Establishment and validation of a dual-analyte high-performance liquid chromatography method for detecting thimerosal...

Preservatives such as thimerosal and phenol are essential for preventing microbial contamination in veterinary vaccines; however, their concentrations must be tightly controlled to minimize toxicological risks to animal health and to ensure formulation stability throughout shelf life. To meet stringent regulatory requirements for batch-release quality control and safety, precise quantification of these preservatives is critical. Accordingly, this study developed a dual-analyte high-performance liquid chromatography (HPLC) method was developed for determining thimerosal and phenol in veterinary vaccines and was validated it in accordance with the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products GL2 guidelines. Chromatographic separation was achieved on a C8 column using an isocratic mobile phase of methanol and 0.1% phosphoric acid buffer (pH 7.0) (40:60, v/v), containing 0.1% (v/v) triethylamine via UV detection at 245 nm. Validation tests assessed the linearity, system suitability, specificity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), and robustness of the method. The method exhibited excellent linearity (R² > 0.995) over 0.01-0.25 mg/mL for thimerosal and over 0.08-2.00 mg/mL for phenol; met the system suitability criteria (retention time ratio 0.90-1.10, theoretical plate number > 2,000, tailing factor < 2, resolution > 2); and demonstrated acceptable accuracy (recoveries within 100%±20%) and precision (relative standard deviation < 5%). The LOQs for thimerosal and phenol were 0.0014 and 0.0045 mg/mL, respectively, and their LODs were 0.0005 and 0.0015 mg/mL, respectively. Robustness testing indicated sensitivity to changes in the mobile phase composition and flow rate. This dual-analyte HPLC method provides a precise and reliable approach for preservative quantification in veterinary vaccines, offering improved suitability for quality control relative to traditional spectrophotometric assays and supporting compliance with international regulatory expectations.