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PreprintCited by 16Doping in SportsPharmacological Effects and AssaysHormonal and reproductive studies

Urinary Elimination of Ecdysterone and its Metabolites following a Single-Dose Administration in Humans

Open access
Ambrosio, Gabriella, Yuliandra, Tasha, Wuest, Bernhard, Mazzarino, Monica, Torre, Xavier de la, Botrè, Francesco, Diel, Patrick, Isenmann, Eduard +1 more·2021·Metabolites
DOI 10.20944/preprints202105.0106.v1PMC PMC8227119

Preprint — published as 10.3390/metabo11060366 · Paper page

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Abstract

Ecdysterone is a phytosteroid widely discussed for its various pharmacological, growth-promoting and anabolic effects mediated by 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 urines 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 urines, detected for more than 2 days with a maximum concentration (Cmax) in the 2.8-8.5 h urines (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. 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.

AI snapshot

Written from the paper’s full text.

TL;DR

In twelve healthy volunteers, a single 50 mg oral dose of pure ecdysterone produced urinary ecdysterone as the most abundant analyte, detectable for more than 2 days, plus two metabolites, 14-deoxy-ecdysterone and 14-deoxy-poststerone, with later detection windows. Cumulative urinary recovery averaged 18%, 2.3%, and 1.5% of the dose, respectively, and ecdysterone/14-deoxy-ecdysterone half-lives were about 3 h.

Key findings

  • Ecdysterone was detected in all subjects, with maximum concentration in the 2.8–8.5 h urine at 4.4–30.0 µg/mL; it appeared after 45 min and remained detectable for more than 2 days (58 h), with maximum excretion rate 0.1–4.8 mg/h in the 0.4–4.4 h urine.
  • 14-Deoxy-ecdysterone was detected in all 12 subjects, with maximum concentration in the 8.5–39.5 h urine at 0.1–6.0 µg/mL and detection for about 3 days (75 h); 14-deoxy-poststerone was detected in 10 of 12 subjects, with maximum concentration in the 23.3–41.3 h urine at 0.1–1.5 µg/mL and detection from 8.50 to 97 h.
  • Mean cumulative urinary excretion was 18% ± 13% for ecdysterone, 2.3% ± 1.74% for 14-deoxy-ecdysterone, and 1.5% ± 2.1% for 14-deoxy-poststerone; total recovery of parent drug plus metabolites was up to 50.3% (mean 21.1%, n = 12).
  • Half-lives were about 3 h for ecdysterone (3.4 and 3.0 h by rate-of-excretion and sigma-minus methods) and 14-deoxy-ecdysterone (3.1 and 2.2 h); 14-deoxy-poststerone gave 9.7 ± 9.5 h versus 4.4 ± 1.0 h. No significant sex differences were found for ecdysterone and 14-deoxy-ecdysterone, while a significant difference (p ≤ 0.05) was reported for 14-deoxy-poststerone.

Methods

  • Single-dose administration study in 12 healthy subjects (7 females and 5 males; mean age 26 ± 3.1 y, weight 74 ± 12.0 kg, height 174 ± 8.3 cm); 50 mg of pure ecdysterone dissolved in alcoholic solution was administered at 9:00 a.m., and urine was collected one day before and five days after administration.
  • Urine samples were analyzed by dilute-and-inject LC–MS/MS with positive electrospray ionization and multiple reaction monitoring; the method was validated according to EMA and ICH M10 guidelines for ecdysterone and 14-deoxy-ecdysterone, including selectivity, linearity, LOD/LOQ, accuracy and precision, matrix effect, stability, and carryover.
  • Excretion profiles, cumulative urinary amounts, and half-lives were calculated using the rate-of-excretion and sigma-minus methods; data acquisition and analysis used MassHunter Quant software, with Origin Pro 9.1 for visualization and statistical treatment.

