title: Capillary zone electrophoresis method for the direct determination of amino acids in recombinant human erythropoietin preparations used for the treatment of anemia.
abstract: A method based on CZE for the determination of glutamic acid, glycine, and alanine in a biopharmaceutical formulation containing recombinant human erythropoietin was developed. The separation was achieved within less than 5 min, using a fused-silica capillary column (55 cm × 50 μm id) and 30 mmol/L phosphate buffer at pH 11.5, containing 0.6 mmol/L CTAB and 10% v/v methanol, as BGE solution. Applied potential of -25 kV, temperature of 15°C and hydrodynamic injection time of 15 s, at 50 mbar, were employed. The detection of the analytes was carried out without any derivatization reaction, at 220 nm using an UV-DAD detector. Linear ranges from 50 to 2500 mg/L and quantification limits of 40, 39, and 37 mg/L were obtained for glutamic acid, glycine, and alanine, respectively. Sample preparation required only a dilution step. Considering peak area and migration time values, the method presented good repeatability (RSD <1.7%; n = 9) and intermediate precision (RSD <1.0%; n = 6). Recovery evaluation using a commercial sample led to values between 97.5 ± 5.2% and 101.5 ± 4.6%, demonstrating the feasibility of the method, which was successfully applied in the quantification of the amino acids of interest in biopharmaceutical samples.
authors.affiliation: Instituto de Tecnologia em Imunobiológicos, Bio-Manguinhos, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.|Programa de Pós-Graduação em Química, Instituto de Química, Universidade Federal Fluminense, Niterói, RJ, Brazil.
authors.forename: Izabel Cristina
authors.initials: IC
authors.lastname: de Souza Crespo
authors.affiliation: Laboratório de Química Analítica Fundamental e Aplicada, Departamento de Química Analítica, Niterói, RJ, Brazil.
authors.forename: Matheus Troina
authors.initials: MT
authors.lastname: de Resende
authors.affiliation: Programa de Pós-Graduação em Química, Instituto de Química, Universidade Federal Fluminense, Niterói, RJ, Brazil.|Laboratório de Química Analítica Fundamental e Aplicada, Departamento de Química Analítica, Niterói, RJ, Brazil.
authors.forename: Annibal Duarte
authors.initials: AD
authors.lastname: Pereira Netto
authors.affiliation: Programa de Pós-Graduação em Química, Instituto de Química, Universidade Federal Fluminense, Niterói, RJ, Brazil.|Laboratório de Química Analítica Fundamental e Aplicada, Departamento de Química Analítica, Niterói, RJ, Brazil.
authors.forename: Flávia Ferreira
authors.initials: FF
authors.lastname: de Carvalho Marques
mesh_terms: D000596:Amino Acids / Q000032:analysis*; D000740:Anemia / Q000188:drug therapy; D019075:Electrophoresis, Capillary / Q000379:methods; D004921:Erythropoietin / Q000032:analysis / Q000737:chemistry* / Q000627:therapeutic use; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D016014:Linear Models; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry* / Q000627:therapeutic use; D015203:Reproducibility of Results
chemical_list: D000596:Amino Acids; C554270:EPO protein, human; D011994:Recombinant Proteins; D004921:Erythropoietin
keywords: Amino acids; Anemia; CZE; Excipients; Recombinant human erythropoietin