| Thiol compounds can regulate redox balance in cells,and participate in protein synthesis and regulate proliferation,differentiation and apoptosis of cardiomyocytes and pancreatic?-cells.At present,numerous reports for detection techniques of thiol compounds has ignored the importance of chirality,but most endogenous thiol compounds contain one or more chiral centers,and pharmacological and toxicological effects of optical isomers are sometimes completely opposite.Therefore,development of a novel highly sensitive and selective method for enantiomeric separation of chiral endogenous thiol compounds has important significance and clinical value in elucidation of physiological roles of chiral thiols in transsulfurization pathway,methionine and?-glutamyl cycle in vivo,and in-depth exploration of pathogenesis of oxidative stress diseases.Given this,we designed and developed a novel mass spectrometry derivatization reagent,(R)-(5-(3-isothiocyana-topyrrolidin-1-yl)-5-oxopentyl)triphenylphosphonium(NCS-OTPP),with triphenyl-phosphine carrying a permanent positive charge and isothiocyanate structures for diastereomeric separation of chiral thiol compounds.Furthermore,a highly sensitive and selective UHPLC-HRMS method was established for simultaneous determination and chiral separation of 3 kinds of thiol enantiomers based on NCS-OTPP derivatization,and applied it to monitor dynamic changes of chiral thiol compounds in serum,urine and saliva of healthy human at oxidative stress state.(1)Firstly,a novel mass spectrometry derivatization reagent with triphenylphosphonium as the core structure,NCS-OTPP for separation of chiral thiol compounds was developed.The ionization efficiency and detection sensitivity of the reagent can be improved in electrospray mass spectrometer due to the existence of permanent positive charge.Besides,the reagent contains isothiocyanate and a chiral center that can targeted recognize thiol compounds and form a pair of diastereomers,which can achieve good chiral separation on inexpensive reversed-phase chromatography columns.And then,we used 3 kinds of chiral thiols as model to evaluate efficiency of chiral resolution.The results showed that 3 kinds of thol diastereomers achieved completely separation with resolution(Rs)of 1.52-1.88 on YMC Triart C18(2.0×150 mm,1.9?m)column after labeled with NCS-OTPP.In addition,characteristic fragment ions m/z 473.18 and m/z 75.95(R-S=C-S-R')of NCS-OTPP with triphenylphosphonium as parent nucleus and chiral thiols were observed in mass spectrum,respectively,and limit of detection(S/N=3)was 2.4-7.2fmol.(2)A highly sensitive and selective UHPLC-HRMS method for simultaneous detection and chiral separation of 3 thiol enantiomers in human serum was established based on NCS-OTPP derivatization method,and applied it to monitor dynamic changes of chiral thiols in healthy volunteers at normal state(heart rate:81.80±9.07 bpm,blood pressure:79.40±15.10/117.00±19.90 mm Hg)and stress state after strenuous exercise(heart rate:126.40±21.09 bpm,blood pressure:73.90±14.73/128.80±23.16 mm Hg).The results indicated that a good linear relationship was obtained in the range of 0.12-250 pmol(R~2?0.9995),inter-day and intra-day precisions were 0.83-4.06%and 0.95-3.11%,mean recoveries were 83.73-103.35%.Additionally,D-Hcy was discovered in human serum for the first time,and free L-Cys,total(oxidized and reduced)L-Cys and L-Hcy levels increased significantly with changes of oxidative stress state(p<0.05).(3)An UHPLC-HRMS method for simultaneous detection and chiral separation of 3 thiol enantiomers were established,and applied it to investigate dynamic changes of chiral thiols in healthy volunteers at normal state,stress state after strenuous exercise and steady state after restoring calm,meanwhile,fitted curves for dynamic monitoring of chiral thiols in urine and saliva was constructed.D-Hcy was found in urine for the first time,and changes of free and total DL-Hcy was significantly different in urine(p<0.05).Besides,fitted curves for ratio of total D/L-Cys peak areas(y=-0.0035x~2+0.015x+0.0088)and D/L-Hcy peak areas(y=-0.0371x~2+0.1724x-0.0122)were basically consistent with change curves of oxidative stress state after exercise in urine.(4)Development of a method for simultaneous detection and chiral separation of 3 thiol enantiomers in Chinese Korean rice wine and monitoring those dynamic changes before and after drinking in vivo.D-Hcy was found in rice wine for the first time.Moreover,L-Cys levels and ratio of D/L-Hcy peak areas could be used as different markers to identify raw materials of rice wine due to varies significantly with different manufacturers and raw materials(p<0.05).Meanwhile,PCA and PLS-DA analysis of chiral thiol enantiomers can be used to separate and identify different rice wine manufacturers and raw materials.On the other hand,fitted curves for 3 kinds of chiral thiols levels and ratio of D/L-Cys and D/L-Hcy peak areas were in accord with changes of the oxidative stress state after drinking in urine.(5)An UHPLC-HRMS method for simultaneous detection and chiral separation of 3 kinds of thiol enantiomers in serum of patients with acute myocardial infarction(AMI)was develped,and applied it to dynamic monitoring of chiral thiols in AMI patients during treatment period.There were significant differences in down-regulated L-GSH,up-regulated L-Cys and DL-Hcy,ratio of L/D-Cys and L/D-Hcy peak areas in AMI patients and healthy human's serum(p<0.05),and L-Hcy levels gradually decreases with extension of therapy period.In summary,a novel mass spectrometry derivatization reagent for resolution of chiral thiol compounds,NCS-OTPP was developed.Moreover,a highly sensitive and selective UHPLC-HRMS method for simultaneous determination and resolution of 3 chiral thiol enantiomers were established in this research.It was found that ratio of L-Hcy and D/L-Hcy peak areas in serum and urine of healthy human can reflect dynamic changes of 3 types of oxidative stress states including normal state,stressed state and steady state.In addition,fitted curves for ratio of D/L-Cys and D/L-Hcy peak areas in urine were consistent with change curve for 3 kinds of chiral thiol compounds under oxidative stress.Moreover,there are significant differences in ratio of L/D-Cys and L/D-Hcy peak areas in AMI patients and healthy human's serum,and dynamic monitoring for L-Hcy was conducive to evaluate the treatment and recovery states of AMI patients.This work provides a novel mass spectrometry derivatization reagent for targeted identification of chiral thiol compounds and research on metabolic pathways in vivo.Meanwhile,this study also affords a novel idea and approach for early screening and warning of oxidative stress-related diseases. |
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