Limitations

  • Stated: the analytical method was fully validated only for ecdysterone and 14-deoxy-ecdysterone; no full validation was performed for 14-deoxy-poststerone, and no investigation of phase II metabolite excretion was carried out.
  • Stated: 14-deoxy-poststerone was detected in only 10 of 12 subjects and an excretion profile curve could be obtained for only 5 volunteers; high inter-individual variability was observed, the half-life of 14-deoxy-poststerone needs further evaluation, and up to 50.3% of the dose was recovered in urine, with the remainder possibly unabsorbed or excreted by other pathways or as undetected metabolites.
  • Evident from the text: the study has a small sample (n = 12), uses a single dose, and describes no placebo or control group; the abstract states that sex-specific differences were not observed, whereas the results and discussion report a significant sex difference (p ≤ 0.05) for 14-deoxy-poststerone.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.20944/preprints202105.0106.v1

Cites

39 of 39 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

2 of 24 cited sources checked · 0 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    The WADA decision to include ecdysterone was based mainly on a controlled administration trial in humans demonstrating performance-enhancing effects in power training.

    “This decision has been based mainly on the results of a controlled administration trial in humans, which demonstrated its performance-enhancing effects in power training [29].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [29] Isenmann E. Ambrosio G. Joseph J.F. Mazzarino M. de la Torre X. Zimmer P. Kazlauskas R. Goebel C. Botre F. Diel P. Ecdysteroids as non-conventional anabolic agent: Performance enhancement by ecdysterone supplementation in humans Arch. Toxicol. 2019 93 1807 1816 10.1007/s00204-019-02490-x 31123801 source

      The Abstract (page unavailable) describes a 10-week strength-training study in young men and reports greater gains in muscle mass and bench-press performance with ecdysterone. It recommends inclusion on the prohibited list, but our copy does not say WADA made a decision or that this trial was the main basis for one. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Findingcitation

    The LOQ was 1 ng/mL for both ecdysterone and 14-deoxy-ecdysterone.

    “The LOQ was determined based on the lowest concentration in which the % error of accuracy was within ±–20% and coefficient of variation (CV) ≤ 20% [37], and corresponded to 1 ng/mL for both ecdysterone and 14-deoxyecdysterone.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [37] International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Draft Version. M10: Bioanalytical Method Validation ICH Geneva, Switzerland 2019

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Findingown data

    Ecdysterone maximum concentration was detected in the 2.8-8.5 h urine, ranging from 4.4-30.0 μg/mL.

    “Its maximum concentration was detected in the 2.8–8.5 h urine, ranging from 4.4–30.0 µg/mL.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The detected analytes in post-administration urine are attributable to the administered ecdysterone dose.
  • Findingown data

    14-deoxy-ecdysterone maximum excretion rate was determined in the 10.1-29.5 h urine, ranging from 0.02-0.24 mg/h.

    “The maximum excretion rate was determined in the 10.1–29.5 h urine, ranging from 0.02–0.24 mg/h.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The detected analytes in post-administration urine are attributable to the administered ecdysterone dose.
  • Findingown data

    14-deoxy-poststerone maximum concentration was detected in the 23.3-41.3 h urine, ranging from 0.1-1.5 μg/mL.

    “The maximum concentration was detected in the 23.3–41.3 h urine, ranging from 0.1–1.5 µg/mL.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The 14-deoxy-poststerone detected in post-administration urine is a metabolite of ecdysterone.
    • Rests on an assumption: The analytical method is suitable for quantitation of 14-deoxy-poststerone despite validation only for ecdysterone and 14-deoxy-ecdysterone.
    • Rests on an assumption: The detected analytes in post-administration urine are attributable to the administered ecdysterone dose.
  • May combine two claims. 21
  • The quote only partly states this claim. 3
  • Classified as probably not a substantial claim. 11

  • May combine two claims.

    Study design and conductcitation

    Concentration data below LOQ but not below LOD were included to evaluate the excretion profile because inclusion can result in a better fit and excluding or replacing them with zero could lead to biased pharmacokinetic parameters.

