LC-MS/MS Method for Confirmation of Recombinant Human Erythropoietin and Darbepoetin in Equine Plasma
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- 作者：Fuyu Guan ; Cornelius E. Uboh ; Lawrence R. Soma ; Eric Birks ; Jinwen Chen ; Janis Mitchell ; Youwen You ; Jeffrey Rudy ; Fran Xu ; Xiaoqing Li ; Gustave Mbuy
- 刊名：Analytical Chemistry
- 出版年：2007
- 出版时间：June 15, 2007
- 年：2007
- 卷：79
- 期：12
- 页码：4627 - 4635
- 全文大小：150K
- 年卷期：v.79,no.12(June 15, 2007)
- ISSN：1520-6882
文摘
Recombinant human erythropoietin (rhEPO) and darbepoetin 
(DPO) are protein-based drugs for the treatment of anemia by stimulating red blood cell production.Consequently, they are abused in human and equinesports. To deter their abuse in the horse racing industry,a sensitive and reliable method for confirmation of theseagents in equine plasma has been in urgent need. Such amethod by LC-MS/MS is described in this paper. Themethod involved analyte enrichment by immunoaffinityseparation using anti-rhEPO antibody linked to magneticbeads, digestion by trypsin, and analysis by LC-MS/MS.Two specific proteotypic peptides, 46VNFYAWK52 and144VYSNFLR150 from rhEPO and DPO were employed forconfirmation of the analytes based on chromatographicretention times and major product ions. The limit ofconfirmation of this method was 0.2 ng/mL, and the limitof detection was 0.1 ng/mL for rhEPO and DPO in equineplasma. This method was successful in confirming thepresence of rhEPO and DPO in plasma samples collectedfrom research horses to which rhEPO or DPO wasadministered and from racehorses following competitionand in noncompetition samples in North America. To ourknowledge, this is the first LC-MS method with adequatesensitivity and specificity in providing unequivocal confirmation of rhEPO and DPO in equine plasma samples.This method provides a powerful enforcement tool thatwas lacking in the fight against the abuse of rhEPO andDPO in the horse racing industry.
(DPO) are protein-based drugs for the treatment of anemia by stimulating red blood cell production.Consequently, they are abused in human and equinesports. To deter their abuse in the horse racing industry,a sensitive and reliable method for confirmation of theseagents in equine plasma has been in urgent need. Such amethod by LC-MS/MS is described in this paper. Themethod involved analyte enrichment by immunoaffinityseparation using anti-rhEPO antibody linked to magneticbeads, digestion by trypsin, and analysis by LC-MS/MS.Two specific proteotypic peptides, 46VNFYAWK52 and144VYSNFLR150 from rhEPO and DPO were employed forconfirmation of the analytes based on chromatographicretention times and major product ions. The limit ofconfirmation of this method was 0.2 ng/mL, and the limitof detection was 0.1 ng/mL for rhEPO and DPO in equineplasma. This method was successful in confirming thepresence of rhEPO and DPO in plasma samples collectedfrom research horses to which rhEPO or DPO wasadministered and from racehorses following competitionand in noncompetition samples in North America. To ourknowledge, this is the first LC-MS method with adequatesensitivity and specificity in providing unequivocal confirmation of rhEPO and DPO in equine plasma samples.This method provides a powerful enforcement tool thatwas lacking in the fight against the abuse of rhEPO andDPO in the horse racing industry.