Determination of Ecdysterone in Dietary Supplements and Spinach by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Ecdysterone is a naturally occurring steroid hormone of the ecdysteroid class. This group is widely marketed to athletes in dietary supplements as a “natural anabolic agent”, advertised to increase strength and muscle mass during resistance training, reduce fatigue and ease recovery. The aim of the study was to develop and validate a straightforward approach for identifying ecdysterone in dietary supplements by means of ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). Furthermore, due to the fact that ecdysterone is one of the compounds naturally occurring in spinach, the fit-for-purpose method for extraction and identification of ecdysterone in spinach is proposed. The validity of the developed method was confirmed with the use of a reference standard and the limit of detection (LOD) for ecdysterone was established at 1 mg/g supplement. The presence of ecdysterone was confirmed in all tested supplements at estimated concentrations ranging between 5 mg/g and 383 mg/g.

Urinary Excretion of Ecdysterone and Its Metabolites Following Spinach Consumption

“…[2][3][4][5][6][7][8][9][10] Abnormally, Grucza et al have reported ecdysterone amount in fresh spinach as 0.1 µg g −1 f.w. [11] The levels of ecdysterone reported in spinach suggest that the normal amount of ecdysterone from spinach in a daily diet is rather low.…”

Quinoa as Functional Food? Urinary elimination of ecdysterone after consumption of quinoa alone and in combination with spinach

“…Quinoa and spinach are two edible plants rich in ecdysterone in varying amounts (Bajkacz et al, 2020;Bakrim et al, 2008;Chen & Feng, 2021;Gorelick et al, 2020;Graf et al, 2014;Graf et al, 2016;Grebenok & Adler, 1991;Grucza et al, 2021;Kumpun et al, 2011). Since its inclusion in the WADA monitoring program (World Anti-Doping Agency, 2020), the urinary excretion of ecdysterone from food became a topic of interest.…”

“…Fresh, cooked, and frozen leaves of spinach also revealed a significant variation in the content of ecdysterone, 80 to 428 µg/g dry mass reported by Fang et al (Fang et al, 2022), 17 – 885 µg/g dry mass based on the study of Bajkacz (Bajkacz et al, 2020), and 24 µg/g as investigated by Chen et al (Chen & Feng, 2021). On the other hand, only a small amount of ecdysterone (0.1 µg/g FW) was quantified from fresh spinach as reported by Grucza et al (Grucza et al, 2021). The high variation in the content of ecdysterone in spinach is related to the location of the growing plants, the genetic information, the growing season, the development stage of plants, and plant variety (Bakrim et al, 2008; Gorelick et al, 2020; Grebenok & Adler, 1991).…”

Development and Validation of High-Performance Liquid Chromatography for Identification and Quantification of Phytoecdysteroids Ecdysterone and Turkesterone in Dietary Supplements

“…Moreover, 20-hydroxyecdysterone is usually marked as "natural anabolic agent" [27]. Although many researchers suggest that ecdysterone supplementation has been used by Soviet athletes since the 1980s and it was sometimes called the "Russian secret" (R. carthamoides naturally grows in Russia), nowadays supplementation with R. carthamoides and other products containing ecdysterone is common practice among athletes from all over the world, because of the high adaptogenic potential of the molecule [8,18,[27][28][29]. The intake of R. carthamoides extracts and its contained secondary metabolites, ecdysterone, and turkesterone, has been associated with multiple athletic performance benefits [30][31][32][33], as shown in Figure 2.…”