    “Concentration data below the LOQ (but not below the LOD) were included to evaluate the excretion profile, as their inclusion can result in a better fit of the excretion profile model, while excluding or replacing them with zero could lead to biased pharmacokinetic parameters [39,40,41,42].”
    Quote in the paperSubstantial claim1 of 4 sources checked
    • cites [39] Keizer R.J. Jansen R.S. Rosing H. Thijssen B. Beijnen J.H. Schellens J.H. Huitema A.D. Incorporation of concentration data below the limit of quantification in population pharmacokinetic analyses Pharmacol. Res. Perspect. 2015 3 e00131 10.1002/prp2.131 26038706 PMC4448983 source

      The Materials and Methods section says the paper included detectable concentrations below the LLOQ but discarded those below the LOD. The Results and Conclusion report improved bias and precision in population PK analyses compared with discarding such data. The paper does not evaluate an excretion-profile model or replacing concentrations with zero, so those parts of the claim are unsupported. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [40] Dorababunn M. Pharmacokinetic Modeling of Data with Below Quantification Limit J. Bioequiv. Bioavailab. 2012 4 2 3 10.4172/jbb.10000e12 source
    • cites [41] Hecht M. Veigure R. Couchman L. CI S.B. Standing J.F. Takkis K. Evard H. Johnston A. Herodes K. Leito I. Utilization of data below the analytical limit of quantitation in pharmacokinetic analysis and modeling: Promoting interdisciplinary debate Bioanalysis 2018 10 1229 1248 10.4155/bio-2018-0078 30033744 source
    • cites [42] Senn S. Holford N. Hockey H. The ghosts of departed quantities: Approaches to dealing with observations below the limit of quantitation Stat. Med. 2012 31 4280 4295 10.1002/sim.5515 22825800 source

  • The quote only partly states this claim.

    Findingown data

    For 5 volunteers, it was possible to obtain an excretion profile curve for 14-deoxy-poststerone.

    “For 5 volunteers, it was possible to obtain an excretion profile curve.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The 14-deoxy-poststerone detected in post-administration urine is a metabolite of ecdysterone.
    • Rests on an assumption: The analytical method is suitable for quantitation of 14-deoxy-poststerone despite validation only for ecdysterone and 14-deoxy-ecdysterone.
    • Rests on an assumption: The detected analytes in post-administration urine are attributable to the administered ecdysterone dose.

  • May combine two claims.

    Findingown data

    Mean cumulative urinary excretion percentages were 18% ±13% for ecdysterone, 2.3% ±1.74% for 14-deoxy-ecdysterone, and 1.5% ±2.1% for 14-deoxy-poststerone.

    “The mean values correspond to 18% with an SD of ±13%, 2.3% ± 1.74%, and 1.5% ± 2.1%, respectively.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The 14-deoxy-poststerone detected in post-administration urine is a metabolite of ecdysterone.
    • The authors state this as an assumption: After 12 h, ecdysterone reaches a plateau in cumulative urinary excretion, indicating almost complete elimination and that the maximum amount of ecdysterone in urine is reached.
    • Rests on an assumption: The analytical method is suitable for quantitation of 14-deoxy-poststerone despite validation only for ecdysterone and 14-deoxy-ecdysterone.
    • Rests on an assumption: The detected analytes in post-administration urine are attributable to the administered ecdysterone dose.

  • May combine two claims.

    Findingown data

    The presence of 14-deoxy-ecdysterone was observed in post-administration urine samples and its formation was confirmed for all 12 subjects.

    “In this study, the presence of the metabolite 14-deoxy-ecdysterone was observed in the post-administration urine samples and its formation was confirmed for all 12 subjects considered.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The detected analytes in post-administration urine are attributable to the administered ecdysterone dose.

  • May combine two claims.

    Findingown data

    Ecdysterone was found to have a short urinary half-life of 3.4 h using the rate of excretion method and 3.0 h using the sigma-minus method.

    “Ecdysterone was found to have a short urinary half-life, which corresponds to 3.4 and 3.0 h, when using the rate of excretion and the sigma-minus method, respectively.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Urinary excretion profiles reflect the systemic pharmacokinetics and elimination of ecdysterone and its metabolites.
    • Rests on an assumption: The detected analytes in post-administration urine are attributable to the administered ecdysterone dose.

  • May combine two claims.

