Doping control analysis of filgrastim in equine plasma and its application to a co-administration study of filgrastim and recombinant human erythropoietin in the horse.

Researchers have developed a method to detect the abuse of a specific drug, filgrastim, in equestrian sports through testing horse plasma. This method was effective at identifying the drug’s earlier use.

Overview of the Research

• This research aimed to come up with an effective way to identify a drug known as filgrastim being used in equestrian sports. Filgrastim is a version of a hematopoietic growth factor called recombinant human granulocyte colony-stimulating factor (rhG-CSF) which is responsible for regulating granulopoiesis – a process that leads to the formation and development of granulocytes, certain white blood cells necessary for fighting off infections.
• While this drug is used for medical purposes such as treatment of granulopenia in humans—a condition characterized by low levels of granulocytes—it is banned in sports because of the potential for misuse.

The Testing Methods

• First, the researchers used a screening method known as enzyme-linked immunosorbant assays (ELISA) to check for the presence of filgrastim in horse plasma samples.
• For the cases that tested positive in the ELISA screening, a nano-liquid chromatography-tandem mass spectrometry (LC/MS/MS) confirmation method was used. Post the plasma’s immunoaffinity purification and trypsin digestion, LC/MS/MS was applied for analysis.
• The LC/MS/MS confirmation method was designed to identify a highly specific tryptic peptide associated with filgrastim’s presence.
• The study revealed that filgrastim could be detected and confirmed at a concentration as low as 0.2 nanograms per milliliter in horse plasma.

Field Tests and Findings

• For a practical test, post-administration plasma samples collected from horses were involved. These horses were co-administered with two substances: filgrastim (as rhG-CSF) and epoetin alfa, a version of recombinant human erythropoietin (rhEPO).
• The analysis indicated that both substances could be detected in the horses’ plasma even after a substantial period – 57 hours for rhEPO and 101 hours for filgrastim.
• These findings not only validated the ELISA screening method and the LC/MS/MS confirmation process but also marked the first-ever identification of filgrastim in post-administration samples from horses.