A simple and rapid pre-confirmation method to distinguish endogenous human haemoglobin from synthetic haemoglobin-based oxygen carriers in doping control

A specific, sensitive and rapid analytical procedure based on capillary electrophoresis with UV/Vis detection at 405 and 415 nm was developed and validated to detect human haemoglobin and haemoglobin-based oxygen carriers (Hemopure (R), Oxyglobin (R) and Polyheme (R)) in blood samples collected for doping control. The electrophoretic separation, based on capillary dynamic coating, was achieved in less than 10 min. The effects of capillary temperature, injection conditions and initial ramping were investigated. The optimum separation voltage was 25 kV with a capillary temperature of 20 degrees C, initial ramping of 1 kV/s and an injection pressure of 0.5 psi for 10 s. The removal of haptoglobin using anti-human haptoglobin antibody prior to the analysis was mandatory to increase the specificity of the analysis. Sufficient resolution between endogenous haemoglobin variants and the three haemoglobin-based oxygen carriers here investigated was obtained, thus allowing discrimination between a normal haemolysed sample and a sample in which Oxyglobin (R), Hemopure (R) or Polyheme (R) is present. Good repeatability of migration times (CV% less than 1), peak resolution and adequate sensitivity (limit of detection: 2.5 mg/mL) was obtained.