Development of UPLC-MS/MS methods for quantitation of glutathione, cysteine, 14,15-epoxyeicosatrienoic acid, and 15-hydroxyeicosatetraenoic acid and application in paraquat poisoning cells

Abstract

Glutathione (GSH) and cysteine are important antioxidants in maintaining the oxidation-reduction balance. 14,15-epoxyeicosatrienoic acid (14,15-EET) and 15-hydroxyeicosatetraenoic acid (15-HETE) are biological active compounds metabolized from arachidonic acid. To dynamically monitor GSH, cysteine, 14,15-EET, and 15-HETE, an ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed. The derivative GSH, cysteine, and underivatized 14,15-EET, 15-HETE were determined by electrospray ionization source with multiple reaction monitoring. The developed method was well validated and applied to monitor the extracellular concentration of GSH, cysteine, 14,15-EET, and 15-HETE in paraquat (PQ) poisoning LO2 cells. It was shown that the derivation of GSH and cysteine is complete and specific. There was a good linear relationship among GSH, cysteine, 14,15-EET, and 15-HETE. The relative standard deviations of precision and accuracy were all within 15%. After PQ poisoning, the extracellular concentrations of GSH and cysteine were increased at 72 h. 14,15-EET and 15-HETE started to increase at 24 h, earlier than GSH and cysteine. Irreversible pathological alterations appeared at 48 h. In conclusion, a fast and specific UPLC-MS/MS method for the determination of GSH, cysteine, 14,15-EET, and 15-HETE has been developed and validated. PQ induced the increase of 14,15-EET and 15-HETE earlier than that of GSH and cysteine in LO2 cells culture medium.

Graphical Abstract

Keywords:

• Cysteine
• 14,15-EET
• glutathione
• 15-HETE
• paraquat
• UPLC-MS/MS

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the fund of Wenzhou Science and Technology Bureau (Y20210106), Major projects of Wenzhou Science and Technology Bureau (ZY2022023), the Health Department of Zhejiang Province (2021KY781), State Administration of Traditional Chinese Medicine and Zhejiang Administration of Traditional Chinese Medicine jointly build a science and technology plan project (GZY-ZJ-KJ-23031) and the Key Specialty of Traditional Chinese Medicine of Zhejiang Provincial in the 13th Five-Year Plan period (Emergency Department).