Determination of enzyme kinetics and glutathione conjugates of chlortetracycline and chloroacetanilides using liquid chromatography–mass spectrometry

Glutathione S-transferases (GSTs) isolated from chlortetracycline (CTC)-treated maize catalyzed the conjugation of glutathione (GSH) with CTC, producing stable conjugates that were structurally characterized using liquid chromatography–ion-trap mass spectrometry (LC–IT-MS). Enzyme-mediated dechlorination of CTC resulted during GSH conjugation as revealed by the mass spectra of the CTC–GSH conjugate, which was characterized by the loss of the chlorine isotopic signature, and shorter chromatographic retention time relative to the chlorinated parent compound. Several fragmentation patterns in the mass spectrum of the CTC–GSH conjugate can be used to verify the identity of the enzyme reaction products. The expected molecular ion [M + H]+ of the CTC–GSH conjugate (m/z 751) with chlorine removal was not observed in the positive electrospray ionization. Instead, a base peak of m/z 677, corresponding to the loss of glycine (MW = 75 Da), was observed. When m/z 677 was subjected to further fragmentation, characteristic peaks corresponding to the loss of glutamic acid (m/z = 129) and water (m/z 18) were observed in the MS/MS spectrum. The catalytic activity of the CTC-induced GST towards dechlorination of chloroacetanilide herbicides (alachlor, metolachlor and propachlor), which are known to be detoxified in plants via the glutathione pathway, was also evaluated in vitro. Glutathione conjugates of chloroacetanilides also showed the losses of m/z 129 and m/z 18 that are characteristic of GSH conjugates when characterized by LC–IT-MS. Interestingly, the sensitivity of LC–IT-MS made it possible, for the first time, to detect chloroacetanilides that are conjugated with two GSH molecules, in addition to the known single GSH conjugates. This research demonstrates a more sensitive and specific method of measuring enzyme reaction products using LC–IT-MS.