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Analytical and bioanalytical chemistry2026; doi: 10.1007/s00216-026-06566-3

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

Abstract: 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 (R > 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.
© 2026. The Author(s).
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Publication Date: 2026-05-27 PubMed ID: 42203927PubMed Central: 7071031DOI: 10.1007/s00216-026-06566-3Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study developed a rapid, simple, and sensitive flow injection analysis-tandem mass spectrometry (FIA-MS/MS) method without derivatisation for simultaneously measuring atypical myopathy (AM) biomarkers and acylcarnitine profiles in dried blood spots (DBS) from horses.
  • The assay enables quick, reliable screening to detect toxin-induced AM, a severe and often fatal disease in horses, using minimal sample amounts and straightforward preparation.

Background

  • Atypical myopathy (AM) is a toxin-induced disease in horses caused by ingestion of seeds or seedlings from certain maple tree species.
  • The toxins responsible are hypoglycin A (HGA) and methylenecyclopropylglycine (MCPrG), which cause acute disease progression with high mortality rates (>70% within 72 hours).
  • Early diagnosis is critical and relies on detecting these toxins and their metabolites in biological samples.
  • Existing liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays require chemical derivatisation steps and are generally dependent on serum or urine samples, limiting speed and convenience.
  • Dried blood spots (DBS) offer practical advantages for sample collection and transport, especially from remote locations, but sensitive and efficient assays for AM biomarkers in DBS were lacking.

Objective

  • To develop a derivatisation-free FIA-MS/MS method for rapid, simultaneous quantification of nine analytes related to AM: HGA, methylenecyclopropylacetyl-carnitine (MCPA-carnitine), and seven acylcarnitines.
  • The method aimed to utilize small volumes of whole blood dried on filter paper (DBS), reduce sample prep time, and deliver results quickly (<1 minute per sample) for high-throughput screening.

Methodology

  • Analytes targeted included HGA and MCPA-carnitine as specific toxins/metabolites involved in AM, plus seven acylcarnitines indicative of disease-related metabolic disruption.
  • Only 50 µL of whole blood was required, spotted onto filter paper to create DBS samples.
  • Sample preparation was minimal and did not involve chemical derivatisation, simplifying the process significantly.
  • The FIA-MS/MS technique enabled direct injection of the prepared extracts, speeding analysis.
  • Validation tests assessed limits of quantification (LOQ), linearity of measurements, and analyte stability under varied storage conditions.
  • The method’s clinical relevance was evaluated using DBS samples from horses diagnosed with AM and healthy controls.
  • Long-term routine testing included over 250 DBS samples collected over two years from suspected cases, confirming real-world utility.

Key Findings

  • The assay achieved low LOQs ranging from 0.01 to 0.1 µmol/L, suitable for detecting clinically relevant concentrations of toxins and metabolites.
  • Excellent linearity (correlation coefficients R > 0.994) ensured reliable quantification across concentration ranges.
  • Analytes remained stable in DBS at room temperature for short periods and under freezing (-20 °C) for prolonged storage, supporting flexible sample handling and transport.
  • Clinical samples showed high specificity of HGA and MCPA-carnitine detection for identifying AM cases.
  • Acylcarnitine profiles matched known metabolic disruptions caused by AM, providing additional diagnostic insight.
  • The rapid, derivatisation-free FIA-MS/MS approach integrated well with DBS sample collection, enabling practical remote sampling and efficient lab workflows.

Implications and Conclusions

  • This novel assay offers a robust, high-throughput platform for simultaneous multi-analyte screening of AM biomarkers in horses.
  • Its minimal sample volume requirements, lack of derivatisation, and rapid analysis improve diagnostic turnaround and accessibility.
  • The combination of DBS sampling with FIA-MS/MS facilitates easy remote sample collection and transport, expanding diagnostic capacity to field environments.
  • Adoption of this method can enhance early detection of AM, improve clinical decision-making, and potentially reduce mortality through timely intervention.
  • The approach may serve as a model for developing similar assays for other diseases requiring multi-analyte detection from minimal, easily collected samples.

