Detection and confirmation of α-cobratoxin in equine plasma by solid-phase extraction and liquid chromatography coupled to mass spectrometry

Abstract

α-Cobratoxin (CTX) is a large peptide (71 amino acids) with strong analgesic effect and may be misused in sports such as horse racing. To prevent such misuse, a sensitive method is required for detection and confirmation of the toxin in equine samples. CTX was extracted from equine plasma using an optimized mixed-mode solid-phase extraction (SPE) procedure. Extracted CTX was reduced with dithiothreitol and alkylated with iodoacetamide, and then was digested by trypsin at 56°C for 30min. The digest was analysed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and tryptic peptides T2 (3CFITPDITSK12) and T4 (24TWCDAFCSIR33) were monitored for detection and confirmation of CTX. The limit of detection (LOD) was 0.05ng/mL for CTX in plasma, and the limit of confirmation (LOC) 0.2ng/mL. Unlike small peptides consisting of the 20 canonical amino acids, CTX was stable in equine plasma at ambient temperature for at least 24h. The developed analytical method was successfully applied to analysis of incurred plasma samples; CTX was detected in plasma collected 15min through 36h post subcutaneous administration of CTX (2.0mg dose) to a research horse, and confirmed 30min through 24h. Additionally, an approach named "reliable targeted SEQUEST search" has been proposed for assessing the specificity of T2 at product ion spectrum level for confirmation of CTX. T2 is uniquely specific for CTX, as evaluated with this approach and BLAST search. Furthermore, the effect of dimethyl sulfoxide (DMSO) as a mobile phase additive on electrospray (ESI) response of T2 and T4, background noise level and signal to noise ratio (S/N) was examined; DMSO increased signal intensity of T2 and T4 by a factor of less than 2. It is the first report that DMSO raised background noise level and did not improve S/N for the peptides, to the authors' knowledge. The developed analytical method may be applicable for analysis of CTX in plasma from other species such as greyhound dogs or even human beings.