title: Quantitative analysis of erythropoietin in human plasma by tandem mass spectrometry.
abstract: The extended use of protein drugs in therapeutics has created the need for their quantification in human plasma. A methodology using the therapeutic protein itself as internal standard for quantitative analysis by multiple reaction monitoring (MRM) has been designed and applied to epoetin beta, a recombinant human erythropoietin (rhEPO). After depletion of major proteins, plasma samples were desalted and enriched in rhEPO by reversed phase liquid chromatography prior to tryptic cleavage. Differential isotopic labeling of peptides was performed by derivatization with 2-methoxy-4,5-dehydro-imidazole. A light version (four hydrogen atoms) of this reagent was used for plasma peptides. Tryptic peptides obtained from pure rhEPO were derivatized with a heavy version (four deuterium atoms) of the same reagent and used as internal standards. Two rhEPO tryptic peptides with three MRM transitions per peptide were selected for quantification. This strategy provided a quantification limit close to 50 amol of epoetin beta per microliter of plasma (equivalent to 1.7 ng/mL), i.e., well below the expected therapeutic concentrations in plasma (around 100-500 amol/μL).
authors: EA 2968 Laboratoire de Pharmacocinétique et de Pharmacie Clinique, Université Victor Segalen Bordeaux 2, 146 Rue Léo-Saignat, 33076, Bordeaux Cedex, France. fabien.xuereb@chu-bordeaux.fr
authors: Fabien
authors: F
authors: Xuereb
authors: Stéphane
authors: S
authors: Chaignepain
authors: Dominique
authors: D
authors: Breilh
authors: Frédéric
authors: F
authors: Godde
authors: Marie-Claude
authors: MC
authors: Saux
authors: Christof
authors: C
authors: Lenz
authors: Matthias
authors: M
authors: Glueckmann
authors: Jean-Marie
authors: JM
authors: Schmitter
mesh_terms: D002853:Chromatography, Liquid; D004921:Erythropoietin / Q000097:blood* / Q000737:chemistry; D006801:Humans; D010449:Peptide Mapping; D011994:Recombinant Proteins; D012015:Reference Standards; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D004921:Erythropoietin; D014357:Trypsin