Quantitative analysis of alachlor protein adducts by gas chromatography-mass spectrometry

This study examined the potential use of hemoglobin (Hb)- and serum-protein adducts of alachlor as potential biomarkers of alachlor exposure, a genotoxic and carcinogenic herbicide. The method developed was based on the observation that cleavage of S-cysteinyl alachlor-protein adducts by methanesulfonic acid gave the rearrangement product 3-(2',6'-diethylphenyl)-1, 3-thiazolidine-4-one (TZO). The structure of TZO was confirmed by mass spectroscopy, NMR spectroscopy, and independent synthesis. In the assay, treatment of alachlor-cysteinyl protein adducts by methanesulfonic acid was followed by extraction and analysis. TZO was detected and quantitated by electron-impact GC/MS in the single ion-monitoring mode. [ring-13C6]Alachlor-N-acetylcysteine was added as an internal standard prior to treatment and was converted to [ring-13C6]TZO, allowing response factors to be used to quantitate TZO concentrations. Incubations of alachlor (0-1000 microM) with human albumin and bovine serum albumin (BSA) resulted in linear adduct formation with both proteins. Maximal adduction levels of 613-1130 pmol alachlor-albumin adducts/mg protein were observed, with BSA binding close to twice that of human albumin. A linear concentration response of alachlor-Hb adducts was observed when whole blood from female CD rats was incubated with alachlor in vitro at concentrations up to 300 microM. Maximal binding was 1860 pmol alachlor-Hb adducts/mg globin. Male CD rats treated with alachlor at 150 mg/kg body wt/day ip for 0, 1, 2, and 3 days were sacrificed 4 days after final dosing. A maximal binding of 2250 pmol alachlor-Hb adducts/mg globin was observed. This assay provides a new approach for biomonitoring alachlor levels in experimental animals and has the potential for use in humans.