Identification of α-Cobratoxin in Equine Plasma by LC-MS/MS for Doping Control

Abstract

Cobra venom (Naja kaouthia) contains a toxin called α-cobratoxin (α-Cbtx). This toxin is a natural protein containing 71 amino acids (MW 7821 Da) with a reported analgesic potency greater than morphine. In 2007, in USA, this substance was found in the barns of a thoroughbred trainer and since then till date, the lack of a detection of this molecule has remained a recurring problem for the horseracing industry worldwide. To solve this problem, the first method for the detection of α-cobratoxin in equine plasma has now been developed. Plasma sample (3 mL) was treated with ammonium sulfate at the isoelectric point of α-Cbtx, and the pellet was dissolved in a phosphate buffer and mixed with methanol for precipitation. The supernatant was then concentrated prior to its extraction on WCX SPE cartridges. The eluate was concentrated with two consecutive filtration steps before the trypsin digestion. The samples were analyzed using a LC-MS/MS Q Exactive instrument at 70,000 resolution on the product ions of the doubly charged precursor of the target peptide ((24)TWCDAFCSIR(33)). The method was validated (n = 18) at 5 μg/L (640 pmol/L) according to the Association of Official Racing Chemists (AORC) requirements. The lower limit of detection was 1 μg/L (130 pmol/L). The present method has made it possible for us to confirm the presence of α-Cbtx in a horse plasma sample 24 h post the administration of α-Cbtx. Thus, the present method provides the first sensitive, specific, and reliable analytical method to confirm the presence of α-Cbtx in equine plasma.

Use of split‐free nano‐liquid chromatography–mass spectrometry/high resolution mass spectrometry interface to improve the detection of α‐cobratoxin in equine plasma for doping control

“…The split‐free or splitless nLC system hyphenated to HRMS method combined with a robust sample preparation is established to be a reliable technology to confirm the presence of α‐ Cbtx in plasma samples spiked at 100 pg/mL (12.8 fmol/mL). The LOD based on the single ion y 8 + (m/z 912.3702) was 50 pg/mL (6.4 fmol/mL).…”

“…According to the previous method, the improvement of the α‐ Cbtx detection in terms of sensitivity and specificity focused on the 24 TWCDAFCSIR 33 peptide . The peptide standard was directly introduced with a concentration of 10 ng/μL in water/acetonitrile with 0.2% FA (50/50) into a Q Exactive mass spectrometer.…”

“…A minor ion was also found at m/z 375.2340 corresponding to y 3 + . These results are in accordance with the fragmentation previously described resulting from major product ions at each side of the disulfide bridge (Figure ). The comparison between the experimental mass to charge ratio (m/z) obtained from the 3 specific product ions of α‐Cbtx peptide ion [M+2H] 2+ (m/z 600.7527) to the theoretical data obtained from their elemental composition was performed (Table ).…”

“…The method was previously described by Bailly‐Chouriberry et al Briefly, 3 mL of plasma and 15 mL of Tris/HCl buffer (0.2 M, pH 8.6) were added and mixed. Protein precipitation was carried out by adding a saturated solution of ammonium sulfate (pH 8.6) at 40% and incubating overnight at 4°C.…”

“…Analytes were eluted with 3 mL 25/75 water/acetonitrile containing 0.1% TFA. The filtration step and the trypsinolysis were described previously by Bailly‐Chouriberry et al The peptide mixture (40 μL) was carefully transferred into a glass HPLC insert and completed with 5 μL acetonitrile and the internal standard solution (5 μL, 80 pM). Before analysis, samples were diluted 10 times with a solution of water/acetonitrile (90/10) with 0.2% FA and analysed within 2 days.…”