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<p>Atypical myopathy (AM) is a frequently fatal, toxin-induced disease in horses, characterised by acute progression and mortality rates exceeding 70% within 72&#xa0;h. The disease is caused by ingestion of seedlings or seeds of certain maple species that contain the toxins hypoglycin A (HGA) and methylenecyclopropylglycine (MCPrG). Early diagnosis depends on reliable detection of these compounds and their metabolites. However, most available LC-MS/MS assays are limited by derivatisation requirements and dependence on serum and urine matrices. Here, a rapid derivatisation-free flow injection analysis (FIA)-tandem mass spectrometry (MS/MS) method was developed for the simultaneous quantification of nine analytes: HGA, methylenecyclopropylacetyl-carnitine (MCPA-carnitine), and seven diagnostically relevant acylcarnitines, from dried blood spots (DBS). The procedure requires only 50&#xa0;µL of whole blood, minimal sample preparation, and enables results within one minute. The assay provides low limits of quantification (0.01–0.1&#xa0;μmol/L) with excellent linearity (<i>R</i><sup>2</sup> &gt; 0.994), exhibiting analytical performance suitable for rapid screening of AM biomarkers. Moreover, analytes showed adequate stability in DBS during short-term storage at ambient temperature and prolonged storage at −20&#xa0;°C. Clinical performance of the workflow was evaluated using 17 DBS samples from horses with AM and 15 control DBS samples. AM cases were defined based on the high specificity of HGA and MCPA-carnitine, with accompanying acylcarnitine profiles reflecting disease-associated metabolic disruption. Over 2 years, more than 250 DBS samples from horses suspected of AM were routinely analysed. Overall, the results establish a robust, high-throughput FIA-MS/MS platform integrating DBS sampling with non-derivatised analysis, offering a practical screening tool for remote sample collection, efficient transport, and multi-analyte diagnosis of AM.</p> Graphical abstract <p></p>

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Derivatisation-free FIA-MS/MS assay for rapid simultaneous screening of atypical myopathy biomarkers and acylcarnitine profiles from dried blood spots

  • 期刊论文

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  • 发表日期：

    2026年5月27日

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  • Martina Kadláčková,
  • Dana Dobešová,
  • Eliška Ivanovová,
  • Richard Masař,
  • Petr Jahn,
  • Eva Šamonilová,
  • David Friedecký,
  • Radana Brumarová

摘要

Atypical myopathy (AM) is a frequently fatal, toxin-induced disease in horses, characterised by acute progression and mortality rates exceeding 70% within 72 h. The disease is caused by ingestion of seedlings or seeds of certain maple species that contain the toxins hypoglycin A (HGA) and methylenecyclopropylglycine (MCPrG). Early diagnosis depends on reliable detection of these compounds and their metabolites. However, most available LC-MS/MS assays are limited by derivatisation requirements and dependence on serum and urine matrices. Here, a rapid derivatisation-free flow injection analysis (FIA)-tandem mass spectrometry (MS/MS) method was developed for the simultaneous quantification of nine analytes: HGA, methylenecyclopropylacetyl-carnitine (MCPA-carnitine), and seven diagnostically relevant acylcarnitines, from dried blood spots (DBS). The procedure requires only 50 µL of whole blood, minimal sample preparation, and enables results within one minute. The assay provides low limits of quantification (0.01–0.1 μmol/L) with excellent linearity (R2 > 0.994), exhibiting analytical performance suitable for rapid screening of AM biomarkers. Moreover, analytes showed adequate stability in DBS during short-term storage at ambient temperature and prolonged storage at −20 °C. Clinical performance of the workflow was evaluated using 17 DBS samples from horses with AM and 15 control DBS samples. AM cases were defined based on the high specificity of HGA and MCPA-carnitine, with accompanying acylcarnitine profiles reflecting disease-associated metabolic disruption. Over 2 years, more than 250 DBS samples from horses suspected of AM were routinely analysed. Overall, the results establish a robust, high-throughput FIA-MS/MS platform integrating DBS sampling with non-derivatised analysis, offering a practical screening tool for remote sample collection, efficient transport, and multi-analyte diagnosis of AM.

Graphical abstract