Measurement of S-methylcysteine and S-methyl-mercapturic acid in human urine by alkyl-chloroformate extractive derivatization and isotope-dilution gas chromatography-mass spectrometry

S‐methylcysteine (SMC) is a minor amino acid naturally excreted in human urine, a protective agent against oxidative stress and a biotransformation product of the fumigant biocide methyl bromide and of nicotine. A metabolic source of SMC is catabolism of the repair catalytic protein MGMT (EC 2.1.1.37), which specifically removes the methyl group from the modified DNA nucleotide O‐6‐methyl‐guanine to revert the normal GC base pairing. To assess the value of SMC and of S‐methylmercapturic acid (SMMA) as candidate biomarkers of proliferative phenomena, a sensitive analytical method by GC‐MS was applied in a pilot study of healthy subjects to assess their urinary elimination and the intra‐ and inter‐individual variability. Extractive alkylation with butylchloroformate‐n‐butanol‐pyridine (Husek technique) was employed for sample derivatization and isotope dilution GC‐MS with S‐[CD3]‐SMC and ‐SMMA was applied for specific and sensitive detection. To resolve the target analytes from the main coeluting interferents in the derivatized urine extract a medium‐polarity stationary phase was employed. SMMA was not detected in the morning urine of three healthy fertile‐age women followed for one month above the minimum detectable level of approx. 500 µg/L while SMC concentrations were in the 0.02–0.7 µg/mL range (n = 61) with large inter‐day and inter‐individual variations. In a young healthy male urine samples taken throughout a few days yielded concentrations in the same 90–810 µg/L range (n = 11). These preliminary results points at SMC as a candidate biomarker for the study of methylation turnover in several biochemical processes. Copyright © 2010 John Wiley & Sons, Ltd.