Rapid and subnanomolar assay of recombinant human erythropoietin by capillary electrophoresis using NanoOrange precolumn labeling and laser-induced fluorescence detection.
Because of less functionally critical carbohydrate sectors that contributed to the stability, efforts have been made to quantify intact recombinant human erythropoietin. A simple, rapid capillary electrophoresis with laser-induced fluorescence method for the assay of recombinant human erythropoietin was developed, with a limit of detection of intact recombinant human erythropoietin at subnanomolar concentration (up to 10 ng/mL or 3 × 10(-10) M), which is among the lowest reported. High sensitivity was accomplished by precolumn derivatization with the noncovalent dye NanoOrange. Capillary electrophoresis separation and reaction conditions were carefully manipulated for avoiding microheterogeneity of glycoforms and inhomogeneity of multiple labeling products. The fluorescence signal was linear over the range of 10 ng/mL-10 μg/mL, corresponding to the detection requirement of recombinant human erythropoietin in biofluids and pharmaceutical samples, as demonstrated by a real sample analysis. Although the salt in reaction mixtures showed a detrimental effect on the fluorescence of the derivatives, this method could tolerate a certain amount of salt, extending its application in biofluid analysis. In addition, zero-order fluorescence emission kinetics was obtained, indicating that the rapid decay of recombinant human erythropoietin was derived from a self-quenching effect.
School of Chemical and Biological Engineering, University of Science and Technology, Beijing, P. R. China; Beijing National Laboratory for Molecular Sciences, the Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Institute of Analytical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing, P. R. China.
Nannan
N
Pang
Yu
Y
Bai
Yu
Y
Zhou
Xia
X
Yang
Zhengxiang
Z
Zhang
Honggang
H
Nie
Xiaofang
X
Fu
Huwei
H
Liu
D019075:Electrophoresis, Capillary; D004921:Erythropoietin / Q000032:analysis* / Q000097:blood / Q000652:urine; D005453:Fluorescence; D006801:Humans; D007834:Lasers; D009930:Organic Chemicals / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D012492:Salts / Q000737:chemistry; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
C433144:NanoOrange; D009930:Organic Chemicals; D011994:Recombinant Proteins; D012492:Salts; D004921:Erythropoietin
Capillary electrophoresis; Laser-induced fluorescence; NanoOrange; Recombinant human erythropoietin