Confirmatory Analysis of Continuous Erythropoietin Receptor Activator and Erythropoietin Analogues in Equine Plasma by LC−MS for Doping Control
Citations Over TimeTop 18% of 2010 papers
Abstract
Continuous erythropoietin receptor activator (CERA) is the third generation of recombinant human erythropoietin (rhEPO) medication that retains the effect of promoting red blood cell production but has longer duration of action in the body. CERA, rhEPO, and darbepoetin alpha (DPO) can be misused to enhance performance in both human and equine athletes. To deter such misuse, a very selective and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has now been developed for identification of CERA, rhEPO, and DPO in equine plasma. The method employs a new signature tryptic peptide, T8 ((54)MEVGQQAVEVWQGLALLSEAVLR(76), common to the three proteins), and improved immunoaffinity extraction. The analytes were extracted by anti-rhEPO antibodies from plasma samples that were pretreated with polyethylene glycol (PEG) 6000. The extracted analytes were digested by trypsin and analyzed by LC-MS/MS. The limit of identification was 0.5 ng/mL for CERA, 0.2 ng/mL for rhEPO, and 0.1 ng/mL for DPO in equine plasma; the limit of detection was 0.3 ng/mL for CERA, 0.1 ng/mL for rhEPO, and 0.05 ng/mL for DPO. Specificity of the method was assessed via BLAST and SEQUEST protein database searches, and the T8 is extremely specific at both peptide and product ion levels for the identification of CERA, rhEPO, and DPO. This method was successful in identifying CERA and DPO in plasma samples collected from research horses post the drug administrations. It provides a useful tool in the fight against blood doping with CERA, rhEPO, and DPO in racehorses. Additionally, the following two technical approaches adopted in this study may also be helpful in protein identifications and biomarker discoveries in a broad scope: precipitating plasma proteins with PEG 6000 to improve immunoaffinity extraction efficiency of the target proteins and making a large and more lipophilic peptide detectable at low concentrations by increasing its solubility in the sample solvent.
Related Papers
- → Determination of Ochratoxin A in wine at sub ng/mL levels by solid-phase microextraction coupled to liquid chromatography with fluorescence detection(2006)86 cited
- → Highly Sensitive Determination and Validation of Gabapentin in Pharmaceutical Preparations by HPLC with 4-Fluoro-7-Nitrobenzofurazan Derivatization and Fluorescence Detection(2011)16 cited
- → Analysis of ochratoxin A in milk after direct immunoaffinity column clean-up by high-performance liquid chromatography with fluorescence detection(2007)28 cited
- → Fast high-performance liquid chromatographic screening method for the presence of trenbolone and its major metabolite in urine of slaughter cattle(1985)16 cited
- → ChemInform Abstract: Silacrown Ethers: Synthesis of Macrocyclic Diphenyl Polyethylene Glycol Mono‐ and Disilanes.(1994)