Development and validation of an ultrahigh performance liquid chromatography‐high resolution tandem mass spectrometry quantification method for hypoglycin A and methylene cyclopropyl acetic acid carnitine in horse serum in cases of atypical myopathy

Abstract

Atypical myopathy (AM) is a fatal disease in horses presumably caused by hypoglycine A (HGA) from ingested maple seeds and its active metabolite methylene cyclopropyl 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 ultra high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with full-scan/data-dependent MS/MS. Chromatographic separation was performed by isocratic elution on a hydrophilic interaction liquid chromatography (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 to 2000 ng/mL for HGA and from 10 to 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%). The limit of quantification (LOQ) was defined as the 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 10 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.

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

“…The current work reveals that detection of HGA and MCPA‐carnitine in serum and muscle do not require derivatisation to produce reliable, reproducible and sensitive results. Indeed, we achieved a significantly lower limit of quantitation for both analytes when compared with a recent published method 35 . Moreover, the recoveries obtained in serum by Rudolph et al method (around 20% for both analytes) were strikingly low when compared with the method presented in this work (97%‐100%) 35 .…”

“…Rudolph et al method (around 20% for both analytes) were strikingly low when compared with the method presented in this work (97%-100%). 35 Perhaps the use of a different chromatographic column might account for the previously reported results: ion suppression or poor ionisation of analytes might occur when samples are not sufficiently clean, affecting the recovery process. 32 Besides, the current assay allows detection of both compounds simultaneously while previous methods required the use of 2 different techniques and/ or analyses to detect HGA and MCPA-carnitine in serum/plasma.…”

“…We describe new methodology for reliable quantification of HGA and MCPA-carnitine in equine serum, plasma and muscle and lower the previously reported limits of quantitation. 10,34,35 This novel method also avoids use of a derivatising agent thereby reducing sample analysis time (to under 2 hours) and costs.…”

“…Indeed, we achieved a significantly lower limit of quantitation for both analytes when compared with a recent published method. 35 Moreover, the recoveries obtained in serum by…”

“…36 MCPA-carnitine seems to be the most prevalent HGA metabolite in equine serum while MCPA-glycine is usually very low or undetectable, although is present at higher concentrations in urine. 3,7,35,44 Furthermore, MCPA-glycine and HGA are commonly excreted in urine of healthy equines grazing areas affected by AM, 3 suggesting that HGA can be successfully eliminated in urine as a free molecule and/or via glycine conjugation to some extent without associated clinical signs in horses and human subjects. 3,45 We could not detect MCPA-carnitine in liver tissue of AMaffected horses, which might be related to low metabolism of HGA in this tissue.…”