Abstract

The analysis of erythropoietin (EPO) is mainly based on the detection of isoelectric profiles and their differentiation in endogenous or recombinant patterns. While the first- and second- generation of recombinant EPO were detectable in urine, the third generation of erythropoiesis stimulating agents (ESAs) CERA (Continuous Erythropoietin Receptor Activator), marketed under the name MIRCERA® favors the analysis in blood. This is caused by the low excretion in urine, which relies on the high molecular mass (approximately 6OkDa), limiting the glomerular filtration. The plasma half-life of 120-140h is 15 to 20 times longer compared to other ESAs. Therefore CERA has a prolonged timeframe for detecting its misuse. In WADA accredited laboratories an enzyme-linked immunosorbent assay (ELISA) is used as screening method to detect CERA-misuse in blood. False negative results and false positive results in our lab have shown less reliability of this procedure. Confirmation analysis is performed by isoelectric focusing (IEF). Due to the high protein concentration in serum a purification step is necessary which is performed by immunoaffinity chromatography, liquid chromatographic separation, by magnetic beads combined with a specific anti-hEpo antibody or by protein precipitation with perchloric acid. This presentation shows a further procedure to remove most of serum proteins, using a precipitation by acetonitrile, while CERA and other EPOs stay solubilized. The method was optimized for 0.1 mL of serum. Einleitung