Doping Control Analysis of Bovine Hemoglobin-Based Oxygen Therapeutics in Human Plasma by LC−Electrospray Ionization-MS/MS

Since January 2000, hemoglobin-based oxygen carriers, such as Hemopure, belong to the list of prohibited substances of the International Olympic Committee. Hemopure is based on bovine hemoglobin, which is intra- and intermolecularly cross-linked by glutaraldehyde units causing an average molecular weight of approximately 250,000. Bovine and human hemoglobins differ by 15% in amino acid sequence; hence, tryptic digestion of these proteins generates species-common and -unique peptides. Those specific fragments originate from the alpha- and beta-subunits of hemoglobin, such as bovine Hb peptides alpha(69-90) (2367.2 Da) or beta(40-58) (2089.9 Da). By means of LC-MS/MS, peptides of human and bovine hemoglobin can be separated and identified, enabling the determination of compounds based on Hb of bovine origin and thus the administration of oxygen carriers such as Hemopure. Blank plasma samples were spiked with Hemopure or human or bovine hemoglobin, filtered, enzymatically digested, and analyzed on an Agilent 1100 Series HPLC interfaced to an Applied Biosystems API 2000 triple quadrupole mass spectrometer. In plasma aliquots of 50 microL containing 50 microg of Hemopure (1 mg/mL), peptides of bovine hemoglobin were confirmed, and blank plasma samples as well as 68 specimens of high-performance athletes were tested with the developed procedure.