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A gradient reversed phase high performance liquid chromatography (RP-HPLC) method has been developed and validated for the determination for related substances of Dapagliflozin drug substance. Chromatographic separation of Dapagliflozin from its process and degradation related substances was achieved on YMC Pack Pro C18, 250mm × 4.6mm 5 i.e A stainless steel column 250 mm long, 4.6 mm internal diameter filled with Octadecyl silane chemically bonded to porous silica particles of 5 m diameter maintained column oven temperature at 25°C. Orthophosphoric acid buffer is mobile phase A and acetonitrile is mobile phase B. Wavelength for UV detection: 225nm, flow rate: 0.8 ml/min and Injection volume: 20µl. The developed method suitability was checked and validated as per ICH guidelines for specificity, linearity, accuracy, precision, limit of quantification, limit of detection robustness and ruggedness experiments. Dapagliflozin drug substance was subjected to stress conditions of thermal, hydrolysis, humidity, peroxide and photolytic to observe the degradation products. Limit of detection of each RS is less than 0.008%w/w indicating that the developed method is highly sensitive. The experiment results are given in detailed in this research article.

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The interaction of tylophorinidine (TPD) with lysozyme-a model protein-with biological activity, was investigated by determining its fluorescence and by assessing its activity under various conditions. The results indicated that TPD associated with lysozyme at pH 9.2 efficiently with an association constant K,, of 3.3 X lo4 M-' at 26OC. K,, increased with the increasing temperature in the range 26 to 5 5 T ; the calculated enthalpy change AH was found to be 2.3 kcal/mol. Under the same conditions as above TPD also associated with the free amino acid tryptophan with a K,, of 1.7 X lo4 M-' indicating half the efficiency of its association with protein lysozyme. TPD associated lysozyme was less active than the uncomplexed enzyme in the above temperature range although beyond 45OC the inhibition was more significant. The results imply that TPD binds lysozyme outside the cleft region in the temperature range studied here. However, with increasing temperature the cleft region is gradually widened and/or the whole molecule is expanded such that the accommodation of whole or part of the TPD molecule is facilitated leading to the blockage of lytic activity.

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