title: Determination of amino acid neurotransmitters in rat hippocampi by HPLC-UV using NBD-F as a derivative.
abstract: A simple, rapid and accurate high-performance liquid chromatography method with ultraviolet-visible detection was developed for the determination of five amino acid neurotransmitters - aspartate, glutamic acid, glycine, taurine and γ-aminobutyric acid - in rat hippocampi with pre-column derivatization with 4-fluoro-7-nitrobenzofurazan. Several conditions which influenced derivatization and separation, such as pH, temperature, acetonitrile percentage mobile phase and flow rate, were optimized to obtain a suitable protocol for amino acids quantification in samples. The separation of the five neurotransmitter derivatives was performed on a C18 column using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)-acetonitrile (84:16, v/v) at a flow rate of 1.0 mL/min with the column temperature at 30°C. The detection wavelength was 472 nm. Without gradient elution, the five neurotransmitter derivatives were completely separated within 15 min. The linear relation was good in the range from 0.50 to 500 µmol/L, and the correlation coefficients were ≥0.999. Intra-day precision was between 1.8 and 3.2%, and inter-day precision was between 2.4 and 4.7%. The limits of detection (signal-to-noise ratio 3) were from 0.02 to 0.15 µmol/L. The established method was used to determine amino acid neurotransmitters in rat hippocampi with satisfactory recoveries varying from 94.9 to 105.2%.
authors.affiliation: Department of Sanitary Inspection, School of Public Health, Shandong University, Jinan, China.
authors.forename: Xiaomeng
authors.initials: X
authors.lastname: Wu
authors.forename: Rui
authors.initials: R
authors.lastname: Wang
authors.forename: Qingqing
authors.initials: Q
authors.lastname: Jiang
authors.forename: Shue
authors.initials: S
authors.lastname: Wang
authors.forename: Yao
authors.initials: Y
authors.lastname: Yao
authors.forename: Lihua
authors.initials: L
authors.lastname: Shao
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000737:chemistry; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D005456:Fluorescent Dyes / Q000737:chemistry; D006624:Hippocampus / Q000737:chemistry*; D057230:Limit of Detection; D016014:Linear Models; D008297:Male; D018377:Neurotransmitter Agents / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results
chemical_list: D000596:Amino Acids; D005456:Fluorescent Dyes; D018377:Neurotransmitter Agents; C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; D009327:4-Chloro-7-nitrobenzofurazan
keywords: 4-fluoro-7-nitrobenzofurazan (NBD-F); HPLC-UV; amino acid neurotransmitters; derivatization; hippocampi