Determination of Alanine, Arginine, and Tyrosine in Black, Oolong, Green and White tea by Derivation with Ortho-Phthalaldehyde Using HPLC-FLD

A simple gradient HPLC method utilise a fluorescence detector (FLD) was developed for highly sensitive, rapid and reliable quantitative determination of Alanine, Arginine, and Tyrosine found mainly in commercial tea types of black, oolong, green, and white. The method depends on the derivation of amino acids, with a mixture of O-Phthalaldehyde and 3-Mercaptopropionic acid reagents. The chromatographic separation was achieved using a reversed-phase Agilent Zorbax Exclipe RP18 (C18), 250 mm × 4.6 mm, 5 μm column depending on the gradient HPLC method employs in two solvents A and B as mobile phase. Solvent A contains a mixture of tetrahydrofuran: acetate buffer pH 7.6 (3:97, v/v), and solvent B contains a mixture of acetate buffer pH 7.6 : acetonitrile : methanol (1:2:2,v/v/v). The flow rate of mobile phase was 1.5 mL/min, The FL detector was set at excitation wavelength (λEx) of 340 nm and emission wavelength (λ Em) of 450 nm. The calculated detection limit LOD for amino acids were very low, in the range of 1.214 – 5.592 nmol/L, with very low limit of quantification LOQ range of 3.680 – 16.945 nmol/L and RSD range 1.05 – 2.87%. The developed RP-HPLC method was validated with respect to accuracy, precision and was shown advantageous over the simpler methodology with highly precision, accuracy, sensitive, and negligible interference of natural present in tea infusion. The proposed method was successfully applied for the separation and determination of Alanine, Arginine, and Tyrosine in eleven tea samples of black, oolong, green and white found in the local market.