A new analytical method based on anti-EPO monolith column and LC-FAIMS-MS/MS for the detection of rHuEPOs in horse plasma and urine samples

Abstract

Recombinant human erythropoietin (rHuEPO) is a 30–34 kDa glycoprotein banned by the racing authorities. For some years this molecule has been detected in race horses in USA and in Europe, and even in racing camels. Although direct methods to differentiate horse endogenous EPO and rHuEPO have been developed either by LC-MS/MS or by isoelectric focusing (IEF) with double-blotting, the short confirmation time of such prohibited hormone in plasma remains a problem for horseracing doping control laboratories. In order to improve the rHuEPOs confirmation process in horse plasma or urine in terms of reliability and delay, a small anti-EPO monolith membrane contained in a disposable column (anti-EPO monolith column) has been successfully used and validated (n = 10). This new sample preparation, combined with LC-FAIMS-MS/MS, has been performed on plasma and urine samples collected from one horse which received an Eprex® treatment during six consecutive days and a second one with a single injection of Aranesp®. This inventive technology allowed the possibility to confirm the presence of rHuEPO within one day with a limit of detection validated for both urine and plasma at 250 pg mL⁻¹ by means of a disposable, ready to use immunoaffinity column. The lower limit of detection (LLOD) obtained for each matrix was 100 pg mL⁻¹. These results provide an important improvement for rHuEPO doping control in horseracing especially the possibility to confirm these banned molecules in both matrices, urine and plasma, with a confidence of two specific target peptides.