LC-MS/MS Fingerprinting Analysis of <i>Cyanotis arachnoidea</i> Extracts: Process-Related Artifacts in Anabolic Food Supplements

We report the isolation and complete NMR characterization of four new ecdysteroids and LC-MS/MS fingerprinting of ecdysteroids in two native and two industrially processed <i>Cyanotis arachnoidea</i> extracts along with an autoxidized product mixture of 20-hydroxyecdysone (20E). The autoxidation of 20-hydroxyecdysone leads to the formation of various unknown or uncharacterized ecdysteroid compounds, which can significantly impact the quality and efficacy of commercial ecdysteroid-containing supplements, potentially affecting their regulatory status and consumer safety. A total of 146 ecdysteroids were detected. Among these, 20 were identified using authentic and fully characterized reference standards, including the newly reported compounds. The autoxidative origin of many process-related artifacts was confirmed in the two commercial ecdysteroid extracts. Considering the pharmacological versatility of ecdysteroids and the major pharmacological differences between these compounds and their autoxidized derivatives, our results are of importance regarding the efficacy and safety of commercially available ecdysteroid-containing food supplements.