Quantitation of γ-aminobutyric acid in equine plasma by hydrophilic interaction liquid chromatography with tandem mass spectrometry.

Abstract

γ-Aminobutyric acid is the principal inhibitory neurotransmitter in the central nervous system and regulates the neuronal excitability. There has been anecdotal evidence that γ-aminobutyric acid has been used within a few hours prior to competition in equine sports to calm down nervous horses. However, regulating the use of γ-aminobutyric acid is challenging because it is an endogenous substance in the horse. γ-Aminobutyric acid is usually present at low ng/mL levels in equine plasma; therefore, a sensitive method has to be developed to quantify these low background levels. Measuring low concentrations of endogenous γ-aminobutyric acid is essential to establish a threshold that can be used to differentiate levels attributable to exogenous administrations of γ-aminobutyric acid. A hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method was developed and validated for the quantitation of γ-aminobutyric acid in equine plasma. Calibrators were prepared in artificial surrogate matrix consisting of 35mg/mL equine serum albumin in phosphate buffered saline. Samples were prepared by protein precipitation with acetonitrile. Utilizing this methodology, a total of 403 equine plasma samples collected post-competition from horses participating in equestrian events in Canada were analyzed.