Methodologies for detection of hemoglobin-based oxygen carriers

Blood substitutes based on hemoglobin or hemoglobin-based oxygen carriers (HBOCs) are oxygen-carrying therapeutic agents developed for use in operations and emergencies in place of donated blood. Increased oxygen-carrying capacity through the use of blood substitutes could help elite athletes to lengthen endurance capacity and improve their performance. As blood substitutes become more readily available, it is essential that a qualitative detection method for their abuse in sport is available. Ideally, such a method would be simple and inexpensive. This study investigates methods that could be used as screening procedures to easily detect HBOCs in plasma and develops tests that can unequivocally confirm their presence. The investigation into the screening method indicates that the direct visual screening of plasma discoloration is the most appropriate with detection limits of less than 1% HBOC in plasma. Two methods are shown to confirm the presence of exogenous hemoglobin in plasma samples, size-exclusion chromatography with photodiode array detection and high-performance liquid chromatography analysis of enzymatic digests with detection by electrospray mass spectrometry. This work emphasizes the need for cooperation between drug developers and sports testing laboratories to ensure that methods for the detection of putative doping agents are available prior to product release.