Design Of Quantitative Analysis Method And The Application Of 3 Kinds Of Amino Acid Enantiomers In Saliva Of Diabetics

The third largest diabetes as a threat to human health disease,low in the early diagnosis of invasive methods should be developed.Saliva is one of important body fluids that can be used as a diagnosis of biological sample,but has not been applied to literature reports of diabetes diagnosis.This study to establish and perfect a can be used for the first time of quantitative amino acid enantiomers in human saliva ultra-high performance liquid chromatography(UPLC)with fluorescence(FL)analysis method.With human saliva as the biological sample,this paper designed and developed D,L-Alanine(Ala),D,L-Valine(Val),D,L-Leucine(Leu)3 D,L-amino acids UPLC-FL method.6-Aminocaproic acid as internal standard(IS).The resolution of free three kinds of D,L-amino acids in human saliva was detected by combination of the R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzo xadiazole[R(-)-DBD-PyNCS]derivatives and UPLC-FL.The reaction of the reagent with D,L-amino acids effectively proceeds at 55? for 20 min in the presence of 3%triethylamine(TEA)to produce the corresponding diastereomers.Each pair of the resulting derivatives was efficiently separated by a gradient elution(a mixture of H2O and CH3CN containing 0.2%formic acid(HCOOH))using a reversed-phase ACQUITY UPLCTM BEH C18(1.7 ?m,100 mm × 2.1 mm i.d.)column and sensitively detected by FL(excitation at around 460 nm,fluoresce at around 550 nm).Within 20 min of 3 D,L-the separation of amino acids(Rs)>1.5,up to the complete separation.Scope inside have good(r2 of 0.9988 or higher);The minimum detection limit of 10.0-200 fmol;Precision intra-day and inter-day assays RSD is not greater than 10.44%;The recovery rate of 95.2-109.6%,all meet the requirements of biological sample determination.This method is accurate,sensitive,reliable,strong specificity and can be used in human saliva of 3 D,L-quantitative amino acids.This paper established the healthy volunteer and diabetics saliva three kinds of amino acids in the clinical diagnosis of application solutions.On the choice of clinical samples,collected 17 cases of healthy volunteer of saliva,10 cases of men and women in 7 cases;A diagnosis index for HbA1C,the subjects' saliva collected 28 cases of diabetics,13 cases of 15 cases of men and women.The in healthy volunteer and diabetics are identified in the saliva of 3 D,L-amino acids.Among the diabetics and healthy volunteer,proportion of Ala is much higher than Val and Leu,and Val is slightly higher than Leu.The compare between diabetic patients and healthy volunteer of male has a significant difference except the D-Ala group(p<0.05),so as the compare between diabetics and healthy volunteer except the D-Leu(p<0.01).As a result,we take the concentrations of L-Ala,D-Val,L,Val,L-Leu in saliva as a new biomarkers to the diagnosis of diabetes.This study through of comparison 3 D,L-amino acid quantitative analysis in human saliva,not only to verify the feasibility in the diagnosis of diabetes,but also provide a new low-invasive methods of early warning of diabetes.