caa a22 4500 445803428 CHVBK 20180317145149.0 cr unu---uuuuu 170323e20111001xx s 000 0 eng 10.1007/s10337-011-2119-y doi (NATIONALLICENCE)springer-10.1007/s10337-011-2119-y UPLC-MS/MS Method for the Identification of Recombinant Human Erythropoietin Analogues in Horse Plasma and Urine [Elektronische Daten] [James Scarth, Cathrin Seibert, Pamela Brown, Phil Teale, Gavin Beamon, Clive Pearce, Richard Sams] Recombinant human erythropoietin (rhEPO) analogues are known to have been used in horse sports for their assumed performance enhancing properties. Recently, several authors have published liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods for confirming the presence of rhEPO analogues in horse plasma. In the current study, an improved LC-MS/MS confirmatory procedure for rhEPO, darbepoetin (DPO) and continuous erythropoietin receptor activator (CERA) in horse plasma was developed and validated. The method was also adapted for and applied to urine samples for the first time. Similar to previously published plasma assays, the methods utilise size exclusion and immunoaffinity extraction prior to tryptic cleavage, enzymatic deglycosylation and LC-MS/MS analysis of the resulting signature tryptic peptides (rhEPO/CERA T5, rhEPO/CERA/DPO T6 and DPO T9). However, the novel application of UPLC chromatography significantly improves the run time of the method compared to nano- or micro-LC and its robustness compared to nano-LC. Furthermore, recombinant canine EPO was found to serve as an effective internal standard, thus allowing confidence in interpretation of the success/failure of every step in the procedure. Limits of detection for confirming the presence of rhEPO, CERA and DPO in plasma were 0.1, 0.25 and 0.05ngmL−1, respectively, which were equal to or lower than limits achieved using previously published LC-MS/MS based methods. Limits of detection for confirming the presence of rhEPO, CERA and DPO in urine were 0.05, 0.15 and 0.025ngmL−1 and the analysis of urine samples collected from horses administered rhEPO (Eprex™) or DPO (Aranesp™) demonstrated the use of this matrix as a suitable alternative in situations where plasma is not available. Springer-Verlag, 2011 Column liquid chromatography nationallicence Erythropoietin nationallicence Human nationallicence Horse nationallicence Plasma nationallicence Urine nationallicence Scarth James HFL Sport Science (an LGC company), Newmarket Road, Fordham, CB7 5WW, Cambridgeshire, UK aut Seibert Cathrin HFL Sport Science (an LGC company), Newmarket Road, Fordham, CB7 5WW, Cambridgeshire, UK aut Brown Pamela HFL Sport Science (an LGC company), Newmarket Road, Fordham, CB7 5WW, Cambridgeshire, UK aut Teale Phil HFL Sport Science (an LGC company), Newmarket Road, Fordham, CB7 5WW, Cambridgeshire, UK aut Beamon Gavin HFL Sport Science (an LGC company), Newmarket Road, Fordham, CB7 5WW, Cambridgeshire, UK aut Pearce Clive HFL Sport Science (an LGC company), Newmarket Road, Fordham, CB7 5WW, Cambridgeshire, UK aut Sams Richard HFL Sport Science (an LGC company), Alysheba Way, 40509, Lexington, KY, USA aut Chromatographia Springer-Verlag 74/7-8(2011-10-01), 593-608 0009-5893 74:7-8<593 2011 74 10337 https://doi.org/10.1007/s10337-011-2119-y text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10337-011-2119-y text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Scarth James HFL Sport Science (an LGC company), Newmarket Road, Fordham, CB7 5WW, Cambridgeshire, UK aut NATIONALLICENCE 700 1- Seibert Cathrin HFL Sport Science (an LGC company), Newmarket Road, Fordham, CB7 5WW, Cambridgeshire, UK aut NATIONALLICENCE 700 1- Brown Pamela HFL Sport Science (an LGC company), Newmarket Road, Fordham, CB7 5WW, Cambridgeshire, UK aut NATIONALLICENCE 700 1- Teale Phil HFL Sport Science (an LGC company), Newmarket Road, Fordham, CB7 5WW, Cambridgeshire, UK aut NATIONALLICENCE 700 1- Beamon Gavin HFL Sport Science (an LGC company), Newmarket Road, Fordham, CB7 5WW, Cambridgeshire, UK aut NATIONALLICENCE 700 1- Pearce Clive HFL Sport Science (an LGC company), Newmarket Road, Fordham, CB7 5WW, Cambridgeshire, UK aut NATIONALLICENCE 700 1- Sams Richard HFL Sport Science (an LGC company), Alysheba Way, 40509, Lexington, KY, USA aut NATIONALLICENCE 773 0- Chromatographia Springer-Verlag 74/7-8(2011-10-01), 593-608 0009-5893 74:7-8<593 2011 74 10337 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer