Simultaneous determination of caffeine, gallic acid, theanine, (-)-epigallocatechin and (-)-epigallocatechin-3-gallate in green tea using quantitative ¹H-NMR spectroscopy

Green tea consists of the dried leaves of Camellia sinensis, and enjoys great popularity all over the world due to its pleasant taste and positive impact on human health. It has also been regarded as a natural medicine containing great amounts of caffeine (CA), gallic acid (GA), theanine (TH), and tea polyphenols, mainly including epicatechin (EC), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epigallocatechin-3-gallate (EGCG). In the present study, ¹H-NMR spectroscopy is utilized for chemical characterization along with simultaneous determination of CA, GA, TH, EGC and EGCG in commercial green tea. Signal assignment for representative samples was facilitated by reference compounds and comparing with information in the literature. On the other side, the diagnostic singlet signals at δ 7.68, 7.14, 6.59 and 6.62 in the ¹H-NMR spectra were selected as quantitative peaks for CA, GA, EGC and EGCG, respectively, while the triplet signal at δ 1.12 with a coupling constant (J) of 7.26 Hz was chosen for the determination of TH. TSP-d₄ was adopted as the internal standard (IS) and the reference chemical shift of δ 0.00. The limits of detection (LODs) and limits of quantitation (LOQs) were measured as 28.9 and 57.8 μg mL^-1 for CA, 18.7 and 37.4 μg mL^-1 for GA, 23.4 and 46.8 μg mL^-1 for TH, 28.1 and 56.2 μg mL^-1 for EGC, and 28.1 and 56.2 μg mL^-1 for EGCG, respectively. The relative standard deviation of both precision and repeatability assays were lower than 4.5%. The mean recoveries of high, medium and low concentration levels for each analyte were among the range of 93.1-106.3%. The contents of CA, GA, TH, EGC and EGCG were measured among the ranges of 3.72-8.38 mg g^-1, 0.34-1.88 mg g^-1, 1.56-4.48 mg g^-1, 2.96-8.50 mg g^-1 and 2.78-10.60 mg g^-1, respectively, in 9 batches of green tea. Above all, ¹H-NMR spectroscopy is proven as a reliable tool not only for metabolic characterization, but also for simultaneous determination of effective components in green tea.