Capillary electrophoretic analysis of whole blood samples for hemoglobin‐based oxygen carriers without the use of immunoprecipitation

Abstract

Hemoglobin-based oxygen carriers (HBOCs) are blood substitutes, synthesized by polymerizing hemoglobin, which are being developed and investigated as alternatives to blood for medical purposes. However, due to their ability to increase the oxygen carrying capacity when taken by healthy individuals, HBOCs have been used as a doping agent among endurance athletes and are included in the World Anti-Doping Agency's Prohibited List. To maintain the fairness of competitions and continue the battle against doping, it is essential to be able to detect HBOCs if present in an athlete's blood. To achieve this goal, it is necessary to differentiate HBOCs from the native hemoglobin and to do so in a cost and time effective manner. We have developed a rapid capillary zone electrophoresis (CZE), UV absorbance, method capable of detecting HBOCs, in whole blood samples, at levels below those considered necessary to provide a performance enhancement. Our approach to the analysis for HBOCs utilizes the whole blood sample, not just the plasma, and does not require the use of immunoprecipitants to ensure accurate analysis. By lysing the red blood cells and using centrifugal filtration, followed by our CZE separation, we are able to effectively distinguish between native hemoglobin and HBOCs. Through this method, we have been able to reliably detect concentrations of HBOCs at the equivalent of 5.5 g/L, the equivalent to a 3.5% increase in blood hemoglobin concentration for an athlete.