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 T 6 . All four transitions of T 6 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.