Development And Effectiveness Evaluation Of Procysteine Structure Mass Spectrometry Reagent For Chiral Resolution Of Amino Compounds

Most of amino acids have chiral centers.A pair of chiral amino acid enantiomers sometimes have significant differences in the biological activity of the human body.For example,the content of D-alanine(D-Ala)in the gastric juice of patients with cancer and Helicobacter pylori infection is significantly high.However,the identification,detection and separation of a pair of chiral amino acids enantiomers is significantly difficult due to the high degree of similarity in the structure.Among many of cliral separation methods,liquid-mass spectrometry(LC-MS)has became an advantageous tool for the analysis of chiral amino acid enantiomers due to its high sensitivity and selectivity.In view of this,the study aims to develop a novel chiral mass spectrometry derivatization reagent with capacity of targeted recognizing amino functional groups and establish a high sensitivity and high selectivity analysis method by ultra-high performance liquid chromatography-mass spectrometry(UHPLC-MS).The procysteine(OTZD)with chiral center and carboxyl functional groups was focused as the parent structure in the study.Firstly,the chiral derivatization reagent TPPP-OTZD was synthesized by reacted triphenylphosphine(TPPP)with procysteine(OTZD).And TPPP-OTZD can identify amino functional group and improve the sensitivity of detection in mass spectrometry due to the existence of a positive charge.Then,a novel chiral mass spectrometry derivatization reagent BC-OTZD was synthesized by reacted a smaller molecular weight benzyl with procysteine(OTZD)in order to product stable characteristic ion fragments.And BC-OTZD have the capacity of targeted recognizing the amino functional group and the ability of chiral separation.The reagent can be analyzed and detected by mass spectrometry accurately and stably due to benzyl group(m/z 91)as its characteristic ion fragment.The chiral separation efficiency of BC-OTZD was evaluated by the detection and separation of 10 kinds of amino acids on a C18 column at 40? with 0.1%formic acid aqueous solution and 0.1%fonnic acid acetonitrile as the mobile phase based on LC-MS.The results showed:10 kinds of amino acids(D,L-Ser,D,L-Ala,D,L-Cys,D,L-Thr,D,L-Tyr,D,L-Val,D,L-Phe,D,L-Leu,D,L-Ile,D,L-His)have achieved completely separation and those resolutions were from 1.86 to 7.84.In addition,5 kinds of amino acids(D.L-Ala,D,L-Tyr,D,L-Val,D,L-Phe,D,L-Leu)with higher content than others in saliva was tested by methodology validation,The results showed that the linearity of the five amino acids was good in the range of 0.25-50 pmol.(R2>0.9990);the detection limit(S/N=3)was between 0.01 and 0.50 pmol,the intra-day and inter-day precisions were 0.21-4.13%and 0.10-6.17%,respectively;The mean recovery of amine acids spiked in saliva was from 86.00 to 102.13%,Finally,the quantitative analysis was performed by saliva collected from 6 healthy volunteers(3 males and 3 females).The results showed that the average content of D,L-Tyr,D,L-Val,D,L-Phe,D,L-Leu and D-Ala in male saliva were almost equal to that in female saliva.The average value of L-Ala in female saliva were slightly greater than that in female saliva.And the average value of L-Ala in male and female saliva were 1.48 ± 1.30 fmol/mL and 2.83 ± 1.44 pmol/mL,respectively.In the study,amino compounds chiral mass spectrometry reagents TPPP-OTZD and BC-OTZD with procysteine as the parent structure may be developed.The reagent showed good chiral resolution ability.An effective mass spectrometry reagent for chiral separation of amino compounds will be provided.A novel method for quantitative analysis of amino chiral metabolites should be established.