Analytical strategy for the detection of ecdysterone and its metabolites in vivo in uPA(+/+)‐SCID mice with humanized liver, human urine samples, and estimation of prevalence of its use in anti‐doping samples

Abstract Ecdysteroids are of interest as potential sport performance enhancers, due to their anabolic effects. The current study aimed to analyze levels of the most abundant ecdysteroid, ecdysterone (20‐hydroxyecdysone, 20‐OHE) in easily available dietary supplements, and, outline an analytical strategy for its detection, and that, of its metabolites, (1) following administration of pure 20‐OHE to uPA(+/+)‐SCID mice with humanized liver, (2) in a human volunteer after ingestion of two supplements, one with a relatively low, and the other a high, concentration of 20‐OHE, and, (3) to estimate the prevalence of use of 20‐OHE in elite athletes (n = 1000). Of the 16 supplements tested, only five showed detectable levels of 20‐OHE, with concentrations ranging from undetectable up to 2.3 mg per capsule. Urine of uPA(+/+)‐SCID urine showed the presence of 20‐OHE and its metabolite, 14 deoxy ecdysterone, within 24 hours (hr) of ingestion. In humans, both the parent and the metabolite were detectable within 2 to 5 hr of ingestion, with the metabolite being detectable for longer than the parent. After ingestion of a low dose supplement, the parent and metabolite were detectable for 70 and 48 hr, while following the higher dose it was 96 and 48 hr, respectively. Analysis of urines from athletes (n = 1000) confirmed four positives for 20‐OHE, suggesting a prevalence of use of 0.4%. Prevalence of its use by elite athletes was relatively low, however, this needs to be confirmed in other populations, and with other related ecdysteroids.


| INTRODUCTION
Ecdysteroids are commonly known as arthropods steroid hormone for molting, metamorphosis, embryogenesis and reproduction. 1 They are also identified in some plants species, which can facilitate its extraction and consumption. [2][3][4] Various pharmacological properties of ecdysteroids are recognized, specifically their ability to enhance physical performance through beneficial changes to body composition and anabolic effects. The anabolic effects of ecdysteroids were reported to be mediated by activation of beta estrogen receptor. 4 Thus, it can be considered as a perfect candidate for the use by athletes. [5][6][7][8] As a result, it was recently included in the WADA monitoring program of 2020. 9 There are different classes of ecdysteroids including, ecdysterone, ecdysone and turkesterone2-3, among which 20-hydroxyecdysone (ecdysterone, 20-OHE) is the most abundant and the most studied. 14-deoxy-ecdysterone; 10,11 in calf urine, 14-deoxy-20-hydroxyecdysone, 20,26-dihydroxyecdysone and 14-deoxy-20,26-dihydroxyecdysone; 12 and in mice/rat excretion: 14-deoxy-20-hydroxyecdysone, poststerone and 14-deoxypoststerone. 7,13 The inclusion of ecdysteroids on WADA monitoring list has regenerated interest in investigating its metabolism and prevalence of use in humans. The detection of 20-OHE and its metabolites after administration of high doses in human volunteers (20 and 50 mg) has been reported by Tsitsimpikou et al. 10 and Parr et al, 11 respectively. However, the doses present in the supplements vary considerably and often do not accurately reflect those on the label. 14 Therefore, there is a need to quantitate the 20-OHE levels in the supplements themselves, as well as, detect the fate, both parent and metabolite, after in vivo ingestion.
Thus, aims of the present study were to investigate the analytical strategy for the detection of 20-OHE, and its metabolites: (1)

| Supplement extraction
The contents of five capsules were mixed and two aliquotes of 0.5 gm each were taken for the extraction. Each aliquote was dissolved in 5 mL of carbonate buffer (pH 9-10) by shaking for 30 min.

| Human urine samples
After identifying the presence of 20-OHE in five out of 16 supplements, ( After 45 days, the same volunteer was administered two capsules (recommended dose) of Turkesterone. A blank urine was collected preadministration. Following administration all the urines were collected for the first 3 days, with only the morning urine being collected for further 3 days, as 20-OHE has previously been shown to be rapidly eliminated within the first 24 hr. All the urine samples were stored frozen at −20 C until analysis.

| Prevalence study
Prevalence of 20-OHE use was determined in anti-doping urine sam-  28.36 min). The transfer line was set at 280 C. Helium was used as a carrier gas at a flow rate of 1.0 mL/min. 2 μL was injected into GCQQQMS using a split mode at a ratio of 1:10 with injector temperature at 280 C, the source temperature was set at 230 C. In the QqQ collision cell Helium was used as a quench gas at 2.25 ml/min and N2 as a collision gas at 1.5 ml/min.

| Statistical analysis
No detailed statistical analysis could be carried as only information on sports discipline and gender were available on these athletes. Therefore, the data are shown as a Venn diagram depicting the various disciplines, and also shows the sport in which the positive results were obtained. All basic statistics were carried out with Excel ( Figure 2). analyzed for the presence of 20-OHE. In addition, Turkesterone supplement was also analyzed. Seven supplements, as per label claim, were supposed to contain 20-OHE. However, only five of these were found to have detectable levels of the ecdysteroid. Furthermore, their concentrations varied significantly ( Table 1). The concentration of 20-OHE is given in both as mg/capsule and as mg/g.

| Quantitation of 20-OHE in dietary supplements
The lowest quantity (0.0005 mg/g) was found in Immunectar, confirmed that finding, albeit in different supplements. However, their study did not name the exact supplements, but used study code numbers.

| SCID mice
The analytical strategy adopted for the detection of 20-OHE and its  Figure 4b.
Taking in to account the above constatations, an LCMS QQQ method was developed in order to detect the parent compound and its 14-deoxy metabolite. The Figure 8 shows chromatographic separation for 20-OHE and metabolite, 14-deoxy-ecdysterone eluted at 5.3 and 5.6 min respectively.
The parent compound was detectable up to 48 hr after the ingestion while the metabolite was detectable up to 24 hr (Figure 9a,b).

| Human excretion studies
Based on mass spectrometry data, both high and low resolution, the 20-OHE metabolite 14-deoxy-20-OHE was tentatively identified, as

| Prevalence study findings
The distribution of one thousand samples is given in Figure 2. The sample preparation for the prevalence study (n = 1000) samples was performed according to the conventional screening procedure in antidoping laboratory. The monitoring transitions are summarized in