ISOCRATIC HPLC METHOD DEVELOPMENT AND VALIDATION TO ASSESS THE HOMOGENEITY OF POWDERED BOAR SEMEN EXTENDER FORMULATIONS

The homogeneous mixing of boar semen extenders components is critical for their performance. The complexity of this process lies in the disparity of the components, ranging from antibiotics in very low concentrations to excipients in significantly higher proportions. It’s essential to use robust analytical methods to ensure accurate final concentrations, uniformity, and product homogenization. This work aimed to develop a rapid, reliable HPLC method to simultaneously detect a chelating agent and an antimicrobial in the extender. Analyses were performed on a SHIMADZU HPLC-UV system with a C8 column at 254 nm detection, using a water:methanol (80:20 v/v) mobile phase at 0.3 mL/min and 100 µL injection volume. A 15 minute run enabled quantification of the chelating agent and the antimicrobial, using calibration curves prepared in placebo with standards between 0.5–5 mg/mL and 0.01–0.1 mg/mL respectively, covering their nominal concentrations in the extender (3 mg/mL and 0.04 mg/mL). Results show linearity across the tested ranges with r² of 0.995 for the chelating agent and 0.999 for the antimicrobial. Limits of detection (LOD) and quantification (LOQ) were 1.06 mg/mL and 3.21 mg/mL for the chelating agent, and 0.002 mg/mL and 0.006 mg/mL for the antimicrobial. Relative standard deviation (RSD) was below 10% for both components. Quantified extender samples showed chelating agent concentrations of 2.4–3.6 mg/mL and antimicrobial concentrations of 0.032–0.048 mg/mL, within ±20% of their nominal values in the extender. The validated HPLC-UV method ensures homogeneity of the extender, meeting the validation criteria for linearity, sensitivity, and reproducibility.