Urinary Elimination of Ecdysterone and Its Metabolites Following a Single-Dose Administration in Humans.

Ecdysterone is a phytosteroid widely discussed for its various pharmacological, growth-promoting, and anabolic effects, mediated by the activation of estrogen receptor beta (ERbeta). Performance-enhancement in sports was demonstrated recently, and ecdysterone was consequently included in the Monitoring Program, to detect potential patterns of misuse in sport. Only few studies on the pharmacokinetics of ecdysterone in humans have been reported so far. In this study, post-administration urine samples in twelve volunteers (single dose of 50 mg of ecdysterone) were analyzed using dilute-and-inject liquid-chromatography-tandem mass spectrometry. Identification and quantitation of ecdysterone and of two metabolites, 14-deoxy-ecdysterone and 14-deoxy-poststerone, was achieved. Ecdysterone was the most abundant analyte present in post-administration urine samples, detected for more than 2 days, with a maximum concentration (Cmax) in the 2.8-8.5 h urine (Cmax = 4.4-30.0 µg/mL). The metabolites 14-deoxy-ecdysterone and 14-deoxy-poststerone were detected later, reaching the maximum concentrations at 8.5-39.5 h (Cmax = 0.1-6.0 µg/mL) and 23.3-41.3 h (Cmax = 0.1-1.5 µg/mL), respectively. Sex-specific differences were not observed. Cumulative urinary excretion yielded average values of 18%, 2.3%, and 1.5% for ecdysterone, 14-deoxy-ecdysterone, and 14-deoxy-poststerone, respectively. Ecdysterone and 14-deoxy-ecdysterone were excreted following first-order kinetics with half-lives calculated with three hours, while pharmacokinetics of 14-deoxy-poststerone needs further evaluation.

Detection and quantitation of ecdysterone in human serum by liquid chromatography coupled to tandem mass spectrometry.

The polyhydroxylated phytosteroid ecdysterone is present in various plants (e.g. spinach). It is widely marketed as the active component of dietary supplements, due to its reported health and performance promoting effects. For evaluation of its actual bioavailability, a fast and sensitive method was developed, optimized and validated for human serum. Instrumental analysis was performed utilizing liquid chromatography-tandem mass spectrometry with positive electrospray ionization and acquisition in multiple reaction mode. Solid phase extraction and dilute-and-inject (following protein precipitation) were tested to isolate ecdysterone from human serum. Both methods were compared in the light of the preset analytical target profile. The limit of detection (LOD) and quantitation (LOQ) for ecdysterone in human serum after SPE extraction corresponded to 0.06 ng/mL and 0.14 ng/mL, respectively, meeting the requested sensitivity of the method. The assay was linear over the range of 0.10 ng/mL to 20.83 ng/mL. As expected, the sensitivity of the SPE method was better than that of the dilute-and-inject procedure, which did not allow for quantitation of all post administration serum samples. Accuracy (relative error; %) and precision (coefficient of variation; %), were both within acceptance criteria (<15%). The developed method was successfully applied to a ten week intervention study conducted in young men performing regular resistance training. Different doses of supplements containing ecdysterone from spinach extract have been administered during the study and the quantitation of ecdysterone in serum samples has been successfully conducted. Ecdysterone could be quantified in all post-administration samples using solid phase extraction (SPE) for sample pretreatment.

How reliable is dietary supplement labelling?-Experiences from the analysis of ecdysterone supplements.

