Detection of Hemoglobin-Based Oxygen Carriers in Human Serum for Doping Analysis: Confirmation by Size-Exclusion HPLC

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2004-04, Vol.50 (4), p.723-731
• Main Authors: Varlet-Marie, Emmanuelle, Ashenden, Michael, Lasne, Francoise, Sicart, Marie-Therese, Marion, Benedicte, de Ceaurriz, Jacques, Audran, Michel
• Format: Article
• Language: English
• Subjects: Adult; Analytical, structural and metabolic biochemistry; Animals; Athletes; Bioengineering; Biological and medical sciences; Blood; Blood Substitutes; Blood Substitutes - analysis; Cattle; Chromatography; Chromatography, Gel; Chromatography, High Pressure Liquid; Doping in Sports; Drug Stability; Drug Storage; Fundamental and applied biological sciences. Psychology; Hemoglobin; Hemoglobins; Hemoglobins - analysis; Humans; Investigative techniques, diagnostic techniques (general aspects); Laboratories; Life Sciences; Liquid chromatography; Male; Medical sciences; Methods; Oxygen; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Polyethylene glycol; Sensitivity and Specificity; Substance Abuse Detection; Substance Abuse Detection - methods
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2003.026591

Hemoglobin-based oxygen carriers (HBOCs) are being developed as potential substitutes for the oxygen-carrying functions of erythrocytes, but athletes may obtain and experiment with HBOCs as an illicit means of enhancing oxygen transport. An electrophoretic technique has been developed to screen for the presence of HBOCs in blood samples (Lasne et al. Clin Chem 2004;50:410-5). Interest has focused on complementary methods that can provide legally defensible scientific evidence for the presence of HBOCs in blood samples collected for doping control. The aim of this research was to develop a size-exclusion SEC-HPLC technique to identify in plasma or serum samples the presence of HBOCs that are currently under development. This method was also used to detect a polymerized bovine hemoglobin (Hemopure) after infusion in 12 healthy males. The chromatograms of all HBOCs tested were clearly separated from the 54-min peak associated with human hemoglobin dimers. It was possible to differentiate between the different HBOC products based solely on their chromatographic profiles, provided they were at high concentrations. Differences were discernible not only based on the presence (or absence) of peaks, but also the separation between respective peaks. The profiles for serum samples collected from the men immediately after infusion of Hemopure showed a distinctive profile. The shape of the chromatographic profile remained consistent for at least 48 h. Under the analytical conditions reported here, SEC-HPLC was able to separate native hemoglobin from the modified hemoglobin molecules present in each of the HBOC products studied. In tandem with electrophoretic screening, SEC-HPLC provides evidence of the presence of HBOCs and can therefore be regarded as a method that satisfies the criteria for use in an antidoping control setting.