Different approaches to the identification of a cortisol isomer compound in horse urine.

The research body is an examination of methods used to identify a cortisol isomer compound in horse urine, with the aim of understanding the discrepancies observed between labs and the structural changes happening to the compound.

Objective of the study

• The scientists’ goal was to understand the mysterious change in cortisol’s structure in horse urine. Racing authorities set a limit for cortisol concentration in horse pee at 1.0 microgram per milliliter, but discrepancies between labs and noticeable structural modifications in cortisol drove the need for this research.

Methodology

• The researchers used High-Performance Liquid Chromatography-Atmospheric Pressure Chemical Ionization-Mass Spectrometry (HPLC-APCI-MS) and Mass Spectrometry-Mass Spectrometry (MS-MS) methods. These techniques were applied to diluted cortisol derived from urine samples, with the dilution carried out in a methanol-water mixture or in labelled methanol-water.
• The team studied stereochemical effects under these conditions and tracked deuterium-hydrogen exchange using HPLC-APCI-MS.

Findings

• Even though the modified cortisol shared the same molecular weight as cortisol, it exhibited a shorter retention time under cortisol quantification conditions. From this information and the MS and MS-MS data, the research concluded that the cortisol wasn’t getting isomerized at C11 into 11 alpha-cortisol.
• The scientists ran isotopic labelling experiments to find out the number of movable hydrogen atoms in the modified cortisol. After learning that both cortisol and its modified version have an equal number of movable hydrogens, they ruled out the hypothesis of a reduction taking place at C20 and an oxidation at C11.
• The research suggests deuterium-hydrogen exchanges might be helpful for figuring out the structure of compounds analyzed by HPLC-APCI-MS in complex matrices like horse urine extract.