Screen and confirmation of PEG-epoetin beta in equine plasma

Methods have been developed to screen for and confirm darbepoetin alfa, recombinant human EPO, andmethoxy polyethylene glycol-epoetin beta (PEG-epoetin 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 beta. The ELISA assay was able to detect PEG-epoetin beta at 0.02 ng/mL in 50 mu L 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 beta. In samples collected following the administration of 100 mu g of PEG-epoetin beta by the intravenous (IV), intramuscular (IM) and subcutaneous (SC) routes, PEG-epoetin 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 beta was 1.0 ng/mL in 2mL of horse plasma. The method successfully confirmed the presence of PEG-epoetin beta in a sample collected from a Mircera (R)-treated horse. Compared to PEG-epoetin 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 mu g. Copyright (c) 2010 John Wiley & Sons, Ltd.