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A simple and rapid pre‐confirmation method to distinguish endogenous human haemoglobin from synthetic haemoglobin‐based oxygen carriers in doping control
Author(s) -
Donati Francesco,
Mazzarino Monica,
de la Torre Xavier,
Botrè Francesco,
Islam Nazia,
Cowan David
Publication year - 2011
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201100178
Subject(s) - repeatability , chromatography , capillary electrophoresis , detection limit , capillary action , oxygen , analytical chemistry (journal) , chemistry , haptoglobin , resolution (logic) , electrophoresis , materials science , biology , immunology , organic chemistry , artificial intelligence , computer science , composite material
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 ® , Oxyglobin ® and Polyheme ® ) 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°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 ® , Hemopure ® or Polyheme ® 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.