Detection of extracellular hemoglobin from Arenicola marina in doping control serum samples by means of liquid chromatography and high-resolution tandem mass spectrometry

The manipulation of blood and blood components in sports is prohibited at all times, and besides blood transfusions, also hemoglobin-based oxygen carriers (HBOCs) can be employed to artificially improve the oxygen transport capacity of the blood. But while most drug candidates based on stabilized hemoglobin (Hb) were found to be characterized by serious side effects, the natural giant extracellular Hb from the marine invertebrate Arenicola marina (lugworm) could be another candidate for transfusion medicine and cheating athletes, as it was found to be well tolerated in preclinical animal studies. Within this research project, lugworm Hb was implemented into the existing doping control detection method for bovine HBOCs based on ultrafiltration, tryptic digestion, and liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS). For the mass spectrometric identification of lugworm Hb, two precursor–product ion pairs for a total of four tryptic peptides originating from subunits hbA2 (T₆), hbB1 (T₃ and T₆), and the linker chain (T₁₆) were employed. The modified approach was comprehensively characterized and found to allow for the specific and sensitive detection of lugworm Hb down to concentrations of 10 μg/mL from 50 μL of serum/plasma. Therefore, it can serve as confirmation procedure for lugworm Hb following visual or electrophoretic screening. Moreover, a proof-of-concept rat administration study was conducted, and the observed detection windows of at least 4 (dose: 200 mg/kg) and 8 h (dose: 600 mg/kg) suggest that the approach can be readily employed to efficiently test in-competition doping control samples for the presence of the drug candidate.