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.

The research article is about the development and validation of a method to detect and measure harmful metabolic byproducts in horse serum that are associated with a fatal disease known as Atypical Myopathy (AM).

Objective of Study

The main goal of this study was to develop and validate an assay capable of measuring the concentrations of hypoglycin A (HGA) and methylene cyclopropyl acetic acid carnitine (MCPA-carnitine) in horse serum. They wanted a method that was quick, simple, and applicable to real samples. These substances are known to have a hand in causing Atypical Myopathy (AM), a deadly disease in horses.

• HGA is presumed to enter the horse’s system when they consume seeds from the maple tree.
• MCPA-carnitine is an active metabolite of HGA.

Method Developed

The substance identification and measurement were carried out using ultra-high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with full-scan/data-dependent MS/MS. Serum samples workup was done via protein precipitation.

• The UHPLC-HRMS/MS technology performs fine chromatographic separation through an isocratic elution mechanism.
• The researchers used a specific hydrophilic interaction liquid chromatography (HILIC) column for the experiment.
• The serum samples were around 250 μL in size.

Validation of Method

The developed method was validated according to international guidelines for several parameters:

• Accuracy and precision: The substances HGA and MCPA-carnitine showed acceptable reliability in terms of accuracy and precision.
• Calibration range: The calibration range for HGA was 100 to 2000 ng/mL, and for MCPA-carnitine was 10 to 1000 ng/mL.
• Limit of Quantification (LOQ): It is defined as the lowest measurable concentration of the substance. Despite being low, the LOQ was above the least known serum concentrations in horses affected by AM.
• Matrix effects and recoveries: They ranged from -79% to +20% (RSD 4.2% to 14.4%), and 17.9% to 21.1% (RSD 2.3% to 10.8 %) respectively for low and high-quality control samples.

Application of the Method and Findings

The methodology was applied to ten bona fide cases of Am and in all instances, significant amounts of HGA and MCPA-carnitine were discovered.

• The findings reaffirm the theory that the presence of these substances is tied to the disease.
• Reliable detection and quantification of HGA and MCPA-carnitine are crucial to understand their association with AM better and help in managing this disease.