HPLC-Fluorescence Determination of Amino Acid-like Neurotransmitters in Plasma from Patients with Alzheimer's Disease

A pre-column derivatization RP-HPLC method was developed for the determination of four amino acid-like neurotransmitters including Glu,Gly,GABA and Asp in the plasma samples obtained from patient with alzheimer's disease(AD) using o-phthalaldehyde as derivatization reagent.The analytes were separated on a Hanbon C18 column by gradient elution using tetrahydrofuran-methanol-0.05 mol/L potassium dihydrogen phosphate(pH 6.0)(2∶35∶63,by volume) as mobile phase A and 90% methanol as mobile phase B.The gradient elution procedure was as follows:100%-40%A at 0-13 min,40%-0%A at 13-14 min,and 100%B at 14-23 min.The signals were detected with a fluorospectrophotometer detector.The calibration curves showed good linearity for four amino acid-like neurotransmitters in the investigated concentration range.The correlation coefficients were between 0.997 5 and 0.999 1.The detection limits of Asp,Glu,GABA and Gly were 0.21,0.01,0.05 and 0.02 pmol/L,respectively.The RSDs of peak areas of the four amino acid-like neurotransmitters were 1.08%,1.24%,1.12% and 1.35%,respectively.The recoveries ranged from 98% to 99%.The method was applied in the determination of Glu,Gly,GABA and Asp in the plasma of AD patients.The result indicated that the levels of Asp and Glu in the plasma of AD patient were significantly higher than those in control group(P0.05),but the level of Gly in AD patient's plasma and the control group had no significant difference.