    Findingown data

    Ecdysterone was detectable in urine after 45 min from administration and for more than 2 days (58 h).

    “Ecdysterone was detectable in urine samples, after 45 min from administration and for more than 2 days (58 h).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The detected analytes in post-administration urine are attributable to the administered ecdysterone dose.

  • May combine two claims.

    Findingown data

    Half-lives of ecdysterone and 14-deoxy-ecdysterone in post-administration urine were similar and corresponded to about 3 h.

    “The calculated half-life for ecdysterone and 14-deoxy-ecdysterone were found to be similar and corresponded to about 3 h.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Urinary excretion profiles reflect the systemic pharmacokinetics and elimination of ecdysterone and its metabolites.
    • Rests on an assumption: The detected analytes in post-administration urine are attributable to the administered ecdysterone dose.

  • May combine two claims.

    Findingcitation

    The LOD was 0.24 ng/mL for ecdysterone and 0.34 ng/mL for 14-deoxy-ecdysterone.

    “The LOD was calculated using the standard deviation (SD) of the response and the slope of calibration (LOD = 3.3 × S.D./slope) [38], and corresponded to 0.24 ng/mL for ecdysterone and to 0.34 ng/mL for 14-deoxy-ecdysterone.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] International Conference on Harmonization of Technical Requirements fo Registration of Pharmaceuticals for Human Use Validation of Analytical Procedurs: Text and Methodology Q2(R1) ICH Geneva, Switzerland 2005

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Findingown data

    14-deoxy-ecdysterone was detected after 2.95 h and remained detectable for about 3 days (75 h).

    “14-Deoxy-ecdysterone was already detected after 2.95 h and remained detectable for about 3 days (75 h).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The detected analytes in post-administration urine are attributable to the administered ecdysterone dose.

  • May combine two claims.

    Findingown data

    Results from cumulative excretion of 14-deoxy-ecdysterone showed that the plateau was reached at different times: 58.3% of subjects between 25-40 h, 25% between 15-25 h, and 16.6% between 40-62 h.

    “Specifically, in 58.3% of subjects the plateau was reached between 25–40 h, in 25% it was reached between 15 to 25 h, and in the remaining 16.6% between 40–62 h.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The detected analytes in post-administration urine are attributable to the administered ecdysterone dose.

  • May combine two claims.

    Findingown data

    Cumulative excretion percentages of 14-deoxy-ecdysterone and 14-deoxy-poststerone were much lower than that of ecdysterone, which was excreted faster than the metabolites.

    “These results showed that the cumulative excretion percentages of 14-deoxy-ecdysterone and 14-deoxy-poststerone were much lower than the one obtained for ecdysterone, which was excreted in urine samples, faster than the metabolites (Figure 4).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The 14-deoxy-poststerone detected in post-administration urine is a metabolite of ecdysterone.
    • Rests on an assumption: The analytical method is suitable for quantitation of 14-deoxy-poststerone despite validation only for ecdysterone and 14-deoxy-ecdysterone.
    • Rests on an assumption: The detected analytes in post-administration urine are attributable to the administered ecdysterone dose.

  • May combine two claims.

    Findingown data

    Cumulative urinary excretion percentages ranged from 2.8-47.2% for ecdysterone, 0.4-6.1% for 14-deoxy-ecdysterone, and 0.01-4.9% for 14-deoxy-poststerone.

    “The cumulative excretion percentages range from 2.8–47.2% for ecdysterone, from 0.4–6.1% for the 14-deoxy-ecdysterone, and from 0.01–4.9% for 14-deoxy-poststerone.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The 14-deoxy-poststerone detected in post-administration urine is a metabolite of ecdysterone.
    • Rests on an assumption: The analytical method is suitable for quantitation of 14-deoxy-poststerone despite validation only for ecdysterone and 14-deoxy-ecdysterone.
    • Rests on an assumption: The detected analytes in post-administration urine are attributable to the administered ecdysterone dose.

  • May combine two claims.

    Findingown data

    14-deoxy-ecdysterone was found to have a short half-life of 3.1 h using the rate of excretion method and 2.2 h using the sigma-minus method.

