Determination of Amino Acids of Novel Food in Food by HPLC Coupled with Pre-Column Derivatization

A method for simultaneous determination of Theanine and γ-aminobutyric acid (GABA), as the novel food of amino acids, which was established using pre-column derivatization and high-performance liquid chromatography (HPLC). 4-Dimethylaminoazobe nzene-4'-sulfonyl chloride (DABS-Cl) is employed as the derivatization reagent with chromophore linked to Theanine and GABA, which lacks chromophore for DAD analysis in its pristine structure. After the detection wavelength was confirmed, the chromatographic and derivatization conditions were also optimized, including the chromatographic column, mobile phases and their gradient, derivatization temperature and time, the additive amount of buffer solution and derivatization reagent. Methodological verification showed that the derivant of Theanine (DABS-Theanine) and derivant of GABA (DABS-GABA) have good linearity in the range of 1-100 μg/mL, with correlation coefficients of 0.9996 and 0.9995, respectively. The recoveries for both amino acids were between 93.95% and 103.90%, with RSDs ranging from 0.99% to 3.93%. The limit of detection (LOD) was 0.6 mg/kg for Theanine and 0.2 mg/kg for GABA. The limit of quantification (LOQ) was 1.7 mg/kg for Theanine and 0.6 mg/kg for GABA. Furthermore, five commercial products containing Theanine or GABA in two matrices (candy and beverage) were analyzed by the proposed method for validation. The average contents of Theanine and GABA were in the range of 307.49-1312.13 mg/kg and 22.98-6744.55 mg/kg, respectively. The developed method features easy sample pretreatment, a simple derivatization system, good separation specificity, good repeatability, accuracy and reliability, and can meet the large-scale determination Theanine and GABA in various food substrates.