Detection of hypoglycin A and MCPA-carnitine in equine serum and muscle tissue: Optimisation and validation of a LC-MS-based method without derivatisation.

BACKGROUND: Measurement of hypoglycin A (HGA) and its toxic metabolite, methylenecyclopropylacetic acid (MCPA), in equine serum confirms a diagnosis of atypical myopathy (AM), a pasture-associated toxic rhabdomyolysis with high mortality linked to the ingestion of Acer trees plant material. Supportive diagnostic tests include plasma acyl-carnitine profiling and urine organic acid testing, but these are not specific for AM. Previously reported HGA and MCPA analytical techniques used liquid chromatography-mass spectrometry (LC-MS) with a derivatising step, but the latter prolongs testing and increases costs. OBJECTIVES: To develop a rapid LCMS method for detection of serum and tissue HGA and MCPA that enables expedited diagnosis for horses with AM. STUDY DESIGN: Analytical test validation. METHODS: Validation parameters to industry standards using as criteria precision, accuracy, linearity, reproducibility and stability in analyte-spiked samples were calculated on 9-calibration points and 3 different validation concentrations in both serum and muscle tissue. RESULTS: The test was successfully validated for the detection of HGA and MCPA-carnitine in equine serum and muscle. Test linearity was excellent (r2  = .999), accuracy was very good for both analytes (93%-108%), precision did not exceed 10% coefficient of variation and reproducibility met the requirements of the Horwitz equation. Stability was unaffected by storage at a range of temperatures. MAIN LIMITATIONS: The spectrum of the tested analytes was limited to only two relevant analytes in favour of a quick and easy analysis. Linearity of the muscle method was not evaluated as calibration curves were not produced in this matrix. CONCLUSION: We report an optimised, simplified and validated method for detection of HGA and MCPA-carnitine in equine serum and muscle suitable for rapid diagnosis of suspected AM cases. The serum-based test should also enable risk assessment of toxin exposure in cograzing horses and assessment of horses with undiagnosed myopathies, while the tissue detection test should help to confirm cases post-mortem and to determine toxin distribution, metabolism and clearance across different tissues.

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.