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 ${\bf{\beta}}$ (PEG‐epoetin ${\bf{\beta}}$) 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 ${\bf{\beta}}$. The ELISA assay was able to detect PEG‐epoetin ${\bf{\beta}}$ 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 ${\bf{\beta}}$. In samples collected following the administration of 100 µg of PEG‐epoetin ${\bf{\beta}}$ by the intravenous (IV), intramuscular (IM) and subcutaneous (SC) routes, PEG‐epoetin ${\bf{\beta}}$ 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 ${\bf{\beta}}$ was 1.0 ng/mL in 2 mL of horse plasma. The method successfully confirmed the presence of PEG‐epoetin ${\bf{\beta}}$ in a sample collected from a Mircera®‐treated horse. Compared to PEG‐epoetin ${\bf{\beta}}$, 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. Copyright © 2010 John Wiley & Sons, Ltd. Methods have been developed to screen for and confirm EPO analogs in horse plasma. The 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.