Glutathione is a ubiquitous peptide that traps reactive compounds (mercaptan reactions) and reduces oxidants (glutathione dimers) to prevent damage to important proteins and nucleic acids. The determination of active metabolites based on the reaction of glutathione with active substances has been developed. Glutathione is added to in vitro metabolic stability methods using microsomes or liver cells to provide metabolic CYP enzymes (see Chapter 29). Thiol groups of glutathione react at metabolically activated sites of drugs. The glutathione and n-acetylcysteine adducts were extracted and analyzed by liquid chromatography-mass spectrometry (LC/MS/MS). Adducts are detected as their molecular ions or using a specific neutral loss (NL) MS/MS mode (for example, NL 129 in positive ion mode and NL 272 in negative ion mode). This method can be used to screen candidates without synthesizing radioactive markers. MS/MS and nuclear magnetic resonance (NMR) spectra were used to elucidate the glutathione binding sites to drug candidates.

This method should be validated internally and the results should be confirmed using alternative methods as it may produce false positives. Endogenous compounds can also cause false positives. Alternative approaches include the use of isomolar stably labeled glutathione peptide analogs to produce double MS ions that are clearly visible in MS data and the use of cyanide as a collector. Advanced mass spectrometry scanning and techniques (e.g., quality defects, data correlation experiments, high resolution MS) can be used to further accelerate analysis and provide fewer false positives and quantitative reaction products.

Glutathione, also known as GSH, is an endogenous component of cell metabolism and is a tripeptide composed of glycine, cysteine, and glutamate. It is usually present in the liver at a concentration of 10 mmol L-1. It is an integral part of the bioconversion of isobiomass to protect the body from reducing agents. Glutathione binding (facilitated by the glutathione transferase family) contributes to detoxification by binding electrophiles that may bind to proteins or nucleic acids, resulting in cell damage and genetic mutations. The existence of truly specific binding sites for GSH in the central nervous system has been reported, which meets the main requirement for treating GSH as a neurotransmitter, among other functions.