Journal of Pharmaceutical and Biomedical Analysis Open · Published 2026-02-02 · DOI 10.1016/j.jpbao.2026.100103
Ergothioneine (EGT), a strong natural antioxidant, is a promising healthcare product owing to its anti-aging activity. EGT is found in various mushrooms, and investigations into its quantity and properties, including chirality, are important from a biological perspective. This study aimed to develop an enantioselective analytical method for EGT and histidine (His) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method is developed based on paired enantio-chemical tags, (4-chloro-6-methoxy-1,3,5-triazin-2-yl)-d- and l-leucine, to thiol sites or amine groups. The developed method achieved clear enantioseparation of d- and l‑EGT and d- and l-His with high sensitivity (limits of quantification reached 70–200 fmol). After linearity estimation using a standard reference, the quantification of d- and l-EGT and d- and l-His in 15 mushroom species was performed. The results suggested that the d-EGT and d-His contents were low in all mushroom species tested, whereas sub-milligram-to-milligram-scale l-forms were detected in Pleurotus citrinopileatus, Pleurotus ostreatus, and Pleurotus eryngii var. ferulae species. Although %D was low, the presence of d-EGT in mushroom species was quantified for the first time. Furthermore, docking simulations were performed to investigate the differences in enzymatic interactions between carnitine/organic cation transporter 1 and 3‑mercaptopyruvate sulfurtransferase. The enantiomers of EGT demonstrate different affinities for 3-mercaptopyruvate sulfurtransferase.
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