Glucosylation and Glutathione Conjugation of Chlorpyrifos and Fluopyram Metabolites Using Electrochemistry/Mass Spectrometry

Abstract

Xenobiotics and their reactive metabolites are conjugated with native biomolecules such as glutathione and glucoside during phase II metabolism. Toxic metabolites are usually detoxified during this step. On the other hand, these reactive species have a potential health impact by disrupting many enzymatic functions. Thus, it is crucial to understand phase II conjugation reactions of xenobiotics in order to address their fate and possible toxicity mechanisms. Additionally, conventional methods (in vivo and in vitro) have limitation due to matrix complexity and time-consuming. Hence, developing fast and matrix-free alternative method is highly demandable. In this work, oxidative phase I metabolites and reactive species of chlorpyrifos (insecticide) and fluopyram (fungicide) were electrochemically produced by using a boron-doped diamond electrode coupled online to electrospray mass spectrometry (ESI-MS). Reactive species of the substrates were trapped by biomolecules (glutathione and glucoside) and phase II conjugative metabolites were identified using liquid chromatography (LC)-MS/MS, and/or Triple time of flight (TripleTOF)-MS. Glutathione conjugates and glucosylation of chlorpyrifos, trichloropyridinol, oxon, and monohydroxyl fluopyram were identified successfully. Glutathione and glucoside were conjugated with chlorpyrifos, trichloropyridinol, and oxon by losing a neutral HCl. In the case of fluopyram, its monohydroxyl metabolite was actively conjugated with both glutathione and glucoside. In summary, seven bioconjugates of CPF and its metabolites and two bioconjugates of fluopyram metabolites were identified using electrochemistry (EC)/MS for the first time in this work. The work could be used as an alternative approach to identify glutathione and glucosylation conjugation reactions of other organic compounds too. It is important, especially to predict phase II conjugation within a short time and matrix-free environment.

Keywords: EC/MS; bioconjugation; glucosylation; glutathione; oxidative metabolism; pesticide.

Figure 1

Mass voltammograms of CPF (1800→2300 mV, 10 mV/s) and GSH (0 mV) (a) and intensity of possible conjugates (b) measured by EC/MS using BDD in µPrepCell^TM.

Figure 2

Total ion chromatogram (TIC) of GSH and CPF oxidative products in the control (black), in 2100 mV DC potential (red), and in RLM incubates (blue) (a), and extracted ion chromatogram (EIC) of bioconjugates at 2100 mV DC potential (b) using BDD WE recorded by LC-MS/MS on (+) ESI.

Figure 3

Mass spectra of product ion m/z 314 from C3 in MS³ (a) and C6 with its suggested fragmentation measured in MS² (b) using LC-QTRAP MS/MS.

Figure 4

Proposed molecular structures of GSH-conjugates with CPF oxidative products.

Figure 5

Mass voltammograms of CPF oxidative products and Glc after scanning in 1800–2100 mV using EC/MS equipped with BDD WE (a) and EIC chromatograms with and without applied potentials measured by LC-MS/MS (b).

Figure 6

Proposed structures of CPF TPs glucosylation products in online EC/MS.

Figure 7

EIC of FLP oxidative products effluent incubated with GSH (a) or n-Glc (b), and peak area ratios of selected conjugates shown at different applied potentials (c), measured by LC-MS/MS on (+) ESI.

Figure 8

GSH (C10) and glucoside (C11) conjugates of monohydroxyl FLP.