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

Abstract

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 (<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.

Structural Identification of a Non-Glycosylated Variant at Ser126 for O-Glycosylation Site from EPO BRP, Human Recombinant Erythropoietin by LC/MS Analysis

“…Several HPLC methods have been developed for quantification of rHu-EPO ( Dudkiewicz-Wilczyńska et al, 2002 ; Rane et al, 2012 ; Wilczyńska et al, 2005 ) with the presence of different stabilizers. Variant peaks were also identified during development but any analytical evidence was not provided for those variants.…”

UPLC Technique in Pharmacy—An Important Tool of the Modern Analyst

“…The results of HPLC and UPLC analyses for piracetam were also compared, recording 10 times lower LOD and LOQ values for the same assay in favor of UPLC and a six-times shorter analysis time in isocratic mode [128]. By using UPLC instead of HPLC for the determination of erythropoietin, the total analysis time was reduced from 20 to 4 min while obtaining a greater range of linearity of the method [168]. Precisely because of the speed, resolution, and sensitivity of the apparatus, UPLC methods are very well suited for use with a mass spectrometer, which increases the possibilities of this technique and makes it a practical and reliable tool for more laboratories, allowing for precise solvent administration, perfect reproducibility, and minimal sample transfer.…”

UHPLC: The Greening Face of Liquid Chromatography

“…column: ambient Detection: UV Injection volume: 5.0 μL [ 30 ] The determination of endogenic substance Determination of erythropoietin in human serum albumin Stationary phase: C18 (50 mm × 2.1 mm, 1.7 μm) Mobile phase: 0.1 % TFA in acetonitrile: 0.1 % TFA in water (gradient elution) Flow rate: 0.35 mL/min Temp. column: 60 °C Detection: UV Injection volume: 2.0 μL [ 43 ] …”

“…Applications of UHPLC methods concerned assays of compounds with similar chemical structures, for example analogues from the same therapeutic group. Such methods can also be applied in the presence of related substances, including impurities and degradation products (Table 3 ) [ 31 – 43 ]. The most common applications of UHPLC methods in pharmaceutical analysis in recent years have dealt with: Stability studies of API in bulk substance as well as in its pharmaceutical forms, especially during the development of stability-indicating analytical methods [ 37 – 39 , 44 – 48 ] Profiles of impurities [ 49 , 50 ] Dissolution studies [ 41 , 51 , 52 ].…”