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.

Grazing Mares on Pasture with Sycamore Maples: A Potential Threat to Suckling Foals and Food Safety through Milk Contamination

“…In addition to raw milk, aflatoxins were found in milk products like cheese and yogurts. One of the toxin responsible for AM—i.e., HGA—was described as heat resistant in aqueous solution for half an hour at 60 °C [ 23 ] and stable over time in dry vegetal matrix [ 24 ]. Therefore, HGA could also represent a threat to food safety.…”

Atypical myopathy‐associated hypoglycin A toxin remains in sycamore seedlings despite mowing, herbicidal spraying or storage in hay and silage

“…In Europe, the main sources of HGA are the seeds and seedlings of Acer pseudoplatanus (sycamore tree) . Although spring outbreaks are less common than those in autumn (they account for between 4–12% of all cases seen annually ), sycamore seedlings have the highest HGA content when compared with seeds and leaves . Consequently, horse owners have legitimate concerns about the risk that sycamore seedlings pose to their grazing horses, particularly given that seedlings often grow in profusion .…”

“…Outbreaks of AM are highly variable and seasonal, with most cases occurring in the autumn (when seeds are present on pasture) or in the spring (when seedlings germinate and grow) . In Europe, the main sources of HGA are the seeds and seedlings of Acer pseudoplatanus (sycamore tree) . Although spring outbreaks are less common than those in autumn (they account for between 4–12% of all cases seen annually ), sycamore seedlings have the highest HGA content when compared with seeds and leaves .…”

Potential new sources of hypoglycin A poisoning for equids kept at pasture in spring: a field pilot study

“…So, samaras of these box elder trees were incomplete and thus sterile. HGA was not detected in samaras of common ash as recently described 18 In a previous study, Westermann et al (2016) measured HGA in sycamore maple samples that have been stored at −20°C within 24 hours of collection up to analysis. The concentrations found varied highly between 97 mg/kg to 2539 mg/kg in seedlings and from 0 mg/kg to 3683 mg/kg in samaras of sycamore maple taken in spring.…”