title: An ion exchange liquid chromatography/mass spectrometry method for the determination of reduced and oxidized glutathione and glutathione conjugates in hepatocytes.
abstract: A rugged LC-MS/MS method was developed to quantify reduced and oxidized glutathione (GSH and GSSG, respectively) in rat hepatocytes. In addition, GSH conjugates can be detected, characterized and measured in the same analysis. Samples were treated with acetonitrile and iodoacetic acid to precipitate proteins and trap free GSH, respectively. These highly polar analytes were separated by ion exchange chromatography using conditions that were developed to be amenable to electrospray ionization and provide baseline chromatographic resolution. A solvent gradient with a total run time of 13 min was used to elute the analytes, as well as any highly retained components in the samples that would otherwise accumulate on the HPLC column and degrade the chromatography. The analytes were detected using either selected ion monitoring (SIM) using an ion trap mass spectrometer or selected reaction monitoring (SRM) using a triple quadrupole mass spectrometer. The ranges for quantification of GSH and GSSG using an ion trap were 0.651-488 microM and 0.817-327 microM, respectively. Using SRM with the triple quadrupole instrument, the ranges of quantification for GSH and GSSG were 0.163-163 microM and 0.0816-81.6 microM, respectively. The accuracy and precision for both methods were within 15%. The utility of the method was demonstrated by treating rat hepatocytes with model compounds menadione and precocene I. Menadione, which contains a quinone moiety that undergoes redox cycling and induces concentration- and time-dependent oxidative stress in hepatocytes, resulted in decreased GSH concentrations with concomitant increase in concentrations of GSSG, as well as a GSH-menadione conjugate. When hepatocytes were incubated with precocene I, a time-dependent decrease in GSH concentrations was observed with concomitant increase in a GSH-precocene conjugate. GSSG concentrations did not increase in the presence of precocene I, consistent with its lack of redox activity. This analytical method has general utility for simultaneously investigating the potential of test compounds to induce both oxidative stress from redox cycling in vitro and the formation of GSH conjugates.
authors.affiliation: Biochemical and Investigative Toxicology, Department of Safety Assessment, Merck Research Laboratories, WP45A-201, Sumneytown Pike, West Point, PA 19486, USA.
authors.forename: A F
authors.initials: AF
authors.lastname: Loughlin
authors.forename: G L
authors.initials: GL
authors.lastname: Skiles
authors.forename: D W
authors.initials: DW
authors.lastname: Alberts
authors.forename: W H
authors.initials: WH
authors.lastname: Schaefer
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005978:Glutathione / Q000032:analysis*; D019803:Glutathione Disulfide / Q000032:analysis*; D008099:Liver / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D051381:Rats; D014812:Vitamin K / Q000378:metabolism
chemical_list: D014812:Vitamin K; D005978:Glutathione; D019803:Glutathione Disulfide