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Rapid and sensitive reverse phase high performance liquid chromatography (RP-HPLC) and ultra performance liquid chromatography (UPLC) methods with UV detection for quantification of erythropoietin (EPO) in presence of human serum albumin (HSA) as a stabilizer in a pharmaceutical formulation of EPO have been developed and validated. Chromatography was performed with mobile phase containing 0.1% Trifluoroacetic acid (TFA) in MilliQ water and 0.1% TFA in acetonitrile with gradient program and a flow rate of 1.5 mL/min for HPLC and 0.35 mL/min for UPLC. Quantification was accomplished with internal reference standard (qualified using EP reference standard). The methods were validated for linearity (correlation coefficient=0.99), accuracy, precision and robustness. Robustness was confirmed by considering three factors; percentages of TFA in mobile phase, age of test sample and mobile phase and column temperature. Intermediate precision was confirmed by different analysts, different equipments and on different days. The relative standard deviation (RSD) value (&lt;2%, n =30) indicated good precision of the developed method. The proposed RP-HPLC method had retention time less than 20 min while the developed UPLC method had retention time less than 4 min. Both the RP-HPLC and UPLC methods were simple, highly sensitive, precise and accurate, suggesting that the developed methods are useful for routine quality control.

Development and validation of RP-HPLC and RP-UPLC methods for quantification of erythropoietin formulated with human serum albumin