Differentiation and identification of recombinant human erythropoietin and darbepoetin Alfa in equine plasma by LC-MS/MS for doping control.
Recombinant human erythropoietin (rhEPO) and darbepoetin alfa (DPO) are protein-based drugs for the treatment of anemia in humans by stimulating erythrocyte production. However, these agents are abused in human and equine sports due to their potential to enhance performance. This paper describes the first method for differentiation and identification of rhEPO and DPO in equine plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method comprised analyte extraction and enrichment by immunoaffinity separation with anti-rhEPO antibodies, dual digestion by trypsin and peptide-N-glycosidase F (PNGase F), and analysis by LC-MS/MS. Two unique deglycosylated tryptic peptides, (21)EAENITTGCAEHCSLNENITVPDTK (45) (T 5) from rhEPO and (77)GQALLVNSSQVNETLQLHVDK (97) (T 9) from DPO, were employed for differentiation and identification of rhEPO and DPO via LC retention times and major product ions. The limit of identification was 0.1 ng/mL for DPO and 0.2 ng/mL for rhEPO in equine plasma, and the limit of detection was 0.05 ng/mL for DPO and 0.1 ng/mL for rhEPO. Analyte carryover problem encountered was solved by adding 20% acetonitrile to the solvent of the sample digest to increase solubility of the peptides. This method was successfully applied to identification of DPO in plasma samples collected from a research horse following DPO administration and from racehorses out of competition in North America. Thus, it provides a powerful tool in the fight against blood doping with rhEPO and DPO in the horse racing industry.
Guan
Fuyu
F
University of Pennsylvania School of Veterinary Medicine, New Bolton Center Campus, 382 West Street Road, Kennett Square, Pennsylvania 19348, USA.
Uboh
Cornelius E
CE
Soma
Lawrence R
LR
Birks
Eric
E
Chen
Jinwen
J
You
Youwen
Y
Rudy
Jeffrey
J
Li
Xiaoqing
X
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D000068256:Darbepoetin alfa; D004300:Doping in Sports; D004921:Erythropoietin / Q000031:analogs & derivatives* / Q000097:blood*; D006736:Horses / Q000097:blood*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D053719:Tandem Mass Spectrometry / Q000379:methods*
D011994:Recombinant Proteins; D004921:Erythropoietin; D000068256:Darbepoetin alfa