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.

Recent advances in amino acid analysis by capillary electromigration methods: June 2015–May 2017

“…Glu, Gly and Ala are found in these formulations. CZE was used to analyse the AAs without any derivation with a UV‐DAD device . The phosphate BGE was also modified with methanol and CTAB.…”

Quantitation of underivatized branched-chain amino acids in sport nutritional supplements by capillary electrophoresis with direct or indirect UV absorbance detection

“…In general, AAs need to be derivatized when direct UV detection is adopted, except one previously report by Crespo et al [21]. We checked the UV absorption spectrum of BCAAs, which owned a certain UV absorbance below 200 nm.…”

“…Contactless conductivity detector (CCD) is an optional method and the LOD range is from tens to sub μmol/L [15,19,20]. Few reports except Crespo et al have described the glutamic acid, glycine and alanine without derivatization were detected by the direct CE-UV detection (at 220 nm) [21]. In addition, many studies have showed that indirect UV absorbance detection is an important and alternative tool for the analysis of AAs in their native state [22], but has not been successfully applied in the separation of BCAAs.…”

Recent advances in amino acid analysis by capillary electromigration methods, 2013–2015

“…AA are used as excipients in pharmaceutical formulations because they do not interfere with the enzymatic activity or the functional structure of proteins. CE was demonstrated as a good method for the simultaneous determination of Glu, Gly, and Ala, without addition of any other reagent or derivatization reaction, in a biopharmaceutical formulation containing recombinant human erythropoietin, considered as revolutionary treatment of anemia .…”

“…Close to EOF, micelles permeate into a sample zone sweeping and concentrate the analytes into a narrow zone between the Buffer 2 and the sample zone. as a good method for the simultaneous determination of Glu, Gly, and Ala, without addition of any other reagent or derivatization reaction, in a biopharmaceutical formulation containing recombinant human erythropoietin, considered as revolutionary treatment of anemia [83].…”