Validated HPLC Method to Simultaneously Determine Amprolium Hydrochloride, Sulfaquinoxaline Sodium and Vitamin K3 in A.S.K Powder on ZIC-HILIC Column
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A new HPLC method that is based on zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) coupled with ultraviolet detection was developed, optimized and validated for the simultaneous determination of amprolium hydrochloride, sulfaquinoxaline sodium, and Vitamin K3 (as menadione sodium bisulfite) in A.S.K powder. The separation was carried out using ZIC-HILIC column (250 mm × 4.6 mm, 5 mm) and a mobile phase of 0.2 M Ammonium acetate (NH4AC) buffer and acetonitrile (15:85; v/v) with pH adjusted to 5.7 by glacial acetic acid at a flow rate of 0.5 ml/min. The analytes were monitored by UV detection at 263 nm. The effects of the operational chromatographic conditions on retention and resolution were tested. Different concentrations of the organic solvent in the mobile phase, the ionic strength of the NH4AC buffer and pH of the mobile phase were investigated. The optimized method was subjected to validation by examining specificity, accuracy, precision, linearity, range, ruggedness and robustness. The results were evaluated as per the International Conference on Harmonization (ICH) and United States Pharmacopoeia (USP33/NF28) guidelines and it fulfilled the validation criteria. The method is sensitive, specific, fast, accurate, and requires minimum sample manipulation. It was applied on commercial A.S.K batches, to which all the active ingredients were separated from their excipients.