The standard curves in both biofluids ranged up to 1000 ng/ml with R-values > 0.995. The within- and between-run accuracy ranged from 97.1 % to 106.9 % with a precision of <= 10.8 %. Cross-validation of serum samples with another laboratory showed good agreement with a bias of 0.56, -3.0 and -6.5 ng/ml for nicotine, cotinine and 3-OH-cotinine, respectively. Conclusions: The integration of a delay column into the LC-MS/MS method mitigated the interference from environmental nicotine and facilitated the quantification of very low nicotine concentrations and two of its major metabolites in saliva and serum. C18 chromatography and single-step sample extraction make the method stable and suitable for large sample loads.