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Enhancing sensitivity of phenol-sulfuric acid assay using orthophosphoric acid as modifier

Background Phenol-sulfuric acid assay has been widely used for quantification of sugars in biological fluid and industry. The conventional method originally proposed by Dubois et al, was modified several times for enhancing sensitivity of assay such as substituting phenol with other chromogens, by optimizing assay conditions and by adding phenol after dehydration reaction. Both conventional and modified assays have utilized acid catalysis property of sulphuric acid but effect of adding phosphoric acid has not been studied before.

Methods The present study was conducted in Department of Biochemistry, ANIIMS, Port Blair and IGIMS, Patna. The method being developed in our study consisted of adding orthophosphoric acid to the reaction mixture before addition of sulphuric acid. The method was optimized with different amounts of orthophosphoric acid. Statistical analysis was done using SPSS and Microsoft Excel software.

Results The current study found an enhancing effect (on sensitivity) of orthophosphoric acid in optimal concentration of 5.16 mmoles. Comparison among standard curves of methods that were compared showed that the curve was steepest for current study and average absorbance was 0.199±0.017 for conventional, 0.253±0.011 for method by Rasouli et al, and 0.290±0.013 for current study. Pooled serum analysis exhibited absorbance of 0.157±0.015 in conventional method while in modified conventional method absorbance was 0.234±0.010 and highest absorbance was observed in current study at 0.281±0.012.

Conclusions Our results suggest that orthophosphoric acid exerts a positively modifying effect on phenol sulphuric acid assay.