A rapid and eco-friendly method for determination of the main components of gamma-oryzanol in equestrian dietary and nutritional supplements by liquid chromatography—Tandem mass spectrometry

•A novel method for the determination of the main gamma-oryzanol components was developed.•The method is based on LC–MS/MS and ultrasound-assisted extraction.•The proposed procedure obtained an excellent Eco-Scale score (76).•The method was validated and achieved satisfactory results for parameters such as sensitivity, repeatability and recovery.•The proposed protocol was applied to the analysis of thirty equestrian supplements. Gamma-oryzanol (GO) has gained special attention in the equine sports industry in recent years due to its touted properties, including the fact that it may cause anabolic effects on muscle growth and reduce fatigue. Many manufactures offer supplements containing GO as a naturally occurring anabolic substance; however, some producers do not declare its presence in product compositions. Taking into consideration the touted properties of GO, its ambiguous effectiveness and the open character of the Prohibited Substances List established by the Fédération Equestre Internationale, there is an urgent need to elaborate procedures for the estimation of horse exposure to GO during supplementation, as well as during routine analysis of supplements. This work describes the development and validation of the method for determination of the four main GO components, i.e., cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, campesteryl ferulate and β-sitosteryl ferulate, in equestrian supplements based on LC–MS/MS after a simple ultrasound-assisted extraction (Eco-Scale score value of 76). The analytical performance achieved satisfactory results in terms of linearity (R2 > 0.9910), sensitivity (LODs ranged from 0.4 to 1.9 ng/mL), intra- and interday accuracy (from 90.4–115.8%), precision (CV