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Bosch J.,Bioanalysis Group | Ueki M.,Anti doping Research Laboratory | Such-Sanmartin G.,Bioanalysis Group | Segura J.,Bioanalysis Group | And 3 more authors.
Analytica Chimica Acta | Year: 2012

Detecting recombinant human growth hormone (rhGH) abuse in sport remains one of the major challenges in doping control. We have compared two different approaches to detect the hGH (human growth hormone) abuse. The first measures the concentrations of the 22. kDa hGH isoform (rec assay) and pituitary derived isoforms (pit assay) and a ratio rec/pit is obtained. The second measures the concentrations of 22 and 20. kDa hGH isoforms and also a ratio 22/20. kDa is derived. Using a single set (nine healthy male subjects, 7 days, 0.026. mg/kg/day of rhGH, 2 week wash out period) both approaches were compared. To quantify the agreement between the immunoassays, B.A. (Bland-Altman) analysis and P.r. (Pearson correlation) were used. To fully understand the assay readings, all relevant antibodies were characterised by surface plasmon resonance (SPR).In either approach the ratio numerator produces similar results and the denominator determines both signal-amplitude and time-frame of possible application. The rec vs pit approach displays a higher distinctive capacity to detect hGH abuse but the complex binding properties of the capture antibodies make it very difficult to evaluate the precise contributions of the individual hGH variants to the assay result. In the 22 vs 20 approach, the 20 kDa hGH concentration measures determine its applicability. Both approaches are based on a different principle, should be preferably applied within 24 h after rhGH administration, and are perfectly comparable given the results obtained. The reduced time frame of application indicates that their principle application should be preferably in an out-of-competition setting. © 2012 Elsevier B.V. Source

Mallorqui J.,Bioanalysis Group | Mallorqui J.,University Pompeu Fabra | Llop E.,Bioanalysis Group | de Bolos C.,Gastric Carcinogenesis Group | And 6 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2010

A method is described to isolate human erythropoietin (hEPO) from plasma using an EPO-specific immunoaffinity micro well plate (IAP). The operating conditions of the method (binding, blocking and elution) were optimised to avoid isoform discrimination and cross-contamination with other glycoproteins. The overall hEPO recovery was ca. 56% and significant clean-up for plasmatic hEPO was achieved. Polyvinylpyrrolidone (PVP) was used as a blocking reagent and elution took place at pH 11.0. Under these conditions all isoforms from recombinant human EPOs (rhEPOs) and analogues were uniformly recovered guaranteeing lack of discrimination. The resulting procedure allowed isolating erythropoietin from plasma in conditions amenable to hEPO analysis by other techniques such as SDS-PAGE or IEF. Moreover, avoiding contamination with other glycosylated material allowed the identification in human plasma samples of the non-human N-glycolyl-neuraminic acid (Neu5Gc) using HPLC-FLD. Neu5Gc is present as 1-2% of the sialic acid content in rhEPO so this approach could be used to unequivocally detect abuse of rhEPOs or analogues as part of the doping control. © 2010 Elsevier B.V. Source

Mallorqui J.,Bioanalysis Group | Mallorqui J.,University Pompeu Fabra | Gutierrez-Gallego R.,Bioanalysis Group | Gutierrez-Gallego R.,University Pompeu Fabra | And 5 more authors.
Journal of Pharmaceutical and Biomedical Analysis | Year: 2010

A screening method able to differentiate recombinant human erythropoietins (rhEPOs) and analogues like CERA from human urinary erythropoietin (uhEPO) is described. The method is based on the discrimination between isoforms observed when the protein is eluted under acidic followed by basic conditions from immunoaffinity microtiter wells. From a comparison with the complex IEF protocol currently applied in anti-doping analysis, the newly developed assay procedure is amenable to wide screening application and presents good resolving power between rhEPOs and uhEPO. © 2009 Elsevier B.V. All rights reserved. Source

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