Development Of An Improved Isocratic Hplc Method For The Determination Of Gallic Acid, Caffeine And Catechins In Tea

ABSTRACT

Polyphenols are the primary compounds responsible for the health benefits of tea,

including its antioxidant and anti-inflammatory properties. Caffeine contributes to tea’s

stimulant properties, while gallic acid and polyphenols contributes to its antioxidant

properties. Most of the High Performance Liquid Chromatography (HPLC) methods used for

the determination of tea biochemicals include gradient elution systems which involve

expensive instrumentation. The aim of this study was to develop an improved sensitive, fast,

cost effective and accurate isocratic HPLC method with photo diode array (PDA) detection

for analysis of gallic acid, caffeine and catechins in tea (Camellia sinensis), using a suitable

internal standard. The developed HPLC analytical method consisted of a C6-phenyl column

(4.6 x 150 x 5μm) and an isocratic elution system of water-acetonitrile-methanol-ortho

phosphoric acid-ethyl acetate (77.5:18:2.0:0.5:2.0 v/v/v/v/v) at a flow rate of 1.0 mL/min.

The detection was done using a PDA detector at a wavelength of 278 nm. This resulted in an

excellent chromatographic separation of the catechins, gallic acid and caffeine in tea in the

presence of guaiacol (2-methoxyphenol) internal standard, which minimized sample matrix

effect and instrumental fluctuations. The total analysis time in the new method was 12.5 min,

more than three times faster than the ISO 14502-2:2005(E) method. The analytical results

obtained for gallic acid, caffeine and catechins in four tea types–green Cut, Tear and Curl

(CTC), black CTC, green orthodox and black orthodox–using this developed method and ISO

14502-2:2005(E) method showed that there was no significant statistical difference between

the two methods. In the CTC and orthodox green tea types the CV was in the range of 2.9 –

14.8 while in the CTC and orthodox black tea types the CV was in the range of 3.7 – 21.4

when comparing the individual catechins, gallic acid and caffeine determined by the two

methods. The method was validated against the Food and Drug Administration (FDA, 2013)

guidelines and proved that the method is efficient, simple and fast for qualitative and

quantitative determination of the biomolecules of interest in tea samples. This method

produced excellent accuracy and precision. Within run precision was less than 2.18 %.

Therefore, the results generated by this developed method can be adequately used as

complimentary alternative to the subjective organoleptic determinations mostly used in the tea industry to determine quality and prices of te