Study And Application Of D-Glu And D-Ser Separation By Reversed-Phase High Performance Liquid Chromatography In Combination With Chiral Derivatizing Reagent

OBJECTIVE:In the past, people believed the mammal, in vivo, aminoacid existence form mainly was the L-amino acid, by the L- amino acid constituting each kind of active proteins and enzymes which can display physiological actions. Along with analysis technology progress,the presence of some free D-amino acids has recently been shown in various living organisms including mammals, and their functions and origins have gradually been determined. Up to now ,the D-serine, the D-aspartic acid and the D-threonine and so on had been discovered already in some animals. The recent researchment discovered that Alzheimer ' s disease, Parkinsiological disease, Schizophrenia and renal diseases had direct or indirect connection with the D- serine density .Therefore, further exist studies of other D-amino acid members in human have the important and significant meaning. The glutanic acid is one kind excitatory amino acid. The amino acid exists in the brain main center, participated in the excited transmission, and involved the study, ponder, memory, cognition and inference abilitity. Indeed, in vivo , majority amino acid metabolism needs through its special aminotransferase catalysis to produce the glutamic , and then the glutamic discharge in vitro by the L-glutamic dehydrase catalysis to produce the urea . The glutanic acid also is one kind of non- essential amino acids. The glutamic acid also may produce the glucose through the blood transportation form the brain and the muscle. It can be said that the glutamic acid almost take part in all physiological activity in the human body.It is one of the most active amino acids.The serine can arrives the acceptor in the central nervous system and takes part in the information transmission. The D- amino acid function in the central nervous system N- the methyl-D-aspartic acid acceptor, which has extremely important affects in the person normal physiology and in the pathology physiological process such as the accepts ability , the schizophrenia, the nephrosis, the brain blood lack and so on.In this article , through the reverse-phase High performance liquid chromatography ,associated with fluorescence detector. 19 free AA have been separated including the DL-Glu and the DL-Ser . This method used in the examination of the micro amino acid froms as blood serum and urine samples. This method used the gradient elution procedure . In this article, DL-Glu and DL-Ser in the human body blood serum and urine has been separated successfully.Method:This method includes the pre-column derivatization of amino acids with OPTA and AcCys, reversed-phase high-performance liquid chromatography and by the fluorescence detector, This research use flows includes two kind of flow solutions .first one: Chromatograph pure methyl alcohol (A component); second one : 50mmol/L NH4Ac fluid solution(PH 6.0) (B component). gradient elution step: 0-13min A 7% B 93%; 13-40min A 4% B to 96 %; 40-50min A 13% B 87%; 50-70min A 90% B 10%; Ultraviolet wave length 345nm, fluorescence wave length:λex =350nm,λem =450nm. flow speed : 1.0ml/min-1; Column warm: Room temperature; sample size: 20 u L.Results:This method includeing the pre-column derivatization of amino acids with OPTA and AcCys, reversed-phase high-performance liquid chromatography and the fluorescence detector, 19 free amino acide be separated . The glutanic acid two isomers and the serine two isomers successfully have been separated at the meantime . The result showed that the method accuracy is good in the blood serum and urine samples, the coefficient above 0.9990. The D-glutanic acid lowest examination limits is 0.5ng, the D- serine lowestexamination limits is 0.6ng Conclusion:We have established the successed method by the gradient elution RP-HPLC colume associated with OPA and NAC to separate 19 AA. The D- glutanic acid and the D- serine was separated in 40min. By the method ,the D-glutanic acid and the D- serine existence has been detected in the person blood serum. Through the researchment we find there are some correlation between D- glutanic acid and the human body age.