    “As for ecdysterone, a short half-life was also observed for 14-deoxy-ecdysterone, calculated as 3.1 and 2.2 h, using the rate of excretion and the sigma-minus method, respectively.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Urinary excretion profiles reflect the systemic pharmacokinetics and elimination of ecdysterone and its metabolites.
    • Rests on an assumption: The detected analytes in post-administration urine are attributable to the administered ecdysterone dose.

  • May combine two claims.

    Findingown data

    A small increase in the excretion rate of 14-deoxy-poststerone after the maximum peak was achieved was observed, similar to the trend of the excretion rate of 14-deoxy-ecdysterone.

    “Similar to the trend of the excretion rate of 14-deoxy-ecdysterone, a small increase in the excretion rate of 14-deoxy-poststerone, after the maximum peak was achieved, was observed.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Brandt reported 14-deoxy-ecdysterone as a urinary metabolite of ecdysterone.

    “Brandt reported the 14-deoxy-ecdysterone [33].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [33] Brandt F. Pharmakokinetik und Metabolismus des 20-Hyroxyecdysons im Menschen Ph.D. Thesis Philipps-Universität Marburg Marburg, Germany 2003 10.17192/z2004.0136 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In a single-dose administration study, 51.5 mg of pure ecdysterone was administered to one healthy volunteer, and the presence of 14-deoxy-ecdysterone was confirmed in post-administration urine.

    “Recently, in a single dose administration study, 51.5 mg of pure ecdysterone has been administered to one healthy volunteer, and the presence of 14-deoxy-ecdysterone was confirmed in the post-administration urine samples, in comparison to the in-house synthesized reference [34].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] Parr M.K. Ambrosio G. Wuest B. Mazzarino M. de la Torre X. Sibilia F. Joseph J.F. Diel P. Botrè F. Targeting the administration of ecdysterone in doping control samples Forensic. Toxicol. 2019 38 172 184 10.1007/s11419-019-00504-y source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Parr et al. reported ecdysterone as the most abundant analyte in post-administration urine, with a wider detection window than 14-deoxyecdysterone, which was excreted later.

    “Parr et al. reported ecdysterone as the most abundant analyte in post-administration urine samples, with a wider detection window than 14-deoxyecdysterone, which was excreted later [34].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] Parr M.K. Ambrosio G. Wuest B. Mazzarino M. de la Torre X. Sibilia F. Joseph J.F. Diel P. Botrè F. Targeting the administration of ecdysterone in doping control samples Forensic. Toxicol. 2019 38 172 184 10.1007/s11419-019-00504-y source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Tsitsimpikou et al. reported 2-deoxy-ecdysterone and deoxy-ecdysone as urinary metabolites of ecdysterone.

    “Indeed, while Tsitsimpikou et al. reported a 2-deoxy-ecdysterone and deoxy-ecdysone as urinary metabolites [32]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Tsitsimpikou C. Tsamis G.D. Siskos P.A. Spyridaki M.H. Georgakopoulos C.G. Study of excretion of ecdysterone in human urine Rapid Commun. Mass Spectrom. 2001 15 1796 1801 10.1002/rcm.433 11565096 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Ecdysterone has been included in the WADA Monitoring Program since 2020 to assess potential patterns of misuse in sport.

    “Only recently (since 2020), ecdysterone has been included in the Monitoring Program of the World Anti-Doping Agency (WADA), under the section “Anabolic Agents, In-and Out-of-Competition” [28], to assess potential patterns of misuse in sport.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [28] World Anti-doping Agency The 2020 Monitoring Program Available online: https://www.wada-ama.org/sites/default/files/wada_2020_english_monitoring_program_.pdf (accessed on 1 December 2019)

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Tsitsimpikou et al. reported that ecdysterone is detected in urine mainly as deoxy-ecdysone metabolite, followed by the parent compound and the 2-deoxy-ecdysterone metabolite.

