Comparison Studies of UPLC and HPLC for Analysis of Tannins from Fruits of Various Species of Terminalia

Terminalia species represent a rich source of tannins and many preparations of these species are used in traditional medicine as a cardiac tonic and diuretic. Different Terminalia species also have different ethno-botanical importance [1]. Terminalia species provide rich sources of secondary metabolites such as cyclic triterpenes and their derivatives, flavonoids, tannins, phenolic acids. The exact chemical classes and levels may vary in different Terminalia species. Gallic acid is the basic unit of hydrolyzable tannins [2–3].

Two new UPLC and HPLC methods were developed for the simultaneous determination of hydrolysable tannins from the fruits of different species of Terminalia (T. chebula, T. arjuna, T. bellirica, T. tomentosa). A separation by LC was achieved by using a reversed phase column, PDA detection, a water/acetonitrile mobile phase, both containing formic acid using a gradient system and a temperature of 40°C. The hydrolysable tannins (gallic acid (1), gallic acid methyl ester (2), corilagin (3), chebulagic acid (4), 1,2,3,4-tetra-O-galloyl-β-D-glucose (5), ellagic acid (6), chebulinic acid (7) and 1,2,3,4,6-penta-O-galloyl-β-D-glucose (8)) could be separated within 13.0 minutes using UPLC method and within 45min using HPLC method. The methods were validated for linearity, repeatability, limits of detection and limits of quantification. The wavelengths used for detection with the diode array detector were 254nm and 275nm. Differences between the techniques are also discussed. The UPLC system reduced the analysis time up to 3-fold compared to that of gradient mode HPLC system using 5µm particle packed analytical columns. Typical chromatograms obtained from final HPLC and UPLC conditions of chemical fingerprint analysis are depicted in Figures 1 and 2. The limits of detection with HPLC method were between 0.1–0.3µg/mL and with UPLC system were in the range 0.05–0.1µg/mL. HPLC analysis requires a long run time for a series of routine analyses. Therefore, modern developments in LC were applied in order to save time and solvent consumption. The sensitivity of UPLC system was good even for such low injection volumes as 2µL. Because UPLC functions according to the same chromatographic principles and separation mechanism of HPLC, method transfer and revalidation were quite easy.

Fig.1: UPLC Chromatograms of a mixture of standards and Fruits of Various Terminalia species

Fig.2: HPLC Chromatograms of a mixture of standards and Fruits of Various Terminalia species

Acknowledgements : This research is supported in part by Science Based Authentication of Dietary Supplements funded by the Food and Drug Administration grant No. 1U01FD004246–01; the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No. 58–6408–2-0009, and the Global Research Network for Medicinal Plants (GRNMP), King Saud University and the authors would like to thank Annette Ford for the extractions of plant samples. References: [1] Kirtikar KR, et al. (2001) Indian Medicinal Plants Oriental Enterprises, 2nd edn, Uttranchal, India, 5: 1415–1439. [2] Ahmad I, et al. (1998)J Ethnopharmacol 62: 183–193. [3] Chattopadhyay RR, et al. (2007) Pharmacog Rev 1: 151–156.