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

Abstract

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.

Fig 1. Results obtained in the extraction experiment, represented as mean ± SD of 6 seed homogenates exposed to 3 different solvents at 2 different temperatures and durations.

MeOH yielded the best results at both temperatures and ethanol showed consistently low extraction performance. Aqueous extraction improved with time. Bars with different lower case letters are statistically significantly different: a-b p = 0.004; a-c p = 0.006; a-d p = 0.04; a-e p = 0.22; b-e: p = 0.01.

Fig 2. Matrix-matched experiments testing HGA-spiked extracts added to seeds from four different trees: C avellana (hazelnut), A. campestre (field maple), A. platanoides (Norway maple) and Fraxious excelxior (ash tree).

Results are presented as mean ±SD of HGA measurements in two different seed matrices/species. There was no statistically significant difference in recoveries obtained from each of the first 3 tree seed homogenates, however ash tree showed poor recovery when spiked with HGA. Hazelnut was finally used for method validation as it is easy to obtain in local markets without need for field collection. Bars with different lower case letters are statistically significantly different (p = 0.001).

Fig 3. Results are presented as mean ±SD of duplicate injections.

Calibration standards were evaluated at day 1 (D1), day 34 (D34) and day 62 (D62) after undergoing 9 freeze-thaw cycles. There was no statistically significant difference between any of the measurements, showing that the calibration standards are stable at -20°C for at least 2 months and that they maintained HGA concentrations after several freeze-thaw cycles.