SEPARATION AND QUANTIFICATION OF PHARMACOLOGICALLY ACTIVE MARKERS p-METHOXY BENZOIC ACID, 3, 4-DIHYDROXYBENZOIC ACID AND GALLIC ACID FROM CAPPARIS SPINOSA AND FROM MARKETED FORMULATION BY HPTLC

A simple, accurate, precise and reproducible High Performance Thin Layer Chromatographic (HPTLC) method for simultaneous quantification of p-methoxybenzoic acid, 3, 4-dihydroxybenzoic acid and Gallic acid in the leaf extract of Capparis spinosa was developed. Chromatography was performed on silica gel 60 F254 pre-coated HPTLC plates with double development using two solvent systems. Using first solvent system, Chloroform: Ethyl acetate: Formic acid: Glacial acetic acid 7.5 : 2 : 0.5 : 0.5 (v/v/v/v) the plate was developed till 80 mm while the second development was carried out till 85 mm using Chloroform: Ethyl acetate: Formic acid: Glacial acetic acid 6 : 2 : 1 : 1 (v/v/v/v) as the mobile phase. After development the HPTLC plate was dried on a hot plate at 40° C for 5 min and scanned at 254 nm. The method was found to give well separated sharp bands of p-methoxybenzoic acid, 3,4-dihydroxybenzoic acid and gallic acid at Rf of 0.87, 0.66, 0.43 respectively. The quantity of p-methoxybenzoic acid, 3, 4- dihydroxybenzoic acid and gallic acid was found to be 0.00666 %, 0.02626 % and 0.01095 %; in plant and 0.01291 %, 0.02423 % and 0.06381 % in formulation respectively. The method was validated in terms of linearity, specificity, precision and recovery. Statistical analysis proved that the proposed method is accurate and reproducible. The developed method can be used as a quality control tool for simultaneous quantification of these markers from raw material as well as marketed formulation. Keywords: Capparis spinosa, p-methoxybenzoic acid, 3, 4-dihydroxybenzoic acid, Gallic acid, HPTLC, simultaneous quantification