Simultaneous determination of selenium containing amino acids and their sulfur-analogues in green tea and gynostemma pentaphyllum infusion with high performance liquid chromatography based on fluorescence labeling
Author: Hui-Juan Zhang and Pan-Feng Gao and Xiao-Feng Guo and Hong Wang
In this study, an analytical method using 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene (TMBB-Su) as the labeling reagent has been developed to simultaneously detect Se-methylselenocysteine (SeMeCys), selenomethionine (SeMet), S-methylcysteine (SMeCys) and methionine (Met) in green tea and gynostemma pentaphyllum infusion by high performance liquid chromatography (HPLC) coupled to fluorescence detection. The derivatization of four targeted amino acids proceeded with 80 μmol/L TMBB-Su in 10 mmol/L mixed acid buffer (pH 7.20) at 25°C for 25 min, which was optimized by orthogonal array design. The baseline separation of these amino acid derivatives was obtained on a C8 column with a binary gradient elution using the mobile phase of 20 mmol/L mixed acid buffer solution (pH 6.40) and methanol with 2.5% tetrahydrofuran (THF). Under the optimal derivatization and separation conditions, the detection limit of each targeted amino acid was achieved to 0.2 nmol/L. A good linear relationship in the range of 0.001–1 μmol/L was obtained with a regression coefficient higher than 0.9912 for each targeted amino acids, and the relative standard deviations (RSDs) ranged from 1.8% to 3.5% for within-day determination (n = 6) and from 3.8% to 4.1% for between-day determination (n = 6). The proposed method can be used to detect selenium- and sulfur-containing amino acids simultaneously, compared to the existing HPLC with element-specific detection methods. Recoveries within 94.4–108.3% from tea and gynostemma pentaphyllum samples indicated that the matrix effect could be negligible in sample analysis. SeMeCys, SeMet, SMeCys and Met were all found in selenium enriched green tea and gynostemma pentaphyllum infusion. SeMeCys was also detected in normal green tea infusion with a mean content of 0.98 μg/g.