DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR DETERMINATION OF BROMELAIN, TRYPSIN, RUTOSIDE AND DICLOFENAC IN BULK AND PHARMACEUTICAL DOSAGE FORM

Abstract

Serine proteases are enzymes that cleave peptide bond in proteins in which serine serves as the nucleophilic amino acid at the active site. Tripsin is apancreatic serine protease with substrate specifically based upon positively charged lysine and arginine side chains. The selected dosage form trypsin, rutoside, bromelain contains natural pancreatic serine protease used to treat pain and inflammatory diseases. The objective of present research work is to develop a novel, simple, accurate and precise reversed phase High Performance Liquid Chromatography (HPLC) stability indicating method for rapid and simultaneous quantification of Bromelain, Trypsin, Rutoside and Diclofenac. Since this is a new combination of pharmaceutical dosage form and no HPLC methods were developed .There is a need to develop a chromatographic method which is cost effective. This chromatographic method is developed which can easily analyze mixture of compounds with less retention time and low solvent consumption. The chromatographic separation was achieved on Inertsil ODS (250x4.6mm, 5µ). Mobile phase contained a mixture of OPA buffer at pH 2.4 and Acetonitrile in the ratio of 50:50 v/v, flow rate 1.0 ml/min and UV detection at 257nm. The proposed method shows a good linearity in the concentration range of 22.5-135 µg/ml for Bromelain, 12-72µg/ml of Trypsin, 25-150µg/ml of Rutoside and 12.5-75 µg/ml for Diclofenac under optimised conditions. Precision and recovery study results are in between 98-102%. In the entire robustness conditions %RSD is below 2.0%. Degradation has minimum effect in stress condition and solutions are stable up to 24 hrs. This method is validated for different parameters. The study was determined according to the ICH Q2B guidelines. All the parameters of validation were found to be within the acceptance range of ICH guidelines. The developed chromatographic method can be used in routine analysis in pharmaceutical industry.