Simultaneous quantitative determination of microcystin-LR and its glutathione metabolites in rat liver by liquid chromatography–tandem mass spectrometry

• Development of a LC/MS/MS method for the analysis of MC-LR and its GSH metabolites in rat liver.
• Pretreatment conditions were optimized to extract analytes efficiently.
• Combined use of SPE columns increases analyte purity when isolated from a complex matrix.
• This method has been validated with excellent results.
• Time- and dosage-effect studies were conducted using rat liver.

The roles of glutathione (GSH) and cysteine (Cys) in the detoxification of Microcystin-LR (MC-LR) have recently become a popular area of research. However, lacking analysis methods for MC-LR-GSH and MC-LR-Cys (two main GSH pathway metabolites) in mammals, elucidation of the detoxification mechanism and metabolic pathway of MC-LR in mammals is difficult. In this study, a novel method for the simultaneous quantitative analysis of MC-LR, MC-LR-GSH and MC-LR-Cys in rat liver was developed and validated. The analytes were simultaneously extracted from rat liver using 3 M sodium chloride solution containing 0.01 M EDTA-Na2-5% acetic acid, followed by solid-phase extraction (SPE) on Oasis HLB and silica cartridges and determination by liquid chromatography–electrospray ionization mass spectrometry (LC–ESI–MS/MS). Under the optimized pretreatment conditions and instrument parameters, good recoveries of MC-LR, MC-LR-GSH and MC-LR-Cys were obtained at three concentrations (0.2, 1.0 and 2.5 μg g−1 dry weight (DW)) with values ranging from 97.7 ± 4.2 to 98.7 ± 5.1%, 70.1 ± 4.8 to 71.1 ± 4.1% and 79.8 ± 3.5 to 81.4 ± 4.0%, respectively. The relative standard deviations (RSDs) of these compounds at 0.2, 1.0 and 2.5 μg g−1 DW were between 4.3% and 6.9%. The limits of detection (LODs) were 0.005, 0.007 and 0.006 μg g−1 DW and the limits of quantification (LOQs) were 0.017, 0.023 and 0.020 μg g−1 DW for MC-LR, MC-LR-GSH and MC-LR-Cys, respectively. Furthermore, this method was successfully applied to both time- and dosage-effect studies of MC-LR, MC-LR-GSH and MC-LR-Cys in vivo.