Investigations into the human metabolism of Ecdysterone

Code: 21C06MT

The possible performance-enhancing effects and medical benefits of ecdysterone (ECD) have been discussed several times throughout the last decades.[1-4] In 2020, WADA decided to include ECD in their monitoring program and continued this prevalence study in 2021.[5] Only little is known about the metabolism of ECD. In calf urine, the intact compound was eliminated rapidly, and three hydroxylated and de-hydroxylated metabolites were identified.[6] In mice, mainly de-hydroxylation and side-chain cleavage at C-20 were detected.[7] The only study performed on human subjects in the field of sports drug testing was already conducted 2001.[8] Besides the parent compound, 2-deoxyecdysterone (2DE)and deoxyecdysone (DE) (Figure 1) were identified in post-administration urine samples.

i. Sample collection from the human participant – details of the interventions. In order to detect and to comprehensively identify urinary human metabolites of ECD, two consecutive administration trials will be performed. During the first investigation, which serves the purpose of qualitatively detect the occurrence of metabolites, 20 mg of deuterium labelled ECD will be administered orally to one person and samples will be collected before the administration (n = 3) and afterwards. During the first two days after administration every urine specimen will be sampled, followed by morning and evening urines throughout days 3 to 7. Then, for the next two weeks, only morning urine specimens will be collected. The collection scheme for the second administration study conducted with 20 mg of unlabelled ECD will be conducted in accordance with the first trial and may be adopted to gather more data at those time points that were found to be important during the first investigation. Here, three study participants will be enrolled. Within both studies, a washout period of at least one month will take place. The volunteer will be recruited from the male population of employees of the German Sports 3University, Cologne. All samples will be collected in 250 ml PET flasks and stored at -20°C without adding any preservative.

ii. Analytical methods
A) Hydrogen isotope ratio mass spectrometry
Pre and post administration samples will be subjected to established sample preparation protocols enabling to separate unconjugated, glucuronidated and sulfoconjugated phase-II-metabolites. The different urinary fractions will be de-conjugated and further purified by high performance liquid chromatography prior to trimethylsilyl-derivatization. During the injection on the GC/TC/IRMS system the hydrogen isotope ratios (HIR) will enable to identify those steroids still showing a deuterium label and therefore attributable to the administered ECD. Simultaneously, low-resolution mass spectra will be acquired using the hyphenated triple quadrupole MS. These low-resolution mass spectra facilitate relocation of metabolites during HR-MS measurements. 

B) Structural elucidation employing HR-MS All samples containing deuterium labelled compounds will be re-injected on a gas chromatography/high resolution mass spectrometry based system in order to gain structural information of possible ECD metabolites. The full scan high-resolution mass spectra obtained will enable to calculate the elemental composition of metabolites and tandem mass spectrometry based experiments will help to identify the chemical structure of metabolites. Those metabolites most 
promising for sports drug testing may be further characterized employing e.g. reverence materials (if available) or partial chemical synthesis (if possible).

iii. Targets/analyses/variablesThe urine samples collected during the administration trial encompassing deuterated ECD will be processes in order to identify possible metabolites of ECD. Known metabolites will of course be considered in a targeted manner to demonstrate the validity of the approach. Samples collected within the non-deuterated study will be investigated regarding the potential to implement found metabolites into current doping control methods. Both samples (deuterated and non-deuterated) will enable structural elucidation of metabolites taking into account the known site of deuteration

Main Findings

In a first trial, one male volunteer was administered with deuterated ECDY to enable the detection and potential identification of all urinary metabolites still comprising the deuterium label by employing hydrogen isotope ratio mass spectrometry and high resolution/high accuracy mass spectrometry. Samples were collected for up to 14 days and metabolites excreted unconjugated, glucuronidated and sulphated were investigated separately.  The detected deuterated metabolites were confirmed in a second administration trial encompassing two male and one female volunteers. After the administration of 50 mg native (i.e. non-deuterated) ECDY, urine samples were collected for up to 7 days. Besides the already described urinary metabolites of ECDY, more than 20 new metabolites were detected encompassing all expected metabolic conversions including the described side chain cleavage at C-21. A significant inter-individual variation in the amounts of excreted ECDY and its metabolites was noted. Defining a urinary threshold for ECDY will become extremely complicated as either acceptable sensitivity or specificity will most probably not be achievable. Considering other detected metabolites will also be challenging as a potential solution for this issue as, also here, a large inter-individual variation was visible and considerable differences in abundances of early- and late-excretion phase metabolites were observed.