Assessment of rhEPO in Pharmaceutical Formulations by a Reversed‐Phase Liquid Chromatography Method and Bioassay

Abstract

A gradient reversed‐phase liquid chromatography (RP‐LC) was developed for the analysis of alpha and beta rhEPO in pharmaceutical formulations. The RP‐LC method was carried out on a Jupiter C4 column (250 mm×4.6 mm I.D., with a pore size of 300 Å). The elution was performed by a gradient at a constant flow rate of 0.5 mL/min. Mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) and mobile phase B consisted of 0.08% TFA:acetonitrile (30∶70, v/v), using a photodiode array (PDA) detection at 280 nm. The chromatographic separation was obtained within 60 min and was linear in the concentration range of 10–150 µg/mL. The parameters validated, such as the specificity, precision, accuracy, and robustness gave results within the acceptable range. The pharmaceutical samples were analysed by the chromatographic method and compared to the normocythaemic mice bioassay, showing the mean difference between the estimated potencies of 11.2%±1.8 higher for the RP‐LC, with significant correlation (r=0.9799) as calculated by the Pearson's coefficient. The proposed RP‐LC method represents an alternative to the bioassay that can be applied for the potency assessment, improving the quality control of rhEPO in pharmaceutical dosage forms.