The present study aimed to design, develop, and optimize an analytical procedure to perform the quantitative determination of ecdysterone in commercially available dietary supplements. The newly developed procedure is based on the extraction of ecdysterone from the supplements and the subsequent analysis by an optimized UHPLC-MS/MS method. Chromatographic separation was performed on an Agilent Eclipse Plus C18 column (2.1 mm x 100 mm, particle size 1.8 µm). The mass spectrometer was operated in positive ionization mode (ESI+) with acquisition in dynamic multiple reaction monitoring (dMRM) mode. Using the protonated molecular ion [M+H]+ ecdysterone (target) and cortisol (internal reference) were detected at m/z 481 and 363, respectively. The assay was fully validated according to ICH guidelines and the method resulted to be fit for purpose in terms of accuracy and precision (CV% and RE% <15). Time-different intermediate precision was found within the reported range according to AOAC guideline for dietary supplements and botanicals. Quantitation has been performed using an external calibration considering the minimal matrix influences, preliminarily assessed following a cross comparison with an elaborate and time consuming standard addition method. The method was successfully applied to 12 different dietary supplements labelled to contain ecdysterone, showing an actual content generally much lower than the labelled one.

Ecdysteroids as non-conventional anabolic agent: performance enhancement by ecdysterone supplementation in humans.

Recent studies suggest that the anabolic effect of ecdysterone, a naturally occurring steroid hormone claimed to enhance physical performance, is mediated by estrogen receptor (ER) binding. In comparison with the prohibited anabolic agents (e.g., metandienone and others), ecdysterone revealed to be even more effective in a recent study performed in rats. However, scientific studies in humans are very rarely accessible. Thus, our project aimed at investigating the effects of ecdysterone-containing products on human sport exercise. A 10-week intervention study of strength training of young men (n = 46) was carried out. Different doses of ecdysterone-containing supplements have been administered during the study to evaluate the performance-enhancing effect. Analysis of blood and urine samples for ecdysterone and potential biomarkers of performance enhancement has been conducted. To ensure the specificity of the effects measured, a comprehensive screening for prohibited performance-enhancing substances was also carried out. Furthermore, the administered supplement has been tested for the absence of anabolic steroid contaminations prior to administration. Significantly higher increases in muscle mass were observed in those participants that were dosed with ecdysterone. The same hypertrophic effects were also detected in vitro in C2C12 myotubes. Even more relevant with respect to sports performance, significantly more pronounced increases in one-repetition bench press performance were observed. No increase in biomarkers for liver or kidney toxicity was noticed. These data underline the effectivity of an ecdysterone supplementation with respect to sports performance. Our results strongly suggest the inclusion of ecdysterone in the list of prohibited substances and methods in sports in class S1.2 "other anabolic agents".

Effect of non-prohibited drugs on the phase II metabolic profile of morphine. An in vitro investigation for doping control purposes.

The potential consequences of drug-drug interaction on the strategies adopted by anti-doping laboratories to report an adverse analytical finding for morphine were investigated. We evaluated in vitro the effects of 14 drugs on the principal metabolic pathways of morphine. The selected drugs are among those most commonly used by the athletes, none of them presently included in the World Anti-Doping Agency (WADA) Prohibited List. The non-prohibited drugs included 4 antifungals (fluconazole, itraconazole, ketoconazole, and miconazole), 6 benzodiazepines (alprazolam, bromazepam, clonazepam, lorazepam, lormetazepam, and triazolam), and 4 non-steroidal anti-inflammatory drugs (diclofenac, ibuprofen, ketoprofen, and nimesulide). The in vitro assays were based on the use of either human liver microsomes or uridine 5'-diphospho-glucuronosyl-transferases. Morphine and its glucuronides were determined by developed liquid chromatography-mass spectrometry procedure after dilution with an aqueous solution containing their deuterated isotopologues as internal standards. Morphine is mainly excreted as phase II metabolites: about 70% of the parent compound is found to be biotransformed by UGT2B7 to morphine-3-glucuronide (6065%) and morphine-6-glucuronide (5-10%). A reduction of the enzymatic activity of the UGT2B7 was recorded in the presence of 9 of the 14 drugs under investigation (ketoconazole, miconazole, itraconazole, diclofenac, ibuprofen, clonazepam, lorazepam, lormetazepam, and triazolam), with a consequent significant reduction of the levels of the glucuronide metabolites. This phenomenon in vivo may affect the rate of the urinary excretion of morphine with the risk of reporting "false negative" results, especially in case of results close to the decision limit value set by WADA.