Determination of Free Neurotransmitter Amino Acids in Legume Food by Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry

A method was developed for the determination of neurotransmitter amino acids in legume food by ultra high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS). The samples were ultrasonically extracted with ultra pure water. The target compounds were separated on a Zorbax SB-Aq RRHT column(3.0 mm×100 mm, 1.8 μm) by gradient elution using methanol and 0.5 mmol/L ammonium acetate solution as mobile phase at a flow rate of 0.3 mL/min. Qualification was achieved by positive electro-spray ionization using multiple reaction monitoring(MRM), and the external standard method was used for quantitation. The calibration curves for neurotransmitter amino acids were linear in their corresponding range with their correlation coefficients(r) between 0.991 and 0.999. The limits of detection(LOD, S/N≥3) were 2~8 μg/kg while the limits of quantitation(LOQ, S/N≥10) were 8~40 μg/kg. The recoveries for neurotransmitter amino acids at three different spiked levels were in the range of 80.30%~109.97%, with relative standard deviations(RSD, n=6) of 0.12%~7.20%. The RSDs of stability, precision and reproducibility were 4.74%~6.98%, 0.63%~7.27% and 0.46%~5.17%, respectively. Owing to easy, high efficiency and sensitivity, the method was suitable for rapid analysis of neurotransmitter amino acids in legume food.