Haemoglobin-based oxygen carriers (HBOC) were developed since 2000s from human or bovine haemoglobins to increase haemoglobin’s half-life and oxygen transport capacities. Due to their potential performance enhanced properties, HBOCs are considered as major threat. Since few years a new promising HBOC from annelids is available for clinical fields. This protein with a high molecular weight (3600 kDa) is extracted and purified from Arenicola marina. Testing on mice caused no allergic reaction and no kidney damage after administration. This new molecule named M101 is commercialized as HEMOXYCarrier® by HEMARINA company and can be misused for doping purposes either in human or equine athletes.
To investigate this threat, an analytical method was developed to detect M101 in horse plasma. Sample preparation was based on protein precipitation followed by reduction, alkylation and digestion by trypsin for 3 hours before solid phase extraction (SPE) on C18. Detection was then performed by targeting specific peptides using liquid chromatography-high resolution mass spectrometry (LC-HRMS). To improve robustness and throughput, the method was developed on 96-well plate SPE. Analyses were carried out on a UHPLC Nexera system (Shimadzu) linked to a QExactive HF mass spectrometer (Thermo Scientific™). Two doubly or triply charged characteristic tryptic peptides were monitored allowing M101 to be detected in spiked plasma samples at a concentration around 1 µg/mL [1]. For comparison, M101 was detected in spiked human plasma around 50 µg/mL using electrophoretic method. Furthermore, with the same technology it was detected until 4 hours in mice plasma after the intravenous administration of 200 mg/kg of HEMOXYCarrier® [1].