title: Doping control analysis of filgrastim in equine plasma and its application to a co-administration study of filgrastim and recombinant human erythropoietin in the horse.
abstract: Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor regulating granulopoiesis. The recombinant human granulocyte colony-stimulating factor (rhG-CSF) is widely used for the treatment of granulopenia in humans. Filgrastim is a rhG-CSF analogue and is marketed under various brand names, including Neupogen(®) (Amgen), Imumax(®) (Abbott Laboratories), Neukine(®) (Intas Biopharmaceuticals) and others. It is banned in both human and equine sports owing to its potential for misuse. In order to control the abuse of filgrastim in equine sports, a method to identify unequivocally its prior use in horses is required. This study describes an effective screening method for filgrastim in equine plasma by enzyme-linked immunosorbant assays (ELISA), and a follow-up confirmatory method for the unequivocal identification of filgrastim by analysing its highly specific tryptic peptide (1)MTPLGPASSLPQSFLLK(17). Filgrastim was isolated from equine plasma by immunoaffinity purification. After trypsin digestion, the mixture was analysed by nano-liquid chromatography-tandem mass spectrometry (LC/MS/MS). Filgrastim could be detected and confirmed at 0.2ng/mL in equine plasma. The applicability of the ELISA screening method and the LC/MS/MS confirmation method was demonstrated by analysing post-administration plasma samples collected from horses having been co-administered with epoetin alfa as recombinant human erythropoietin (rhEPO) and filgrastim as rhG-CSF. rhEPO and filgrastim could be detected in plasma samples collected from horses for at least 57 and 101h respectively. To our knowledge, this is the first identification of filgrastim in post-administration samples from horses.
authors.affiliation: Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, NT, Hong Kong, China. Electronic address: emmie.nm.ho@hkjc.org.hk.
authors.forename: Emmie N M
authors.initials: EN
authors.lastname: Ho
authors.affiliation: Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, NT, Hong Kong, China.
authors.forename: W H
authors.initials: WH
authors.lastname: Kwok
authors.affiliation: Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, NT, Hong Kong, China.
authors.forename: M Y
authors.initials: MY
authors.lastname: Lau
authors.affiliation: Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, NT, Hong Kong, China.
authors.forename: April S Y
authors.initials: AS
authors.lastname: Wong
authors.affiliation: Veterinary Regulation & International Liaison, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, NT, Hong Kong, China.
authors.forename: Kenneth K H
authors.initials: KK
authors.lastname: Lam
authors.affiliation: Veterinary Regulation & International Liaison, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, NT, Hong Kong, China.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Stewart
authors.affiliation: Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, NT, Hong Kong, China. Electronic address: terence.sm.wan@hkjc.org.hk.
authors.forename: Terence S M
authors.initials: TS
authors.lastname: Wan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D004300:Doping in Sports / Q000517:prevention & control; D004921:Erythropoietin / Q000097:blood* / Q000737:chemistry / Q000494:pharmacology; D000069585:Filgrastim; D016179:Granulocyte Colony-Stimulating Factor / Q000097:blood* / Q000737:chemistry; D006736:Horses; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000097:blood / Q000737:chemistry / Q000494:pharmacology; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: C554270:EPO protein, human; D011994:Recombinant Proteins; D004921:Erythropoietin; D016179:Granulocyte Colony-Stimulating Factor; D000069585:Filgrastim
keywords: Erythropoietin; Filgrastim; Granulocyte colony-stimulating factor; Horse; Liquid chromatography–mass spectrometry; Plasma