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

Cobra (Naja naja kaouthia) venom contains a toxin called α-cobratoxin (α-Cbtx) containing 71 amino acids (MW 7821 Da) with a reported analgesic power greater than morphine. In 2013, the first analytical method for the detection of α-Cbtx in equine plasma was developed by Bailly-Chouriberry et al, allowing the confirmation of the presence of α-Cbtx at low concentrations (1-5 ng/mL or 130-640 fmol/mL) in plasma samples. To increase the method sensitivity and therefore to improve the detection of α-Cbtx in post-administration plasma samples, a nano-liquid chromatography-mass spectrometry/high resolution mass spectrometry (nLC-MS/HRMS) method was developed. This new method allowed us to confirm the presence of α-Cbtx in plasma samples spiked at 100 pg/mL (12.8 fmol/mL) and the detection of α-Cbtx was obtained in plasma samples collected 72 hours post-administration (50 pg/mL or 6.4 fmol/mL) which was defined as the limit of detection (LOD). The presented method is 20-fold more sensitive compared to the method previously described.