Rapid method for glutathione quantitation using high-performance liquid chromatography with coulometric...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Cell Biology

Background:

• Reduced glutathione (GSH) is a critical endogenous antioxidant.
• Accurate quantification of GSH is essential for understanding cellular redox homeostasis and disease mechanisms.
• Existing methods for GSH detection can be time-consuming or require derivatization.

Purpose of the Study:

• To develop and validate a rapid, sensitive, and direct high-performance liquid chromatography with electrochemical detection (HPLC-ECD) method for reduced glutathione (GSH) detection.
• To assess the method's applicability for quantifying other compounds involved in GSH metabolism.
• To determine GSH levels in HepG2 cells under conditions of inhibited and induced synthesis.

Main Methods:

• Development of an isocratic HPLC separation using a fused-core column and sodium phosphate mobile phase.
• Validation of the HPLC-ECD method according to FDA guidelines, assessing linearity, LOQ, LOD, precision, accuracy, and recovery.
• Application of the method to quantify GSH in HepG2 cells treated with buthionine sulfoximine (BSO) and α-lipoic acid (α-LA).

Main Results:

• The developed HPLC-ECD method provides a total analysis time of 5 minutes with a GSH retention time of 1.78 minutes.
• The method demonstrated excellent linearity (R(2) >0.99) between 0.01 and 80 μmol/L, with an LOD of 15 fmol.
• Intra- and interday recoveries ranged from 100.7% to 104.6%, indicating high accuracy and precision.
• The method successfully detected GSH, uric acid, ascorbic acid, and glutathione disulfide in HepG2 cells.
• BSO treatment led to a concentration-dependent decrease in GSH, while α-LA treatment resulted in a concentration-dependent increase.

Conclusions:

• The validated HPLC-ECD method is rapid, sensitive, and direct for quantifying GSH in cultured hepatocytes.
• This method is suitable for analyzing other key metabolites in GSH pathways, such as uric acid and ascorbic acid.
• The developed assay is valuable for studying GSH metabolism and redox balance in various biological matrices, including cell cultures and potentially tissue homogenates and blood.