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Development and Validation of a UHPLC-HRMS/MS Quantification Method for Hypoglycin A and MCPA-Carnitine in Horse Serum in Cases of Atypical Myopathy.

  • Rudolph, Wiebke1
  • Remane, Daniela1
  • Wissenbach, Dirk K1
  • Klein, Carmen2
  • Barnewitz, Dirk2
  • Peters, Frank T1
  • 1 Institute of Forensic Medicine, Jena University Hospital, Jena, Germany. , (Germany)
  • 2 Veterinary Clinic of the fzmb GmbH, Bad Langensalza, Germany. , (Germany)
Published Article
Drug Testing and Analysis
Wiley (John Wiley & Sons)
Publication Date
Nov 17, 2017
DOI: 10.1002/dta.2337
PMID: 29148268


Atypical myopathy (AM) is a fatal disease in horses presumably caused by hypoglycin A (HGA) from ingested maple seeds and its active metabolite methylenecyclopropyl-acetic-acid (MCPA). The aim of this study was the development and validation of a rapid and simple assay for HGA and MCPA-carnitine in horse serum and its application to authentic samples. Identification and quantification were carried out by high resolution LC-MS/MS with fullscan/data-dependent MS2 . Chromatographic separation was performed by isocratic elution on a HILIC column (100 x 2.1 mm, 1.7 μm). Serum samples (250 μL) were worked up by protein precipitation. The method was validated according to international guidelines with respect to selectivity, linearity, accuracy, precision, matrix effects, and recovery. The calibration range was from 100 - 2000 ng/mL for HGA and from 10 - 1000 ng/mL for MCPA-carnitine. HGA and MCPA-carnitine showed acceptable accuracy and precision (bias -3.0% to 1.1%; RSD 9.2% to 12.4%). LOQ was defined as lowest calibrator and well below the lowest published serum concentrations in affected horses. Matrix effects ranged from -79% to +20% (RSD 4.2% to 14.4%), recoveries from 17.9% to 21.1% (RSD 2.3% to 10.8 %) for low and high quality control samples respectively. Applicability was tested in ten authentic AM cases. In all specimens, relevant amounts of HGA and MCPA-carnitine were found (570 - 2000 ng/mL; ~8.5 - 150 ng/mL, respectively). The developed assay allows reliable identification and quantification of HGA and MCPA-carnitine in horse serum and will be helpful to further study the association between HGA/MCPA and AM.

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