A Simultaneous Identification And Quantification Strategy For Determination Of Sulfhydryl-containing Metabolites With LC-MS

Sulfhydryl-containing metabolites are important substances in sulfur metabolism pathway.They have an antioxidant effect and can maintain the balance of the redox state.The changes of sulfhydryl metabolites could disrupt the redox homeostasis and cause the occurrence and development of metabolic disorders and diseases.Due to the instability of sulfhydryl-containing metabolites,the analysis of sulfhydryl-containing metabolites in biological samples is still challenging.By optimizing alkylation reaction,reduction reaction and sample preparation,the rapid derivatization of sulfhydryl-containing metabolites in blood and plasma was achieved for reduced（-SH）and total（-SH,-SS-,S-glutathione protein）analysis.The optimal alkylation reaction conditions are p H 7.4,room temperature,30 m M-NEM and reaction for 30 minutes.The optimal reduction reaction conditions are 4°C,30m M-TCEP,120 m M-NEM and reaction for 45 minutes.A pair of isotope labeling reagents,D₀/D₅-N-ethylmaleimide,were used for simultaneous qualitative and quantitative analysis of sulfhydryl-containing metabolites in normal-and high-fat-diet（NFD and HFD）hamsters.Through qualitative analysis and MS²,the differential sulfhydryl-containing metabolites were found between NFD and HFD in the test samples.In quantitative analysis,the results showed that the difference of glutathione and cysteine on concentration and distribution in blood and plasma of hamsters.The correlation of sulfhydryl-containing metabolites with blood lipid and oxidative stress levels was determined.The results showed that GSH could be considered as a biomarker to reflect the lipid levels and oxidative stress status of HFD hamsters and has a high potential for clinical applications.In summary,the developed method of simultaneous identification with inhouse database and MS² and quantification with standards in one batch provides a promising strategy for the analysis of sulfhydryl-containing metabolites in biological samples,which may promote the in-depth investigation on sulfhydryl-containing metabolites and related diseases.