Background: Nicotine is metabolised by cytochrome P450 CYP2A6 to cotinine (COT), then trans-3'-hydroxycotinine (3'-HC), with both primary metabolites undergoing glucuronidation by UDP-glucuronosyltransferase. Genetic differences in CYP2A6 are becoming increasingly recognised as determinants of smoking cessation, but less is known about the importance of UDP-glucuronosyltransferase (UGT2B10 and UGT2B17) genotypes. A simple rapid UPLC-MS/MS assay was developed and used to investigate metabolism phenotypes in a Caucasian population.

Method: Urine samples were diluted 100-fold with water and analysed using a UPLC triple quadrupole mass spectrometer with positive ion electrospray ionisation and multiple reaction monitoring. Levels of urinary nicotine metabolites; COT, 3’-HC and their respective glucuronides were quantified using deuterated internal standards. UDP-glucuronosyltransferase phenotype was assessed in a sample of smokers (n=260) using the proportion of metabolite present as the glucuronide for both COT and 3’-HC.

  Results: The assay demonstrated satisfactory performance (precision RSD 1.6-6.5% at typical biomarker levels with >98% recovery) suitable for application to smoking studies with a run time less than 5 minutes. A correlation was found between nicotine metabolism and glucuronosyltransferase phenotype for COT in both males and females (p<0.0001) and for 3’-HC in females (P<0.0001). 

Conclusion: A rapid and selective UPLC-MS/MS assay was successfully developed and validated for detection and quantification of cotinine, 3'-HC and their glucuronides in urine, suitable for large smoking cessation studies involving nicotine metabolism phenotyping for both CYP2A6 and UDP-glucuronosyltransferase. Nicotine metabolism is correlated with glucuronosyltransferase phenotype suggesting that UGT2B10 /UGT2B17 genotype may also be an important determinant of smoking cessation.