    “while Tsitsimpikou et al. reported that ecdysterone is detected in urine mainly as deoxy-ecdysone metabolite, followed by the parent compound and the 2-deoxy-ecdysterone metabolite [32].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Tsitsimpikou C. Tsamis G.D. Siskos P.A. Spyridaki M.H. Georgakopoulos C.G. Study of excretion of ecdysterone in human urine Rapid Commun. Mass Spectrom. 2001 15 1796 1801 10.1002/rcm.433 11565096 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The analytical method was validated for ecdysterone and 14-deoxy-ecdysterone only.

    “The analytical method was validated for ecdysterone and 14-deoxy-ecdysterone only.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    The CV of ISTD-normalized matrix factor was lower than 15% in all 6 lots at LQC and HQC, except for 14-deoxy-ecdysterone at LQC where it was 20%.

    “The CV of the ISTD-normalized MF calculated was lower than 15% in all 6 lots of matrix at LQC and HQC, except for 14-deoxy-ecdysterone at the LQC, in which it corresponded to 20%.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Study design and conductown data

    Stability results for ecdysterone and 14-deoxy-ecdysterone in urine were within ±15% under tested conditions.

    “The results obtained were all within the ±15%.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    14-deoxy-ecdysterone was excreted later than the parent compound, probably due to its less polar physico-chemical characteristics.

    “The 14-deoxy-ecdysterone was excreted later than the parent compound, probably due to its less polar physico-chemical characteristics.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The detected analytes in post-administration urine are attributable to the administered ecdysterone dose.

  • May combine two claims. The quote only partly states this claim.

    Study design and conductown data

    The calibration curves for ecdysterone and 14-deoxy-ecdysterone were best fitted by weighted quadratic regression with R2 of 0.997 and 0.998, respectively.

    “The weighted quadratic regression was applied after the evaluation of linearity, according to Mandel’s fitting test (F-test), which resulted in a significantly better fit of the second-order calibration function (quadratic), in comparison to the first-order regression function (linear), with p = 99%.”
    • Also stated in: § 2.1. Validation of the Analytical Methodology · hedged
      “The best fit for ecdysterone and 14-deoxy-ecdysterone was indicated by the correlation coefficients (R2) of 0.997 and 0.998, respectively.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    Carry-over was considered irrelevant because no signal higher than 20% of LOQ was detected in blank samples after HQC injection.

    “No signal higher than the 20% of the LOQ for ecdysterone and 14-deoxy-ecdysterone was detected in the blank samples (methanol). Thus, carry-over was considered irrelevant.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    The analytical procedure was validated in terms of selectivity, linearity, LOD, LOQ, precision and accuracy, matrix effect, stability, and carry over.

    “The analytical procedure was validated in terms of selectivity, linearity, limit of detection (LOD), and limit of quantitation (LOQ), precision and accuracy, matrix effect, stability, and carry over.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    Earlier investigations showed no excretion of ecdysterone or 14-deoxy-ecdysterone as conjugates.

    “However, earlier investigations showed no excretion of ecdysterone or 14-deoxy-ecdysterone as conjugates.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In mice, Kumpun et al. reported 14-deoxy-poststerone as a metabolite of ecdysterone.

    “In mice, Kumpun et al. [10] already reported it as metabolite of ecdysterone, as well.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Kumpun S. Girault J.P. Dinan L. Blais C. Maria A. Dauphin-Villemant C. Yingyongnarongkul B. Suksamrarn A. Lafont R. The metabolism of 20-hydroxyecdysone in mice: Relevance to pharmacological effects and gene switch applications of ecdysteroids J. Steroid Biochem. Mol. Biol. 2011 126 1 9 10.1016/j.jsbmb.2011.03.016 21439380 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    14-deoxy-poststerone may be generated by side-chain cleavage, analogous to the formation of progestins from cholesterol in human steroid biosynthesis.

    “Analogous to the formation of progestins from cholesterol in human steroid biosynthesis, 14-deoxy-poststerone may be generated by side-chain cleavage.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    The study was approved by the ethical committee of the German Sport University Cologne and carried out following the regulations of the Helsinki declaration.

