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.

The research focuses on a novel analytical method utilizing an anti-EPO monolith column and LC-FAIMS-MS/MS for detecting the banned substance, recombinant human erythropoietin (rHuEPO), in horse plasma and urine. This new technique enhances the confirmation process for rHuEPO in horse plasma or urine by increasing reliability, reducing delay, and improving detection limits.

Understanding Recombinant Human erythropoietin (rHuEPO)

• Recombinant human erythropoietin (rHuEPO) is a 30-34 kDa glycoprotein used for doping in racehorses and camels.
• The substance is currently prohibited due to its potential to alter performance unfairly in competitive racing.
• Existing detection methods for rHuEPO include LC-MS/MS and isoelectric focusing (IEF) with double-blotting. However, these have limitations in the confirmation time and accuracy.

The New Analytical Method

• The developed method uses a small anti-EPO monolith membrane contained in a disposable column (anti-EPO monolith column), which has been tested and validated.
• This column enables more reliable rHuEPO detection in horse plasma and urine samples.
• The new sample preparation, which combines this immunoaffinity column with LC-FAIMS-MS/MS, effectively detects rHuEPO presence within a day, considerably reducing the delay in current methodologies.

Testing and Results

• The testing process was performed on plasma and urine samples collected from one horse which received an Eprex® treatment for six consecutive days and another one that received a single injection of Aranesp®.
• The study found a limit of detection validated for both urine and plasma at 250 pg mL(-1) by using the ready-to-use immunoaffinity column.
• The methodology achieved a lower limit of detection (LLOD) of 100 pg mL(-1) for each matrix.
• The new detection technique provides an important improvement for rHuEPO doping control in horseracing. Notably, it can confirm the presence of these banned molecules in both matrices, urine and plasma, with a confidence of two specific target peptides.