Quantification of recombinant human erythropoietin by amino acid analysis using isotope dilution liquid chromatography-tandem mass spectrometry.
Herein, we describe an accurate method for protein quantification based on conventional acid hydrolysis and an isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry method. The analyte protein, recombinant human erythropoietin (rhEPO), was effectively hydrolyzed by incubation with 8 mol/L hydrochloric acid at 130 °C for 48 h, in which at least 1 μmol/kg of rhEPO was treated to avoid possible degradation of released amino acids during hydrolysis. Prior to hydrolysis, sample solution was subjected to ultrafiltration to eliminate potential interfering substances. In a reversed-phase column, the analytes (phenylalanine, proline, and valine) were separated within 3 min using gradient elution comprising 20 % (v/v) acetonitrile and 10 mmol/L ammonium acetate, both containing 0.3 % (v/v) trifluoroacetic acid. The optimized hydrolysis and analytical conditions in our study were strictly validated in terms of accuracy and precision, and were suitable for the accurate quantification of rhEPO. Certified rhEPO was analyzed using a conventional biochemical assay kit as an additional working calibrant for the quantification of EPO and improved the accuracy. The optimized protocol is suitable for the accurate quantification of rhEPO and satisfactorily serves as a reference analytical procedure for the certification of rhEPO and similar proteins.
Center for Bioanalysis, Department of Metrology for Quality of Life, Korea Research Institute of Standards and Science, 267 Gajeong-ro, Youseong-gu, Daejeon, 305-340, Republic of Korea.
Jung-Hyuk
JH
Yim
Ina
I
Yoon
Hyo-Jin
HJ
Yang
Sook-Kyung
SK
Kim
Sang-Ryoul
SR
Park
Yong-Moon
YM
Lee
Ji-Seon
JS
Jeong
D000596:Amino Acids / Q000032:analysis*; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D004921:Erythropoietin / Q000032:analysis*; D006801:Humans; D006868:Hydrolysis; D011865:Radioisotope Dilution Technique; D011994:Recombinant Proteins / Q000032:analysis; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014462:Ultrafiltration
D000596:Amino Acids; D011994:Recombinant Proteins; D004921:Erythropoietin