Screen and confirmation of PEG-epoetin β in equine plasma.

This research is about developing methods to identify a substance called PEG-epoetin β in horse plasma using techniques of enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The substance was detectable up to certain hours after being administered via different routes into the animals’ bodies, with the LC-MS/MS method confirmed for effective identification.

Methods Used

• The study researched techniques to identify PEG-epoetin β in horse plasma, including darbepoetin alfa and recombinant human EPO.
• Three methods were employed: A screening method using ELISA, additional screening with LC-MS/MS, and confirmation using LC-MS/MS.
• This research focused on PEG-epoetin β.

Findings

• Using the ELISA assay, PEG-epoetin β was successfully detected at 0.02 ng/mL in 50 µL of horse plasma.
• Some samples had high background levels of immunoreactivity, which was reduced with the introduction of polyethylene glycol 6000 (PEG 6000) before doing ELISA assay.
• The introduction of PEG 6000 not only reduced high background levels but also increased apparent concentrations of PEG-epoetin β.
• PEG-epoetin β was detectable up to 72, 144, and 120 hours after being inserted through intravenous, intramuscular, and subcutaneous routes, respectively.
• LC-MS/MS was used for samples preparation by extraction with anti-rHuEPO-antibodies-coated Dynabeads followed by digestion with trypsin.

Limit of Confirmation

• The LC-MS/MS confirmation used the multiple reaction monitoring (MRM) scan mode to monitor four precursor-product ion transitions of the EPO-derived peptide T₆.
• All four transitions of T₆ were detectable with an S/N greater than 3.
• The limit of identification for PEG-epoetin β using this method was 1.0 ng/mL in 2 mL of horse plasma.

Conclusion

• The method successfully confirmed the presence of PEG-epoetin β in a sample collected from a Mircera®-treated horse.
• Better sensitivity was found for darbepoetin alfa and recombinant human EPO compared to PEG-epoetin β.
• Darbepoetin alfa was detected in horse plasma four days after intramuscular administration of 100 µg.