    “The study was approved by the ethical committee of the German Sport University Cologne, and was carried out following the regulations of the Helsinki declaration.”
    Quote in the paperNot a substantial claim
31 of 31

  • The paper relies on this without support.

    The 14-deoxy-poststerone detected in post-administration urine is a metabolite of ecdysterone.

    “The 14-deoxy-ecdysterone and a new metabolite, the 14-deoxy-poststerone, were detected and confirmed by comparing the retention time and the mass spectra with the reference standard material, using LC–MS/MS.”

  • The paper relies on this without support.

    Urinary excretion profiles reflect the systemic pharmacokinetics and elimination of ecdysterone and its metabolites.

    “The half-lifes of ecdysterone, 14-deoxy-ecdysterone, and 14-deoxy-poststerone in post-administration urine samples were calculated using two different methods—rate of excretion method and the sigma-minus method.”

  • The authors state this as an assumption.

    After 12 h, ecdysterone reaches a plateau in cumulative urinary excretion, indicating almost complete elimination and that the maximum amount of ecdysterone in urine is reached.

    “From the results of the cumulative urinary excretion, we can assume that after 12 h, ecdysterone reaches a plateau, indicating that it is almost completely eliminated, the maximum amount of ecdysterone in urine samples is reached, and that it corresponds to 18%.”

  • The paper relies on this without support.

    The analytical method is suitable for quantitation of 14-deoxy-poststerone despite validation only for ecdysterone and 14-deoxy-ecdysterone.

    “The developed and validated method enabled the quantitation of ecdysterone and 14-deoxy-ecdysterone in post-administration urine samples”

  • The paper relies on this without support.

    The detected analytes in post-administration urine are attributable to the administered ecdysterone dose.

    “Following a single-dose administration of 50 mg of ecdysterone, the parent compound resulted to be the most abundant analyte in all post-administration urine samples.”
5 of 5

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Only 1-3% of 20-hydroxyecdysone and its metabolites are absorbed after consumption of large amounts of spinach and quinoa.

    “only 1–3% of 20E and its metabolites are absorbed.”

    From How reliable is the labeling of a commercial phytosteroid product? A 12-week randomized double-blind training study (2025) · cites this paper as [40]

    The Results (§2.3) report mean urinary excretion of 18% of the administered ecdysterone dose, plus 2.3% and 1.5% as two metabolites. The Discussion (§3) reports a mean of 21.1% recovered in urine overall, contrary to the claim that only 1–3% of the compound and its metabolites were absorbed. The study administered pure ecdysterone, not spinach or quinoa; it did not directly measure absorption. (LLM, conf=0.88)

    Checked by a reasoning model — may be wrong.

    Experimental — accuracy not yet measured

  • Administration of 50 mg pure 20-hydroxyecdysone resulted in approximately 50% recovery in urine.

    “administration of 50 mg of pure 20E resulted in approximately 50% recovery in urine”

    From How reliable is the labeling of a commercial phytosteroid product? A 12-week randomized double-blind training study (2025) · cites this paper as [40]

    The Conclusions say that about 50% of a 50 mg dose was recovered in urine as ecdysterone and two metabolites. The Discussion qualifies this as the maximum recovery among twelve volunteers (50.3%); mean recovery was 21.1%. Without that qualifier, the claim could imply approximately 50% was typical. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • The first urinary concentrations of ECDY were reported in 2021 by Ambrosio et al. and Kraiem et al., showing a dose-dependent maximum concentration between 0.5 and 1.3 μg/mL for administrations below 5 mg ECDY and between 3 and 30 μg/mL for 50 mg ECDY applications.

    “The first urinary concentrations of ECDY were reported in 2021 by Ambrosio et al. and Kraiem et al., showing a dose-dependent maximum concentration between 0.5 and 1.3 μg/mL for administrations below 5 mg ECDY and between 3 and 30 μg/mL for 50 mg ECDY applications.6,7”

    From Investigations into the human metabolism of ecdysterone. (2023) · cites this paper as [7]

    Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

    Experimental — accuracy not yet measured

  • These extremely different excretion patterns have not been described before.

