Simultaneous Determination of Enrofloxacin, Silver Sulfadiazine, Hydrocortisone Acetate, Hydrocortisone Sodium Succinate, and Preservative Excipients in Pharmaceutical Preparations Using HPLC–DAD Method

Abstract

An HPLC–DAD method for simultaneous determination of enrofloxacin (ENR), silver sulfadiazine (SSD), hydrocortisone acetate (HAC), hydrocortisone sodium succinate (HSS), and preservative excipients common in liquid pharmaceutical forms was developed and validated. Dionex® chromatograph and C18 column were used at room temperature. The mobile phase was acetonitrile and water mixture (48:52 v/v) at pH 3.0. Isocratic elution mode was used at flow rate of 1.0 mL min−1 and wavelength detections were performed at 240, 255, 270, and 280 nm. The method was validated by following international guidelines for evaluating selectivity, linearity, accuracy, precision, robustness, and limits of quantification and detection. Symmetrical chromatograph peaks were obtained in less than 6 min. All four calibration curves were linear. Relative standard deviation of drug contents was less than 2.0% with respect to intermediate precision and repeatability. Mean recoveries with respect to accuracy were between 98 and 102% for all four drugs. The limits of detection and quantification were, respectively, 0.024 and 0.072 μg mL−1 for ENR, 0.035 and 0.105 μg mL−1 for SSD, 0.014 and 0.042 μg mL−1 for HAC, and 0.016 and 0.047 μg mL−1 for HSS. Robustness of method was evaluated by Plackett–Burman’s test, showing the method was significantly affected for only one of the 14 parameter variations. Using mass spectrometry, HSS hydrolysis degradation product (i.e., free hydrocortisone) was identified both in standard chemical reference and in commercial formulation. In addition, the proposed method also was able to separate all drug chromatography peaks of one degradation product and two preservative excipients.