Loughlin A F, Skiles G L, Alberts D W, Schaefer W H
Biochemical and Investigative Toxicology, Department of Safety Assessment, Merck Research Laboratories, WP45A-201, Sumneytown Pike, West Point, PA 19486, USA.
J Pharm Biomed Anal. 2001 Aug;26(1):131-42. doi: 10.1016/s0731-7085(01)00402-2.
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.
开发了一种耐用的液相色谱-串联质谱（LC-MS/MS）方法，用于定量大鼠肝细胞中的还原型谷胱甘肽和氧化型谷胱甘肽（分别为GSH和GSSG）。此外，在同一分析中可以检测、表征和测量GSH缀合物。样品分别用乙腈和碘乙酸处理，以沉淀蛋白质和捕获游离GSH。这些高极性分析物通过离子交换色谱分离，所采用的条件适合电喷雾电离，并能提供基线色谱分辨率。使用总运行时间为13分钟的溶剂梯度洗脱分析物以及样品中任何保留时间长的组分，否则这些组分将积聚在HPLC柱上并使色谱性能下降。使用离子阱质谱仪通过选择离子监测（SIM）或使用三重四极杆质谱仪通过选择反应监测（SRM）来检测分析物。使用离子阱对GSH和GSSG进行定量的范围分别为0.651-488微摩尔和0.817-327微摩尔。使用三重四极杆仪器进行SRM时，GSH和GSSG的定量范围分别为0.163-163微摩尔和0.0816-81.6微摩尔。两种方法的准确度和精密度均在15%以内。通过用模型化合物甲萘醌和早熟素I处理大鼠肝细胞，证明了该方法的实用性。甲萘醌含有一个醌部分，可进行氧化还原循环并在肝细胞中诱导浓度和时间依赖性氧化应激，导致GSH浓度降低，同时GSSG浓度升高，以及形成GSH-甲萘醌缀合物。当肝细胞与早熟素I孵育时，观察到GSH浓度随时间下降，同时GSH-早熟素缀合物增加。在早熟素I存在下，GSSG浓度没有增加，这与其缺乏氧化还原活性一致。这种分析方法对于同时研究受试化合物在体外诱导氧化还原循环产生氧化应激以及形成GSH缀合物的潜力具有普遍用途。
