Journal of Chromatography B, 997 (2015) 75–80 Contents lists available at ScienceDirect Journal of Chromatography B jou rn al hom ep age: www.elsevier.com/locate/chromb Quantification of hypoglycin A in serum using aTRAQ ® assay Franc ¸ ois Boemer a,∗ , Michelle Deberg a , Roland Schoos a , Etienne Baise b , Hélène Amory c , Gilbert Gault d , Jeremy Carlier e , Yvan Gaillard e , Christel Marcillaud-Pitel f , Dominique Votion c a Biochemical Genetics Laboratory, Human Genetics, CHU Liege, University of Liege, Belgium b Department of animal Productions: Biostatistics, Economy and animal selection, Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of Liege, Belgium c Equine Pole, Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of Liege, Belgium d Usc 1233 Ediss, VETAGROSUP, Ecole nationale vétérinaire de Lyon, France e LAT LUMTOX, La Voulte-sur-Rhône, France f Réseau d’EpidémioSurveillance en Pathologie Equine (RESPE), Caen, France a r t i c l e i n f o Article history: Received 24 February 2015 Received in revised form 27 May 2015 Accepted 4 June 2015 Available online 10 June 2015 Keywords: Hypoglycin A Atypical myopathy Amino acids Mass spectrometry aTRAQ a b s t r a c t Background: Hypoglycin A has been recently identified has the causal agent of atypical myopathy (AM) in horses. Its identification and quantification in equine’s biological fluids is thus a major concern to confirm maple poisoning and to provide insight into the poorly understood mechanism of hypoglycin A intoxication. Methods: Quantification of hypoglycin A has been achieved with the aTRAQ kit for amino acid analysis of physiological fluids (AB Sciex). Acquisition method on mass spectrometer has been updated to record the hypoglycin A specific MRM transition. Results: Outlined accuracy profiles demonstrated very reliable data. A good linearity was observed from 0.09 to 50 mol/L and precision was very good with coefficient of variation below 8%. Fifty-five samples collected from 25 confirmed AM horses revealed significant hypoglycin A concentrations, while toxin was not found in serum of 8 control animals. Conclusions: The described aTRAQ variant method has been analytically and clinically validated. The reliability of our approach is thus demonstrated into the workup of atypical myopathy. © 2015 Elsevier B.V. All rights reserved. 1. Introduction Recent reports have identified hypoglycin A contains in the seeds of some Acer species as the causal agent of atypical myopa- thy in horses [1–2]. In Europe, the source of hypoglycin A in autumn appears to be the seeds of Acer pseudoplatanus [3]. The toxin, abundant in unripe ackee fruits, is well known in human medicine and is responsible for the Jamaican vom- iting sickness [4]. Toxicity mechanism is partially understood and involves the toxic metabolite of hypoglycin A, namely methylenecyclopropylacetic acid (MCPA). MCPA-CoA is known to induce inhibition of short and medium chain dehydrogenases [5–6], and of the electron transfer flavoprotein and electron transfer flavoprotein dehydrogenase [7]. It also interferes with amino acid metabolism through inhibition of isovaleryl-CoA dehydrogenase ∗ Corresponding author. E-mail address: f.boemer@chu.ulg.ac.be (F. Boemer). and 2-methylbutyryl-CoA dehydrogenase [5,8]. Consequently, fatty and organic acids conjugated with carnitine accumulate in serum and urine. The inhibition of mitochondrial -oxidation secondarily activates a compensatory -oxidation mechanism, leading to the accumulation of short– and medium– chain dicarboxylic acids (glu- taric and ethylmalonic acid) in urine. Accordingly, the profiling of acylcarnitines and urinary organic acids contributes to the diagno- sis of equine acquired multiple acyl-CoA dehydrogenase deficiency (MADD) [9–10]. Considering that, retrieving and quantifying hypoglycin A in horse’s biological fluids is essential to confirm the intoxication. Additionally, such information is also crucial to understand toxin metabolisation into MCPA and to study its effects on energetic parameters in horses. Nowadays, diagnosis workup of inborn errors of metabolism relies inter alia on amino acids, acylcarnitines and urinary organic acids profiling, these three panels being generally routinely deter- mined in the same laboratories. Then, based on our previous assessment of the aTRAQ kit to study physiological amino acid by http://dx.doi.org/10.1016/j.jchromb.2015.06.004 1570-0232/© 2015 Elsevier B.V. All rights reserved.