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 genoto xic and carcinogenic herbicide. The method developed was based on the obser vation that cleavage of S-cysteinyl alachlor-protein adducts by methanesulf onic acid gave the rearrangement product 3-(2',6'-diethylphenyl)-1,3-thiazo lidine-4-one (TZO). The structure of TZO was confirmed by mass spectroscopy , NMR spectroscopy, and independent synthesis. In the assay, treatment of a lachlor-cysteinyl protein adducts by methanesulfonic acid was followed by e xtraction and analysis. TZO was detected and quantitated by electron-impact GC/MS in the single ion-monitoring mode. [ring-C-13(6)]Alachlor-N-acetylcy steine was added as an internal standard prior to treatment and was convert ed to [ring-C-13(6)]TZO, allowing response factors to be used to quantitate TZO concentrations. Incubations of alachlor (0-1000 mu M) with human album in and bovine serum albumin (BSA) resulted in linear adduct formation with both proteins. Maximal adduction levels of 613-1130 pmol alachlor-albumin a dducts/mg protein were observed, with BSA binding close to twice that of hu man albumin. A linear concentration response of alachlor-Hb adducts was obs erved when whole blood from female CD rats was incubated with alachlor in v itro at concentrations up to 300 mu M. Maximal binding was 1860 pmol alachl or-Hb adducts/mg globin. Male CD rats treated with alachlor at 150 mg/kg bo dy wt/day ip for 0, 1, 2, and 3 days were sacrificed 4 days after final dos ing. A maximal binding of 2250 pmol alachlor-Hb adducts/mg globin was obser ved. This assay provides a new approach for biomonitoring alachlor levels i n experimental animals and has the potential for use in humans.