Confirmation and quantification of hemoglobin-based oxygen carriers in equine and human plasma by hyphenated liquid chromatography tandem mass spectrometry

Oxyglobin (OXY) and Hemopure (HMP) are produced from bovine hemoglobin (Hb) and were developed for the treatment of anemia in animal and human patients, respectively. Hemolink (HML) is a blood substitute of human Hb origin under development. The ability of these agents to carry oxygen in circulating blood and their promise to improve oxygen delivery to tissues supports the potential for their abuse in equine and human athletes. To deter athletes from abuse of these agents, a method has been developed for the detection, confirmation and quantification of OXY, HMP, and HML in equine and human plasma. OXY, HMP, and HML were extracted from equine or human plasma by solid-phase extraction using Bond Elut ENV cartridges and were digested by trypsin at 37 degreesC for 3 h. The tryptic digests were analyzed by LC-MS/MS, and tryptic peptides specific for bovine and human Hbs were targeted. OXY and HMP were detected, quantified, and confirmed using the y14 ion and b8 ion of the tryptic peptide from bovine Hb a chain residues 69-90, and HML was quantified using the tryptic peptide from human Hb alpha chain residues 63-91. The limit of detection for OXY in equine plasma and HML in human and equine plasma was 50 and 250 mug/mL for HMP in human and equine plasma. The limit of confirmation was 250 mug/mL for OXY in equine plasma, 500 mug/mL for HML in human and equine plasma, and 1000 mug/mL for HMP in human and equine plasma. The linear range for quantification was 50-5000 mug/mL for OXY in equine plasma and for HML in human and equine plasma, and 250-5000 mug/mL for HMP in human and equine plasma. The intraday and interday CV were less than 17% for quantification of OXY in equine plasma with external calibration. OXY was stable for more than 30 days at -20 and -70 degreesC. OXY was detected and quantified in equine plasma up to 24 h following administration of a very low dose of OXY (32.5 g in 2 x 125 mL per horse), and its presence in equine plasma was confirmed up to 12 h postadministration.