    “These extremely different excretion patterns have not been described before”

    From Investigations into the human metabolism of ecdysterone. (2023) · cites this paper as [7]

    The Discussion (page unavailable) reports high inter-individual variability in ecdysterone and metabolite excretion, including different times at which subjects reached an excretion plateau. It does not say that such different patterns had never been described before, so the novelty claim is unsupported. (LLM, conf=0.80)

    Experimental — accuracy not yet measured

  • Ambrosio et al. noted significantly higher excretion of 14DP in female volunteers compared with males, which was not detectable for 14DE or ECDY.

    “Interestingly, Ambrosio et al. also noted a significantly higher excretion of 14DP in female volunteers compared with males, which was not detectable for 14DE or ECDY.7”

    From Investigations into the human metabolism of ecdysterone. (2023) · cites this paper as [7]

    The Results (§2.3) and Discussion (§3; page unavailable) report a significant female–male difference in urinary 14-deoxy-poststerone (14DP), but no significant difference for 14-deoxy-ecdysterone (14DE) or ecdysterone (ECDY). The passages do not state the direction of the 14DP difference, so they do not establish that excretion was higher in females. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Conclusion presents the maximum recovery as typical recovery

    Major

    The conclusion says about 50% of the administered dose was recovered, but the Discussion identifies 50.3% as the highest recovery and gives a mean of 21.1%.

  • Abstract denies a reported sex-specific difference

    Major

    The abstract says no sex-specific differences were observed, whereas the Results report a significant difference for 14-deoxy-poststerone.

2 of 2

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. [9]Tchoukouegno Ngueu S. Bioactivity of Plants Secondary Metabolites: Estrogenic, Cytotoxic and Anabolic Effects on Estrogen Target Organs of an Extract of Erythrina Excelsa and Ecdysterone Ph.D. Thesis German Sport University Cologne, Germany 2013
  2. [38]International Conference on Harmonization of Technical Requirements fo Registration of Pharmaceuticals for Human Use Validation of Analytical Procedurs: Text and Methodology Q2(R1) ICH Geneva, Switzerland 2005

Cited by

Sorted by citations

Related

Authors
Ambrosio, G., Yuliandra, T., Wuest, B., Mazzarino, M., Torre, X. D. L., Botrè, F., Diel, P., Isenmann, E., & Parr, M. K.
Affiliations
  • Freie Universität Berlin (DE)
  • Agilent Technologies (Germany) (DE)
  • Federazione Medico Sportiva Italiana (IT)
  • German Sport University Cologne (DE)
Corresponding author
Parr, Maria Kristina
ORCID
Venue
Metabolites
Publisher
MDPI AG
Published
2021-05-06
Volume
11
Issue
6
Pages
366-366
ISSN
2218-1989 (ISSN-L)
DOI
10.20944/preprints202105.0106.v1
PMC
PMC8227119
OpenAlex
W3157606085
Cited by
16
References
39
Citations by year
2026: 4 · 2025: 1 · 2024: 3 · 2023: 5 · 2022: 1 · 2021: 2
FWCI
3.82
Type
journal article
Language
en
Open access
Licence
Locations
  • Metabolites (publishedVersion, cc-by)
  • Preprints.org (acceptedVersion, cc-by)
  • PubMed (publishedVersion)
  • SERVAL (Université de Lausanne) (publishedVersion, cc-by)
  • IRIS (submittedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Florence Research (University of Florence) (submittedVersion)
  • Refubium (Universitätsbibliothek der Freien Universität Berlin) (submittedVersion, cc-by)
  • Universitätsbibliothek der FU Berlin Hochschulschriftenstelle u. Dokumentenserver (cc-by)
Funders
  • World Anti-Doping Agency (WADA 18C18MP, 18C18MP)
  • Freie Universität Berlin
Topics
  • Social Sciences › Sociology and Political Science › Doping in Sports
  • Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Pharmacological Effects and Assays
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Hormonal and reproductive studies
Keywords
20-hydroxyecdysone, urinary excretion, pharmacokinetics, phytosteroids, performance-enhancing drugs, first-order elimination kinetics, half-life

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-10.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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