Method development of high-performance liquid chromatography and mass spectrometry for the analysis of glutathione metabolism and acetaminophen oxidation metabolites with applications in toxicology

Abstract

The dissertation focuses on the development and validation of a highly selective and sensitive high‑performance liquid chromatography method coupled with tandem mass spectrometry for the quantification of glutathione and more than 20 related metabolites in cellular samples. The separation is based on hydrophilic interaction chromatography using a zwitterionic column. The method enables sensitive and selective analysis without the need for derivatization, while the use of 5‑sulfosalicylic acid ensures efficient protein precipitation and the stability of thiol compounds. Method validation demonstrated precision ≤ 5 % and accuracy ≤ 15 %. Application to A549 cancer cell lines revealed significant alterations in glutathione metabolism after exposure to cadmium chloride and cisplatin, which induce oxidative stress. The results contribute to a better understanding of antioxidant defense mechanisms and the ability to monitor cellular status under chemical stress conditions. The second part of the dissertation focuses on the development and validation of a high‑performance liquid chromatography method with UV and mass spectrometric detection for the quantification of oxidative metabolites of acetaminophen. The analysis includes acetaminophen conjugates with glutathione, cysteine, and N‑acetylcysteine, which play a key role in the mechanisms of hepatotoxicity during overdose. Preparative liquid chromatography was used to isolate these conjugates with a purity of ≥ 99 % for subsequent applications. The optimized chromatographic separation enabled selective and sensitive analysis within 6.9 minutes. Validation confirmed high precision and accuracy of the method. Application to HK‑2 cell lines showed a predominance of the cysteine conjugate and almost complete acetaminophen metabolism after 24 hours. The developed method represents a reliable analytical tool for studying acetaminophen metabolism and toxicity.