Objective: The objective of the current study was to develop and validate a simple, robust, precise and accurate RP-HPLC (reverse phase-high performance liquid chromatography) method for the quantitative determination of potential degradation products of Difluprednate (DIFL) in the ophthalmic emulsion.Methods: Chromatographic separation was achieved on the YMC pack ODS-AQ (150× 4.6) mm, 3μm column with a mobile phase containing a gradient mixture of mobile phase A (0.02M Ammonium formate buffer pH 4.5 adjusted with formic acid) and Acetonitrile as mobile phase B, at flow rate of 1.5 ml/min and with UV detection at 240 nm.Results: The peak retention time of DIFL was found at about 17.2 min, the RRT of degradation product-1 (DP-1), degradation product-2 (DP-2), and degradation product-3 (DP-3), were found to be about 0.49, 0.65 and 0.79 respectively (calculated with respect to Difluprednate). Stress testing was performed in accordance with an ICH (international council for harmonisation) guideline Q1A (R2) [1]. The method was validated as per ICH guideline Q2 (R1)[2]. The calibration curve was found to be linear in the concentration range of 0.1 to 0.75 µg/ml for Difluprednate, DP-1, DP-2 and DP-3. The LOD (Limit of detection) was found to be 0.1µg/ml and LOQ (Limit of quantification) of 0.15µg/ml for Difluprednate, DP-1, DP-2 and DP-3 respectively. The recovery from LOQ to 150% was within 90-110%. The forced degradation data confirms the stability indicating the nature of the method.Conclusion: A simple, robust, precise and accurate RP-HPLC method for the quantitative determination of potential degradation products of Difluprednate in the ophthalmic emulsion was developed and validated.

Objective: The objective was to develop and validate the stability indicating reverse-phase high-performance liquid chromatography method for the quantification of potential degradation products of regadenoson (REGA) from its injectable dosage form.Methods: YMC-PAK ODS AQ, 150 mm × 4.6 mm, 3 μm composed with hydrophobic high carbon loading and a relatively hydrophilic surface chemically bonded to porous silica particles column was used with the temperature maintained at 40°C. Mobile phase A composed of 0.1% triethylamine buffer having pH 4.5 while mobile phase B is 100 % acetonitrile was used for gradient elution with 1.5 ml/min as a flow rate. The wavelength used for quantification was 245 nm and 20 μl as an injection volume. The suitability of the method has been checked and validated according to the International Council for Harmonization (ICH) guidelines for different parameters, namely, specificity, linearity, accuracy, precision, limit of quantification (LOQ), Limit of detection (LOQ), and robustness studies.Results: The resolution between REGA and its two-degradation product is >8.0 for all pairs of components. The high correlation coefficient (r2>0.990) values are for drug and all potential degradation products from LOQ to 150% of specification limits for impurities calculated based on the maximum daily dose of REGA. LOQ for the drug as well as each degradation product is <0.02% w/w. The % relative standard deviation (RSD) for precision and intermediate precision is in the range of 0.17–0.89, and % RSD for precision at LOQ is 0.86–2.35. The % RSD for robustness study is maximum 2.59.Conclusion: The developed method can quantify the specified and unknown degradation products from 0.1% in the injectable dosage form which indicates that method is sensitive. Method fulfills the ICH criteria for its different validation parameters and demonstrates that the developed analytical method is highly specific, precise, and robust and would have a great value when applied in quality control and stability studies for REGA injection.