Liquid Chromatography–ElectrosprayIonization–TandemMass Spectrometry Quantitation of Urinary Pyridine-D44-hydroxy-4-(3-pyridyl)butanoic Acid a Biomarker of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanoneMetabolic Activation in Smokers

摘要

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, >1) is a potent tobacco-specific lung carcinogen believed to play a key role in the development of lung cancer in smokers. Metabolic activation of NNK to DNA damaging reactive intermediates proceeds via α-hydroxylation pathways. The end products of these pathways are excreted in the urine of smokers as 4-oxo-4-(3-pyridyl)butanoic acid (keto acid, >3) and 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid, >4). The sum of these biomarkers (after NaBH4 treatment), referred to as total hydroxy acid, could potentially be used to measure the extent of NNK metabolic activation in smokers. However, the same metabolites are formed from nicotine; therefore, there is a need to distinguish the NNK- and nicotine-derived keto and hydroxy acid in smokers’ urine. We previously developed a unique methodology based on the use of [pyridine-D4]NNK ([D4]>1), which metabolizes to the correspondingly labeled biomarkers. In this study, we developed a sensitive and reproducible assay for the detection and quantitation of total [pyridine-D4]hydroxy acid ([D4]>4) in human urine. A two-step derivatization approach was used to convert [D4]>4 to [pyridine-D4]methyl 4-hexanoyl-4-(3-pyridyl)butanoate ([D4]>6), and an LC-ESI-MS/MS method was developed for the analysisof this derivative with excellent sensitivity, accuracy, and precision.The robustness and reproducibility of the assay was further confirmedby its application for the analysis of urine samples from 87 smokerswho smoked [D4]>1-containing cigarettes for1 week. The measured level averaged 130 fmol/mL urine. The developedassay can be used in future studies that may require evaluation ofthe relative efficiency of NNK metabolic activation in humans.