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  Home::Development and Validation of RP-UPLC method for quantification of Human serum albumin in Erythropoi
  Development and Validation of RP-UPLC method for quantification of Human serum albumin in Erythropoi

As human serum albumin is used as a stabilizer in many pharmaceutical as well as biopharmaceutical formulation, concentration of stabilizer should be sustain throughout the shelf life of product. So, a new sensitive, accurate, precise and rapid reverse phase ultra performance liquid chromatography method was developed and validated for quantification of human serum albumin (HSA) in pharmaceutical preparation of Erythropoietin (EPO).Chromatography was performed on a RP C-18 (2.1 mm ID 50 mm L, porosity 300 , particle size 1.7 m) using a mobile phase containing 0.1% Trifluoroacetic acid (TFA) in milliQ water and 0.1% TFA in acetonitrile with gradient program at 0.5 mL/min & 0.7 mL/min flow rate. Detection was carried out at 254 nm. Quantification was accomplished with HSA reference standard. The method was validated as per ICH guidelines for linearity (correlation coefficient > 0.99) (n=7), range, accuracy (n=9), precision (n=12) and robustness. Robustness was confirmed by various aspects like wavelengths, column temperatures, sample compartment temperatures, deliberated changes in concentration of TFA in mobile phase and with different lot of columns during method development. The method was linear over the concentration range of 1.0 mg/mL - 4.0 mg/mL. The precision of the method in terms of relative standard deviation was evaluated from intra- and inter-day replicate injections of system suitability standards of human serum albumin using different columns. Components of within and between-sets variances were found to be below 2% (n = 24) and 5%, respectively, which constituted an acceptable level of variation. Retention time was found to be about 1.99 min and 2.52 min for human serum albumin (HSA) and Erythropoietin (EPO) respectively. The developed method is sensitive, linear, accurate, precise, rapid and robust. The short run time (6.0 min) allows the large numbers of samples in a short period of time, therefore should be cost effective for routine analysis in the pharmaceutical industry.

Date: 15, Sep 2012 Posted By: admin
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