Detection of equine atypical myopathy-associated hypoglycin A in plant material: Optimisation and validation of a novel LC-MS based method without derivatisation.

Hypoglycin A (HGA) toxicity, following ingestion of material from certain plants, is linked to an acquired multiple acyl-CoA dehydrogenase deficiency known as atypical myopathy, a commonly fatal form of equine rhabdomyolysis seen worldwide. Whilst some plants are known to contain this toxin, little is known about its function or the mechanisms that lead to varied HGA concentrations between plants. Consequently, reliable tools to detect this amino acid in plant samples are needed. Analytical methods for HGA detection have previously been validated for the food industry, however, these techniques rely on chemical derivatisation to obtain accurate results at low HGA concentrations. In this work, we describe and validate a novel method, without need for chemical derivatisation (accuracy = 84-94%; precision = 3-16%; reproducibility = 3-6%; mean linear range R2 = 0.999). The current limit of quantitation for HGA in plant material was halved (from 1μg/g in previous studies) to 0.5μg/g. The method was tested in Acer pseudoplatanus material and other tree and plant species. We confirm that A. pseudoplatanus is most likely the only source of HGA in trees found within European pastures.