Simultaneous HPLC Determination of Amino Acids in Tea Infusion Coupled to Pre-column Derivatization with 2,4-Dinitrofluorobenzene

Abstract

An HPLC method coupled to pre-column derivatization with 2,4-dinitrofluorobenzene (DNFB) was developed for simultaneous determination of 19 amino acids in tea infusion. The amino acid-derived samples were separated on a reversed-phase C18 column (4.6 × 250 mm) at 35 °C and monitored by an ultraviolet detector at 360 nm. Separation analysis showed a good resolution (Rs > 1.5) for most amino acids with a gradient elution of mobile phases at a flow rate of 1 ml min-1. Precision analysis revealed low repeatability errors with relative standard deviation (RSD) ranging from 0.11 to 1.53 % for retention times and 0.60 to 2.84 % for peak areas, and recovery rate ranged from 70.60 to 108.80 %. Linear calibration curves were well established at the concentrations ranging from 0.57 to 57.38 μg ml−1, with correlation coefficients R 2 ≥ 0.9918 for 19 detected amino acids. The superior stability of the amino acid derivatives at room temperature gave an advantage to simultaneous preparation of many samples and continuous analysis using auto sampler. The established method was successfully applied to separate and quantify amino acids in three tea cultivars, and the results indicated that the albino tea cultivars contained higher contents of total amino acid, theanine, γ-aminobutyric acid (GABA), glutamic acid (Glu), aspartic acid (Asp), and serine (Ser) than normal tea cultivar.