title: Immuno-magnetic beads-based extraction-capillary zone electrophoresis-deep UV laser-induced fluorescence analysis of erythropoietin.
abstract: Erythropoietin (EPO) is an important glycoprotein hormone. Recombinant human EPO (rhEPO) is an important therapeutic drug and can be also used as doping reagent in sports. The analysis of EPO glycoforms in pharmaceutical and sports areas greatly challenges analytical scientists from several aspects, among which sensitive detection and effective and facile sample preparation are two essential issues. Herein, we investigated new possibilities for these two aspects. Deep UV laser-induced fluorescence detection (deep UV-LIF) was established to detect the intrinsic fluorescence of EPO while an immuno-magnetic beads-based extraction (IMBE) was developed to specifically extract EPO glycoforms. Combined with capillary zone electrophoresis (CZE), CZE-deep UV-LIF allows high resolution glycoform profiling with improved sensitivity. The detection sensitivity was improved by one order of magnitude as compared with UV absorbance detection. An additional advantage is that the original glycoform distribution can be completely preserved because no fluorescent labeling is needed. By combining IMBE with CZE-deep UV-LIF, the overall detection sensitivity was 1.5 × 10⁻⁸ mol/L, which was enhanced by two orders of magnitude relative to conventional CZE with UV absorbance detection. It is applicable to the analysis of pharmaceutical preparations of EPO, but the sensitivity is insufficient for the anti-doping analysis of EPO in blood and urine. IMBE can be straightforward and effective approach for sample preparation. However, antibodies with high specificity were the key for application to urine samples because some urinary proteins can severely interfere the immuno-extraction.
authors.affiliation: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China.
authors.forename: Heye
authors.initials: H
authors.lastname: Wang
authors.forename: Peng
authors.initials: P
authors.lastname: Dou
authors.forename: Chenchen
authors.initials: C
authors.lastname: Lü
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liu
mesh_terms: D004300:Doping in Sports; D019075:Electrophoresis, Capillary / Q000379:methods*; D004921:Erythropoietin / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000652:urine; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D007834:Lasers; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000652:urine; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012709:Serum Albumin; D013050:Spectrometry, Fluorescence
chemical_list: C554270:EPO protein, human; D011994:Recombinant Proteins; D012709:Serum Albumin; D004921:Erythropoietin