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

The research article focused on developing and validating a sensitive method to detect and quantify γ-aminobutyric acid (GABA), a principal inhibitory neurotransmitter, in the plasma of horses. The method would help establish a threshold to differentiate between endogenous and exogenously administered GABA – the latter which is allegedly used to calm horses before competitions.

Overview of the Research

• The researchers centered their study on γ-aminobutyric acid (GABA), a neurotransmitter primarily responsible for reducing neuronal excitability in the central nervous system. It is often present in low nanograms per milliliter (ng/mL) levels in horse plasma. Because GABA is a naturally occurring substance in horses, differentiating between its regular (endogenous) levels and those boosted by external supplements (exogenous) was the main challenge for this study.
• The idea to establish a baseline came from anecdotal instances where GABA was reportedly used in equine sports to calm nervous horses right before competitions. Differentiating between its natural and artificially elevated levels is important for ethical competition and horse welfare.
• To overcome this challenge, the team developed a sensitive method using hydrophilic interaction liquid chromatography combined with tandem mass spectrometry. This new method would measure low concentrations of endogenous GABA, enabling the establishment of a threshold to distinguish between natural and artificially boosted levels.

Method and Sample Preparation

• The research team created calibrators using an artificial surrogate matrix consisting of 35 mg/mL equine serum albumin in phosphate buffered saline. These calibrators serve as a reference to measure the amount of GABA present in the plasma samples.
• The preparation of samples involved protein precipitation with acetonitrile, a common method used to remove proteins from biological samples for downstream analysis. This step was crucial to achieve an accurate measurement of GABA, free of other interfering proteins.
• Once prepared, the samples underwent hydrophilic interaction liquid chromatography (a technique to separate compounds based on their affinity to water) followed by tandem mass spectrometry (an analytical technique for precise mass measurement).

Actual Research and Results

• After developing and validating the method, it was put to use on a total of 403 equine plasma samples. The samples were taken from horses after they participated in various equestrian events in Canada.
• The results of this research are not mentioned in the abstract. But, the idea is to use these results to establish a threshold for normal GABA levels in horses. This will enable the identification of any abnormal increases in GABA levels, likely due to external administration.