LC-MS can show the way to measure hypoglycin A

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  • Published: Jan 1, 2018
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: LC-MS can show the way to measure hypoglycin A

Fatal horse disease linked to sycamore seeds

Atypical myopathy is a horse disease with a high mortality rate of 85% so rapid diagnosis is critical in order to save the animal. The disease is caused by eating the seeds of the sycamore (Acer pseudoplatanus) or those of some related species of tree, such as the box elder (Acer negundo), which is native to North America. The toxic compound hypoglycin A (sometimes spelt hypoglycine A) is the cause of the disease; it is converted inside the body to methylene cyclopropyl acetic acid (MCPA, not to be confused with the herbicide with the same abbreviation). MCPA is believed to be the main active toxin and is typically found as its carnitine conjugate in serum.

Existing methods of detecting hypoglyin A and MCPA usually involve lengthy sample preparation and derivatisation steps. However, when dealing with this disease rapid detection is required to diagnose whether or not it is present. The researchers from Jena and Bad Langensalza developed a new method to detect the toxins in serum using UHPLC and tandem MS, without any derivatisation or lengthy sample preparation.

Hypoglycin A and MCPA-carnitine detected by UHPLC and MS/MS

Protein was precipitated from a serum sample by adding cold acetonitrile and placing the samples in a freezer at -20 °C for 1 hour. The solids were removed by centrifugation and the supernatant liquid was evaporated to dryness. The residue was dissolved in an 80:20 mixture of 50 mM aqueous ammonium formate and acetonitrile.

UHPLC was carried out using a Thermo Fisher Dionex UHPLC instrument fitted with a Thermo Fisher Syncronis HILIC column, which gave better separation of hypoglycin A and MCPA-carnitine than the RP-C18 column used by previous workers. Isocratic elution was employed using a 15:85 mixture of 50 mM aqueous ammonium formate and acetonitrile. The flow rate was 0.6 mL/min and the overall run time was only 15 minutes. Mass spectrometry was carried out with a Thermo Fisher Q Exactive Focus instrument using heated electrospray ionisation (ESI). Parallel reaction monitoring (PRM) mode was used to quantify hypoglycin A and MCPA-carnitine, looking at accurate fragments, while full scan mode was used to look at possible metabolites and veterinary drugs.

The peaks for hypoglycin A and MCPA-carnitine were clear of interference from the serum and did not co-elute with common veterinary drugs. The recovery for both species was low, ca. 20%, but consistent between runs. The method gave acceptable precision and accuracy. The limits of quantification (LOQ) were 100 ng/mL for hypoglycin A and 10 ng/ml for MCPA-carnitine, while the limits of detection were 50 ng/mL for hypoglycin A and 2.5 ng/mL for MCPA-carnitine.

The method was applied to retained blood samples from ten affected horses, nine of which had died and one survived. As expected, the surviving horse showed lower levels of hypoglycin A and MCPA-carnitine than the nine that died. The surviving horse was treated with the veterinary drugs flunixin and dexamethanasone, which could be detected by full scan MS/MS with the new method.

New UHPLC method allows quicker detection of atypical myopathy

The new method allows for relatively quick determination of hypoglycin A and MCPA-carnitine, the only disadvantage being the hour in the freezer needed for protein precipitation. The technique can also detect some metabolites and veterinary drugs. The decrease in the analytical time should allow more rapid diagnosis of atypical myopathy, thus enabling more horses to receive the correct treatment promptly enough for them to survive.

Related Links

Drug Testing and Analysis, 2017, Early View Paper. Rudolf et al. Development and validation of a UHPLC-HRMS/MS quantification method for hypoglycin A and MCPA-carnitine in horse serum in cases of atypical myopathy.

International Journal of Molecular Sciences, 2015, 16, 28566-28581. Rauniyar et al. Parallel reaction monitoring: A targeted experiment performed using high resolution and high mass accuracy mass spectrometry.

Equine Veterinary Journal, 2016, 48, 418-421. Żuraw et al. Equine atypical myopathy caused by hypoglycin A intoxication associated with ingestion of sycamore maple tree seeds.

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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