Screen and confirmation of PEG-epoetin β in equine plasma

Methods have been developed to screen for and confirm darbepoetin alfa, recombinant human EPO, and methoxy polyethylene glycol-epoetin β (PEG-epoetin β) in horse plasma. All three methods screen samples with an enzyme-linked immunosorbent assay (ELISA) and confirm by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This report focuses on PEG-epoetin β. The ELISA assay was able to detect PEG-epoetin β at 0.02 ng/mL in 50 µL of horse plasma. Many samples had high background levels of immunoreactivity; however, introducing polyethylene glycol 6000 (PEG 6000) into the samples before the ELISA assay removed the high background and increased the apparent concentrations of PEG-epoetin β. In samples collected following the administration of 100 µg of PEG-epoetin β by the intravenous (IV), intramuscular (IM) and subcutaneous (SC) routes, PEG-epoetin β was detectable up to 72, 144, and 120 h, respectively. The samples were prepared for LC-MS/MS analysis by extraction with anti-rHuEPO-antibodies-coated Dynabeads followed by digestion with trypsin. The LC-MS/MS confirmation method 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 S/N > 3. The limit of confirmation for PEG-epoetin β was 1.0 ng/mL in 2 mL of horse plasma. The method successfully confirmed the presence of PEG-epoetin β in a sample collected from a Mircera®-treated horse. Compared to PEG-epoetin β, better sensitivity was achieved for darbepoetin alfa and recombinant human EPO. Darbepoetin alfa was detected in horse plasma four days after IM administration of 100 µg.