Separation and identification of the epimeric doping agents – Dexamethasone and betamethasone in equine urine and plasma: A reversed phase chiral chromatographic approach.

The research is aimed at developing a reliable method for identifying and separating commonly abused steroids, dexamethasone and betamethasone, in horse urine and blood. The scientists achieved this using a high-resolution accurate mass spectrometer and reversed phase media, with optimal conditions for chiral separation.

Chirality and Doping

• Chirality refers to the phenomenon where molecules have mirror images of each other and cannot be superimposed. This principle is important in doping control as certain molecules and their mirror images might have different biological effects.
• Dexamethasone and betamethasone are corticosteroid drugs that are illicitly used to increase athletic performance in equine sports. The abuse of these substances is a significant problem due to their potency and long-lasting effects.

Development of a New Analytical Method

• The study aimed to create an analytical method which could simultaneously identify and separate these frequently abused co-eluting corticosteroids in horse plasma and urine. The process would greatly aid in doping control by allowing a targeted approach to substance abuse.
• The new method developed in this study used a Thermo Q Exactive high-resolution accurate mass spectrometer, a highly sensitive instrument capable of detecting even very minute concentrations of each isomer in the complex biological matrices of plasma and urine samples.

Chromatographic Separation

• The chromatographic separation of the dexamethasone and betamethasone mixtures was accomplished using chiral columns of amylose and cellulose. These common polysaccharides were chosen due to their effective chiral resolution properties, meaning they can effectively separate molecular mirror images from each other.
• Reversed phase media also showed the potential to successfully provide chiral resolution in liquid chromatography-mass spectrometry (LC-MS) analysis.

Optimization of Method

• In addition to establishing the method, the study also focused on optimizing various factors such as the elution strength, nature of the organic modifier, additives, and column temperature. These variables can significantly impact the effectiveness of the chromatographic separation and were therefore carefully manipulated to achieve the most efficient separation and identification of the two steroids.