Practicing IEF-PAGE of EPO: the impact of detergents and sample application methods on analytical performance in...

Electrophoretic techniques, namely isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are key techniques used for confirming the doping-related abuse of recombinant erythropoietins and analogs. IEF-PAGE is performed on horizontal slab-gels with samples applied to the surface of the gel. Different sample application techniques can be employed, but application pieces and applicator strips are most frequently used. However, defective application pieces cause lane streaking during IEF of erythropoietin (EPO), which is especially pronounced in the acidic region of the gel. The effect is due to an incompatibility of the substance used for enhancing the wettability of the cellulose-based commercial product and is batch-dependent. A detailed mass spectrometric study was performed, which revealed that defective sample application pieces (bought between 2007 and 2010) contained a complex mixture of alcohol ethoxylates, alcohol ethoxysulfates, and alkyl sulfates (e.g. SDS). Anionic detergents, like the sulfates contained in these application pieces, are in general incompatible with IEF. Alternative application techniques proved partly useful. While homemade pieces made of blotting paper are a good alternative, the usage of applicator strips or shims is hampered by the risk of leaking wells, which lead to laterally diffused samples. Casting IEF-gels with wells appears to be the best solution, since sustained release of retained proteins from the application pieces can be avoided. Edge effects do not occur if wells are correctly filled with the samples. The evaluation of EPO-profiles with defects is prohibited by the technical document on EPO-analytics (TD2009EPO) of the World Anti-Doping Agency (WADA).