Screen and confirmation of PEG-epoetin β in equine plasma
| Autor: | S. M. Neades, Y. Chang, G. Matsumoto, D. H. Catlin, G. M. Maylin |
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| Rok vydání: | 2010 |
| Předmět: |
Spectrometry
Mass Electrospray Ionization Darbepoetin alfa Injections Subcutaneous Pharmaceutical Science Administration Oral Enzyme-Linked Immunosorbent Assay Polyethylene glycol Performance-Enhancing Substances Tandem mass spectrometry Injections Intramuscular Analytical Chemistry Polyethylene Glycols Dynabeads chemistry.chemical_compound Liquid chromatography–mass spectrometry Tandem Mass Spectrometry PEG ratio medicine Environmental Chemistry Animals Humans Horses Erythropoietin Spectroscopy Doping in Sports Chromatography Selected reaction monitoring Horse Reproducibility of Results Recombinant Proteins Substance Abuse Detection chemistry Injections Intravenous medicine.drug Chromatography Liquid |
| Zdroj: | Drug testing and analysis. 3(1) |
| ISSN: | 1942-7611 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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