We describe a novel analytical method for quantification of free amino acids in tea using variable mobile phase pH, elution gradient and column temperature of reversed-phase high-performance liquid chromatography (RP-HPLC). The study of mobile phase pH 5.7 was chosen to simultaneous quantification of 19 free amino acids in tea, while it improved maximum resolution of glutamine, histidine and theanine. Elution gradient was adapted for enhancing the solution of free amino acids, mainly because of adjustment of mobile phase A and B. The column temperature of 40 °C was conducive to separate free amino acids in tea. The limit of detection (LOD) and limit of quantitation (LOQ) of this method were in the range of 0.097-0.228 nmol/mL and 0.323-0.761 nmol/mL, respectively. The relative standard deviation of intraday and interday ranged in 0.099-1.909% and 3.231-7.025%, respectively, indicating that the method was reproducible and precise, while recovery ranged between 81.06-112.78%, showing that the method had an acceptable accuracy. This method was applied for the quantification of free amino acids in six types of tea. Multivariate analysis identified serine, glutamine, theanine and leucine as the most influencing factor for classify among analyzed sample.
Keywords: Free amino acids; Glutamine; RP-HPLC; Tea; Theanine.