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.

Metal cation control of electroosmotic flow magnitude in phospholipid‐coated capillaries

“…This necessitated the preparation of small unilamellar vesicles of DLPC. This was achieved using a modification of our previously published approach . Briefly sufficient DLPC was dissolved to prepare 1 mL of a 10 mM DLPC solution in a 20 mM Tris buffer, at pH 7.4, and containing 20 mM of one of the metal cation salts (typically calcium chloride).…”

Analytical chemistry research at primarily undergraduate institutions: training tomorrow's investigators

“…Biomedical applications include continued development of high-performance thin-layer chromatography (HPTLC) for pharmaceutical and tissue sample analysis, 43,44 a capillary electrophoresis method to detect hemoglobin polymers in blood for anti-doping tests, 45 and a demonstration that iontophoresis in tissue may include an electroosmotic component. Biomedical applications include continued development of high-performance thin-layer chromatography (HPTLC) for pharmaceutical and tissue sample analysis, 43,44 a capillary electrophoresis method to detect hemoglobin polymers in blood for anti-doping tests, 45 and a demonstration that iontophoresis in tissue may include an electroosmotic component.…”

“…A number of recent papers also highlight applications-driven research in separation science. Biomedical applications include continued development of high-performance thin-layer chromatography (HPTLC) for pharmaceutical and tissue sample analysis, 43,44 a capillary electrophoresis method to detect hemoglobin polymers in blood for anti-doping tests, 45 and a demonstration that iontophoresis in tissue may include an electroosmotic component. 46 Another novel application of separations from PUIs is a swab-based method for sample acquisition, preparation, and HPLC analysis of small molecules from amphibian skin that allows these compounds to be quantied without sacricing the animal.…”

Role of Capillary Electrophoresis in the Fight Against Doping in Sports

“…Another method undertook the determine of HBOCs in whole blood samples, probing both the plasma and red blood cells lysate for the synthetic protein. 36 This method used UV absorbance detection and relied on centrifugal filtration to selectively retain HBOCs from the protein rich sample. The samples were separated by CZE with a phospholipid bilayer coating to protect the capillary surface from protein adsorption.…”