Abstract

A new high performance liquid chromatography (HPLC) method was developed based on an indirect determination of tannase activity through gallic acid measurement using paracetamol as an internal standard. The separation was performed on a C₁₈ column using a mobile phase consisting of aqueous formic acid solution (1%) and methanol (85:15; v/v) pumped at 1 ml min^-1. Samples (10 μl) were injected and signals were detected at 254 nm. Repeatability was 0.81 and 1.28 for peak normalization values of gallic acid (4.16 x 10^-5 M) and internal standard (2.02 x 10^-5 M). A good linearity was shown in the range of 1.04 x 10^-5 to 8.32 x 10^-5 M. Limit of detection (LOD) and limit of quantification (LOQ) values were calculated to be 2.2 x 10^-6 M and 6.6 x 10^-6 M, respectively. The applicability of the method was tested using Penicillium spinulosumwhich produces tannase. The method was highly sensitive, accurate, reliable, and repeatable and at the same time it is applicable to both pure and crude enzyme.

Key words: Tannase activity, gallic acid, tannic acid, high performance liquid chromatography, enzyme activity, validation.