A rapid screen for four corticosteroids in equine synovial fluid

Area of Science:

• Veterinary Pharmacology
• Analytical Chemistry
• Equine Sports Medicine

Background:

• Equine antidoping methods traditionally focus on plasma and urine.
• Analysis of synovial fluid is gaining importance for detecting intra-articular corticosteroid injections.
• Existing synovial fluid methods are often singleplex, lack sensitivity, and do not cover all relevant corticosteroids.

Purpose of the Study:

• To develop a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) screening method.
• To detect four common intra-articularly administered corticosteroids: betamethasone, methylprednisolone, methylprednisolone acetate, and triamcinolone acetonide.

Main Methods:

• Sample preparation involved protein precipitation and basified liquid-liquid extraction.
• A six-minute isocratic LC-MS-MS separation was performed using a Phenomenex Polar-RP column.
• Detection utilized a triple quadrupole mass analyzer with thermospray ionization and selective reaction monitoring.

Main Results:

• The method achieved high selectivity for the four target corticosteroids.
• Limits of detection ranged from 1-3 ng/mL.
• Extraction efficiency was 50-101%, with matrix effects between 14-31%.

Conclusions:

• The developed LC-MS-MS method is a rapid and sensitive screen for specific corticosteroids in equine synovial fluid.
• The assay is suitable for equine antidoping applications, addressing limitations of previous methods.
• Validation to ISO/IEC 17025 standard ensures reliability for real-world use.