Cite This Article

APA
Kadláčková M, Dobešová D, Ivanovová E, Masař R, Jahn P, Šamonilová E, Friedecký D, Brumarová R. (2026). Derivatisation-free FIA-MS/MS assay for rapid simultaneous screening of atypical myopathy biomarkers and acylcarnitine profiles from dried blood spots. Anal Bioanal Chem. https://doi.org/10.1007/s00216-026-06566-3

Publication

Analytical and bioanalytical chemistry
ISSN: 1618-2650
NlmUniqueID: 101134327
Country: Germany
Language: English

Researcher Affiliations

Kadláčková, Martina
  • Laboratory for Inherited Metabolic Disorders, Department of Medical Genetics, Faculty of Medicine and Dentistry, Palacký University Olomouc, Hněvotínská 3, 779 00, Olomouc, Czech Republic.
  • Laboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital Olomouc, Zdravotníků 248/7, 779 00, Olomouc, Czech Republic.
Dobešová, Dana
  • Laboratory for Inherited Metabolic Disorders, Department of Medical Genetics, Faculty of Medicine and Dentistry, Palacký University Olomouc, Hněvotínská 3, 779 00, Olomouc, Czech Republic.
  • Laboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital Olomouc, Zdravotníků 248/7, 779 00, Olomouc, Czech Republic.
Ivanovová, Eliška
  • Laboratory for Inherited Metabolic Disorders, Department of Medical Genetics, Faculty of Medicine and Dentistry, Palacký University Olomouc, Hněvotínská 3, 779 00, Olomouc, Czech Republic.
  • Laboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital Olomouc, Zdravotníků 248/7, 779 00, Olomouc, Czech Republic.
Masař, Richard
  • Laboratory for Inherited Metabolic Disorders, Department of Medical Genetics, Faculty of Medicine and Dentistry, Palacký University Olomouc, Hněvotínská 3, 779 00, Olomouc, Czech Republic.
  • Laboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital Olomouc, Zdravotníků 248/7, 779 00, Olomouc, Czech Republic.
Jahn, Petr
  • Equine Clinic, Faculty of Veterinary Medicine, University of Veterinary Sciences Brno, Palackého třída 1946/1, 612 00, Brno-Královo Pole, Czech Republic.
Šamonilová, Eva
  • Equine Clinic, Faculty of Veterinary Medicine, University of Veterinary Sciences Brno, Palackého třída 1946/1, 612 00, Brno-Královo Pole, Czech Republic.
Friedecký, David
  • Laboratory for Inherited Metabolic Disorders, Department of Medical Genetics, Faculty of Medicine and Dentistry, Palacký University Olomouc, Hněvotínská 3, 779 00, Olomouc, Czech Republic.
  • Laboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital Olomouc, Zdravotníků 248/7, 779 00, Olomouc, Czech Republic.
Brumarová, Radana
  • Laboratory for Inherited Metabolic Disorders, Department of Medical Genetics, Faculty of Medicine and Dentistry, Palacký University Olomouc, Hněvotínská 3, 779 00, Olomouc, Czech Republic. radana.brumarova@upol.cz.
  • Laboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital Olomouc, Zdravotníků 248/7, 779 00, Olomouc, Czech Republic. radana.brumarova@upol.cz.

Grant Funding

  • DRO (FNOl / Ministerstvo Zdravotnictví Ceské Republiky
  • 00098892) / Ministerstvo Zdravotnictví Ceské Republiky
  • Grant QL24010403 / Národní Agentura pro Zemědělský Výzkum

Conflict of Interest Statement

Declarations. Ethics approval: All procedures performed in this study were conducted in accordance with the national legislation of the Czech Republic (Act No. 246/1992 Coll., on the Protection of Animals Against Cruelty, as amended) and with EU Directive 2010/63/EU on the protection of animals used for scientific purposes. The Animal Experimentation Ethics Committee of the University of Veterinary Sciences Brno was informed about all procedures and approved the study (approval no. ES_6-2025_Jahn). Competing interests: The authors declare no competing interests. Source of biological material: Blood samples were obtained from horses during routine veterinary diagnostic procedures and were used for the purposes of this study. Animal welfare: All procedures were part of routine veterinary practice, and no experimental interventions affecting animal welfare were performed. Informed consent was obtained from the animal owners.

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