Screening and confirmation of recombinant human erythropoietin and darbepoietin-α in spiked plasma samples from drug-free horses

Gupta, Sumit; Sage, Abby M.; Singh, Ashok K.

doi:10.1016/j.aca.2005.07.065

Cited by 11 publications

(24 citation statements)

References 26 publications

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“…Use of an ionene-dynamically coated capillary in a CE-MS system has separated three glycoforms of EPO; molecular weights were verified by MALDI-TOF-MS (Yu et al, 2005a). MALDI-TOF has also been used to differentiate rhEPO (29 kDa from Research Diagnostics, Flanders, NJ) from darbepoietin (36 kDa, a product from Amgen, Thousand Oaks, CA) in spiked horse plasma (Gupta, Sage, & Singh, 2005). Four immunoassay based methods detected both EPOs but could not differentiate them and three also cross-reacted with equine EPO.…”

Section: A Biopharmaceuticalsmentioning

confidence: 99%

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for the period 2005–2006

Harvey

2010

Mass Spectrometry Reviews

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This review is the fourth update of the original review, published in 1999, on the application of MALDI mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2006. The review covers fundamental studies, fragmentation of carbohydrate ions, method developments, and applications of the technique to the analysis of different types of carbohydrate. Specific compound classes that are covered include carbohydrate polymers from plants, N- and O-linked glycans from glycoproteins, glycated proteins, glycolipids from bacteria, glycosides, and various other natural products. There is a short section on the use of MALDI-TOF mass spectrometry for the study of enzymes involved in glycan processing, a section on industrial processes, particularly the development of biopharmaceuticals and a section on the use of MALDI-MS to monitor products of chemical synthesis of carbohydrates. Large carbohydrate-protein complexes and glycodendrimers are highlighted in this final section.

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Section: A Biopharmaceuticalsmentioning

confidence: 99%

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for the period 2005–2006

Harvey

2010

Mass Spectrometry Reviews

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“…The method described previously [18] was used in this study. In brief, plasma samples were mixed with the incubation buffer (3:1 v/v) and the denaturation solution (3:0.25 v/v) supplied with the kit (ProZyme kit).…”

Section: Deglycosylation Of Rhepo and Darmentioning

confidence: 99%

“…Horse albumin-antibody coupled IA columns (661 cm 2 ) were prepared as described previously for anti-rhEPO coupled columns [18]. A 0.5 mL aliquot of the blank or spiked plasma was stored for ELISA.…”

Section: Albumin Depletionmentioning

confidence: 99%

“…(ii) DAR and rhEPO interacts with albumin to form a DAR-or rhEPO-albumin complex that results in coelution of the two proteins when immunoaffinity (IA) methods are used for their extraction [18].…”

mentioning

confidence: 99%

“…(iv) Plasma rhEPO or DAR concentrations in horses may be below the detection limits of the analytical methods. A preliminary study has shown that eEPO concentration in plasma samples from drug-free race horses was 60 6 25 mU/mL [18]. Thus, horses with EPO level .110 mU/mL (mean 1 2 SD) will be called violation.…”

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confidence: 99%

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Analysis of recombinant human erythropoietin and darbepoietin in spiked plasma

Singh

Gupta

2007

Proteomics Clinical Apps

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Darbepoietin (DAR) and recombinant human erythropoietin (rhEPO) stimulate erythropoiesis, leading to an increase in red blood cells. Along with their legitimate clinical use, rhEPO and DAR are also misused in racing horses for performance enhancement. To control the illegal use of DAR and rhEPO, it is important to develop analytical methods for the detection and confirmation of these proteins in plasma. Analysis of rhEPO and DAR in plasma is challenging due to the presence of a number of high abundance proteins including albumin that interferes with their extraction. The present study showed that the extraction of rhEPO or DAR from plasma using anti-EPO-antibody coupled immunoaffinity (IA) extraction yielded low (25-40%) recovery. Albumin-depletion using antialbumin-antibody coupled IA columns also depleted the target proteins and further reduced their recovery. Pre-extraction of spiked plasma using hydroxyapatite (HTP)-ProGel or ConA columns followed by the IA column yielded 65 to 70% recovery. The extracted samples were (i) analyzed directly with or without SDS-PAGE for intact proteins and (ii) analyzed after trypsin hydrolysis, with or without SDS-PAGE, for peptide fingerprinting using MALDI-TOF. Trypsin and enolase were used as internal calibrators for intact protein analysis and a peptide EYEATLEECCAK was used as internal calibrator for fragment analysis. Analysis of extracted sample without SDS-PAGE yielded, along with the target proteins (rhEPO and DAR), albumin and other related proteins. SDS-PAGE separated the target proteins with albumin and yielded clean samples. Inclusion of internal calibrators resulted in a linear dose-response relationship for both intact protein and digested fragments and allowed quantification of the target peptides. Thus, extraction of plasma using a combination of ConA and IA extractions yielded approximately 70% recovery of target proteins with a small amount of albumin and other proteins. SDS-PAGE improved the quality of the MALDI-TOF results. Minimum detection limits for digested fragments were lower than those for intact proteins.

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Blood cells RNA biomarkers as a first long‐term detection strategy for EPO abuse in horseracing

Bailly-Chouriberry¹,

Noguier²,

Manchon³

et al. 2010

Drug Testing and Analysis

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Recombinant human erythropoietins (rHuEPOs) are glycoproteins drugs, produced by the pharmaceutical industry to restore production of red blood cells by stimulating human bone marrow for which this pathology has been diagnosed. It is suspected that these molecules are diverted as doping agents in horseracing to enhance oxygen transport and aerobic power in racehorses. Although indirect double-blotting or direct liquid chromatography-mass spectrometry (LC-MS) methods have been developed to confirm the presence of rHuEPO in a sample, the short detection time (48 h) is still a problem for doping control. In this context, gene profiling investigation through Serial Analysis of Gene Expression (SAGE) has been conducted on seven thoroughbreds treated with Eprex. This functional genomic method has been performed from total blood cells collected from each animal to assess the mRNA expression consecutive to rHuEPO injections. Sample pooling was chosen as a powerful, cost-effective, and rapid means of identifying the most common and specific changes in terms of gene expression profile and to eliminate individual variation. Consequently, three SAGE libraries were constructed, before, during, and after Eprex treatment. More than 71 440 mRNA signatures were observed and subjected to statistical analysis; 49 differentially expressed genes were identified and analyzed by qPCR. From the selected gene list, were defined as potential biomarkers in terms of their low inter-individual variation and capacity as strong markers of rHuEPO administration up to 60 days after the beginning of the doping period. In this paper, a new strategy is proposed to the horseracing industry to prevent rHuEPO abuse.

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Screening and confirmation of recombinant human erythropoietin and darbepoietin-α in spiked plasma samples from drug-free horses

Cited by 11 publications

References 26 publications

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for the period 2005–2006

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for the period 2005–2006

Analysis of recombinant human erythropoietin and darbepoietin in spiked plasma

Blood cells RNA biomarkers as a first long‐term detection strategy for EPO abuse in horseracing

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