Journal
DRUG TESTING AND ANALYSIS
Volume 13, Issue 6, Pages 1158-1168Publisher
WILEY
DOI: 10.1002/dta.3006
Keywords
CEHC and CMBHC; equine; liquid chromatography– mass spectrometry; vitamin E; vitamin E metabolites
Funding
- National Institutes of Health [K01OD015134, L40 TR001136]
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An UPLC-APCI-MS/MS method was developed and validated for quantifying vitamin E and its metabolites in equine urine, allowing for evaluation as a diagnostic tool in the treatment of eNAD/EDM. This method demonstrated accuracy and precision in analyzing the concentrations of vitamin E metabolites in healthy horses.
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 (alpha-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 (alpha-CEHC), alpha-carboxymethylbutyl hydroxychromans (alpha-CMBHC), gamma-carboxyethyl hydroxychroman gamma-CEHC, and alpha-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.
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