Enantio-selective analytical method for ergothioneine using chemical tagging coupled with LC-MS/MS and D/L ratio measurements in mushrooms

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. • Enantioselective analytical methods for ergothioneine (EGT) and the precursor His • A novel enantio-chemical tag, CMT-L-Leu, was used • High-sensitivity enantio-separation of D, L-EGT and D, L-His achieved • D, L-EGT and D, L-His quantified in 15 mushroom species • The simulated affinity of D, L-EGT for functional proteins is potentially different