Analysis of bile acid glutathione thioesters by liquid chromatography/electrospray ionization–tandem mass spectrometry

Abstract

The formation of thioester-linked glutathione (GSH) conjugates of bile acids (BAs) is presumed to occur via trans-acylation reactions between GSH and reactive acyl-linked metabolites of BAs. The present study examines the chemical reactivity of cholyl-adenylate and cholyl-CoA thioester, acyl-linked metabolites of cholic acid (CA), with GSH to form CA-GSH conjugate in vitro. The authentic specimen of CA-GSH was synthesized along with GSH conjugates of four common BAs found in the human body. Their structures were confirmed by proton-nuclear magnetic resonance spectroscopy and electrospray ionization (ESI)–tandem mass spectrometry in positive- and negative-ion modes. Incubation of cholyl-adenylate or cholyl-CoA thioester with GSH was carried out at pH 7.5 and 37 °C for 30 min, with analysis of the reaction mixture by liquid chromatography/ESI–tandem mass spectrometry, where CA-GSH was detected on the product ion mass chromatograms monitored with stable and abundant dehydrated positive-ion [ M + H H 2O] + at m/ z 680.3 and fragmented negative-ion [GSH H] − at m/ z 306.0, and was definitely identified by CID spectra by comparison with those of the authentic sample. The results show that both cholyl-adenylate and cholyl-CoA thioester are able to acylate GSH in vitro.