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Home / Equine Research Bank / Drug Test Anal / Determination of vitamin E and its metabolites in equine uri...
Drug testing and analysis2021; 13(6); 1158-1168; doi: 10.1002/dta.3006

Determination of vitamin E and its metabolites in equine urine using liquid chromatography-mass spectrometry.

Abstract: Equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM) is a hereditary, deteriorating central nervous disease in horses. Currently, the only way to confirm eNAD/EDM is through a postmortem histological evaluation of the central nervous system. Vitamin E, specifically the isoform alpha-tocopherol (α-TP), is known to protect eNAD/EDM susceptible horses from developing the clinical phenotype. While vitamin E is an essential nutrient in the diet of horses, there are no diagnostic tests able to quantitate vitamin E and its metabolites in urine. An ultra-performance liquid chromatography-atmospheric-pressure chemical ionization mass spectrometry (UPLC-APCI-MS/MS) method was developed and validated following acidic hydrolysis and solid phase extraction to quantitate vitamin E and its metabolites in equine urine. A blank control horse urine matrix was used and spiked with different concentrations of analytes to form a standard curve using either alpha-tocopherol-d6 or chlorpropamide as the internal standard. Inter-day and intra-day statistics were performed to evaluate the method for accuracy (90% to 116%) and precision (0.75% to 14%). Matrix effects, percent recovery, and stability were also assessed. The method successfully analyzed alpha-carboxyethyl hydroxychroman (α-CEHC), alpha-carboxymethylbutyl hydroxychromans (α-CMBHC), gamma-carboxyethyl hydroxychroman γ-CEHC, and α-TP concentrations in urine to determine a baseline levels of analytes in healthy horses, and can be used to determine concentrations of vitamin E metabolites in equine urine allowing for its evaluation as a diagnostic approach in the treatment of eNAD/EDM.
© 2021 John Wiley & Sons, Ltd.
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Publication Date: 2021-02-09 PubMed ID: 33527764DOI: 10.1002/dta.3006Google Scholar: Lookup
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  • Journal Article
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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.

This research article discusses the development and validation of a method to measure the levels of Vitamin E and its metabolites in horse urine, aiming to use it as a diagnostic approach for a hereditary neurological disease among horses known as Equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM).

Objective and Background

  • This research was aimed at addressing the lack of a diagnostic test that can quantitatively determine the levels of Vitamin E and its metabolites in horse urine. The ultimate goal was to explore its potential use as a diagnostic means for the detection and treatment of a hereditary central nervous disease in horses, the equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM).
  • Historically, eNAD/EDM diagnosis relied on postmortem histological evaluations of the central nervous system. The disease leads to progressive neurodegeneration in horses.
  • Previous studies have shown that a form of Vitamin E, alpha-tocopherol (α-TP), can protect susceptible horses from developing the clinical features of eNAD/EDM. However, the quantification methods for Vitamin E metabolites were absent in the field.

Method Development and Validation

  • An ultra-performance liquid chromatography-atmospheric-pressure chemical ionization mass spectrometry (UPLC-APCI-MS/MS) method was developed and validated in this research to quantify Vitamin E and its metabolites in equine urine.
  • Necessary procedures such as acidic hydrolysis and solid phase extraction were undertaken before the quantification process.
  • A standard curve was formed by spiking a blank control horse urine matrix with different concentrations of analytes. These were alpha-tocopherol-d6 or chlorpropamide, which were used as internal standards.
  • The method was thoroughly evaluated for accuracy and precision using inter-day and intra-day statistics. Performance indicators ranged between acceptable limits.

Results and Conclusion

  • The study successfully quantified alpha-Carboxyethyl Hydroxychroman (α-CEHC), alpha-Carboxymethylbutyl Hydroxychromans (α-CMBHC), gamma-Carboxyethyl Hydroxychroman (γ-CEHC), and α-TP concentrations in urine. These can be used to determine baseline levels of analytes in healthy horses.
  • Matrix effects, percent recovery, and stability were also assessed during the process.
  • The method developed has potential application for determining concentrations of vitamin E metabolites in equine urine, thus paving a path for its evaluation as a diagnostic approach in the treatment of eNAD/EDM.

Cite This Article

APA
Favro G, Habib H, Gennity I, Puschner B, Hales EN, Finno CJ, Moeller BC. (2021). Determination of vitamin E and its metabolites in equine urine using liquid chromatography-mass spectrometry. Drug Test Anal, 13(6), 1158-1168. https://doi.org/10.1002/dta.3006

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 13
Issue: 6
Pages: 1158-1168

Researcher Affiliations

Favro, Gianna
  • Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California, USA.
Habib, Hadi
  • Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California, USA.
Gennity, Ingrid
  • Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California, USA.
Puschner, Birgit
  • Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California, USA.
Hales, Erin N
  • Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, California, USA.
Finno, Carrie J
  • Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, California, USA.
Moeller, Benjamin C
  • K. L. Maddy Equine Analytical Chemistry Laboratory, California Animal Health and Food Safety Laboratory, School of Veterinary Medicine, University of California, Davis, California, USA.

MeSH Terms

  • Animals
  • Chromatography, High Pressure Liquid / methods
  • Horse Diseases / diagnosis
  • Horse Diseases / drug therapy
  • Horses
  • Neuroaxonal Dystrophies / diagnosis
  • Neuroaxonal Dystrophies / drug therapy
  • Neuroaxonal Dystrophies / veterinary
  • Solid Phase Extraction
  • Tandem Mass Spectrometry / methods
  • Vitamin E / analysis
  • Vitamin E / metabolism
  • Vitamin E / urine

Grant Funding

  • K01OD015134 / NIH HHS
  • L40 TR001136 / NIH HHS
  • K01OD015134 / NIH HHS
  • L40 TR001136 / NIH HHS

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Citations

This article has been cited 1 times.
  1. Hales EN, Habib H, Favro G, Katzman S, Sakai RR, Marquardt S, Bordbari MH, Ming-Whitfield B, Peterson J, Dahlgren AR, Rivas V, Ramirez CA, Peng S, Donnelly CG, Dizmang BS, Kallenberg A, Grahn R, Miller AD, Woolard K, Moeller B, Puschner B, Finno CJ. Increased α-tocopherol metabolism in horses with equine neuroaxonal dystrophy. J Vet Intern Med 2021 Sep;35(5):2473-2485.
    doi: 10.1111/jvim.16233pubmed: 34331715google scholar: lookup
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