Determination of benzethonium chloride in anthrax vaccine adsorbed by HPLC

A novel and sensitive HPLC method for the determination of benzethonium chloride (BZC) in anthrax vaccine was developed. Adjuvant Alhydrogel was removed by syringe filter after a simple sample pretreatment—acidification prior to injection. Chromatography was performed by isocratic reverse phase separation with methanol/262 mM ammonium acetate (80/20, v/v) on an endcapped C18 column with diode array detector (DAD). The method showed excellent recovery (100 ± 1.5%). The results indicated that this method could accurately determine BZC at the limit of detection (LOD) of 0.5 ppm and the limit of quantitation (LOQ) of 1.5 ppm with dynamic range up to 100 ppm. The comparison of analysis between new HPLC and old titrimetric methods is also reported. The HPLC method is proven to be more accurate and precise with much less vaccine sample and human labor required.