Resolution and quantitation of mercapturic acids derived from crotonaldehyde, methacrolein, and methyl vinyl ketone in the urine of smokers and non-smokers

Using improved HPLC analysis conditions, we report the separation of 3 isomers of mercapturic acid conjugates previously assigned in the literature only to 3-hydroxy-1-methylpropylmercapturic acid (HMPMA-1), a human urinary metabolite of crotonaldehyde. The new conditions, employing a biphenyl column cooled to 5 °C and eluted with a gradient of formic acid, acetonitrile, and methanol allow the analysis of human urinary mercapturic acids derived not only from crotonaldehyde, but also from its isomers methacrolein (3-hydroxy-2-methylpropyl mercapturic acid, HMPMA-2) and methyl vinyl ketone (3-hydroxy-3-methylpropyl mercapturic acid, HMPMA-3). The mercapturic acids were detected and quantified by LC-ESI-MS/MS using the corresponding stable isotope labelled mercapturic acids as internal standards. The analysis was validated for accuracy and precision and applied to urine samples collected from cigarette smokers and non-smokers. Smokers had significantly higher levels of all three mercapturic acids than did no...