Determination of Ecdysterone in Dietary Supplements and Spinach by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry
Abstract
:1. Introduction
2. Materials and Methods
2.1. Spinach, Supplements, Chemicals, and Reagents
2.2. Sample Preparation
2.2.1. Supplement Extraction
2.2.2. Spinach Extraction
2.3. Instrumental Analysis
2.3.1. Liquid Chromatography Separation
2.3.2. Mass Spectrometry
3. Results
3.1. Method Validation
3.1.1. Selectivity
3.1.2. Matrix Effects, Precision, LOD, and LOQ
3.1.3. Carry-Over
3.2. Method Application to Dietary Supplement Samples
3.3. Spinach Sample
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Isenmann, E.; Ambrosio, G.; Joseph, J.F.; Mazzarino, M.; de la Torre, X.; Zimmer, P.; Kazlauskas, R.; Goebel, C.; Botrè, F.; Diel, P.; et al. Ecdysteroids as non-conventional anabolic agent: Performance enhancement by ecdysterone supplementation in humans. Arch. Toxicol. 2019, 93, 1807–1816. [Google Scholar] [CrossRef] [PubMed]
- Parr, M.K.; Ambrosio, G.; Wuest, B.; Mazzarino, M.; de la Torre, X.; Sibilia, F.; Joseph, J.; Diel, P.; Botrè, F. Targeting the administration of ecdysterone in doping control samples. Forensic Toxicol. 2020, 38, 172–184. [Google Scholar] [CrossRef]
- Hunyadi, A.; Herke, I.; Lengyel, K.; Báthori, M.; Kele, Z.; Simon, A.; Tóth, G.; Szendrei, K. Ecdysteroid containing food supplements from Cyanotisarachnoidea on the European market: Evidence for spinach product counterfeiting. Sci. Rep. 2016, 6, 373322. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Cheng, D.M.; Yousef, G.G.; Grace, M.H.; Rogers, R.B.; Gorelick-Feldman, J.; Raskin, I.; Lila, M.A. In vitro production of metabolism-enhancing phytoecdysteroids from Ajuga turkestanica. Plant Cell Tiss. Organ Cult. 2008, 93, 73–83. [Google Scholar] [CrossRef]
- Guibout, L.; Mamadalieva, N.; Balducci, C.; Girault, J.P.; Lafont, R. The minor ecdysteroids from Ajuga turkestanica. Phytochem. Anal. 2015, 26, 293–300. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Parr, M.K.; Botre, F.; Nas, A.; Hengevoss, J.; Diel, P.; Wolber, G. Ecdysteroids: A novel class of anabolic agents? Biol. Sport 2015, 32, 2. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Grucza, K.; Kowalczyk, K.; Wicka, M.; Szutowski, M.; Bulska, E.; Kwiatkowska, D. The use of a valid and straightforward method for the identification of higenamine in dietary supplements in view of anti-doping rule violation cases. Drug Test Anal. 2019, 11, 912–917. [Google Scholar] [CrossRef] [PubMed]
- World Anti-Doping Agency (WADA). Minimum Criteria for Chromatographic-Mass Spectrometric Confirmation of The Identity of Analytes for Doping Control Purposes. Available online: https://www.wada-ama.org/sites/default/files/resources/files/td2021idcrfinaleng0 (accessed on 25 October 2021).
- World Anti-Doping Agency (WADA). WADA2021 Prohibited List. Available online: https://www.wada-ama.org/sites/default/files/resources/ (accessed on 25 October 2021).
- Feng, S.; Jiang, J.; Hu, P.; Zhang, J.; Liu, T.; Zhao, Q.; Li, B. A phase I study on pharmacokinetics and pharmacodynamics of higenamine in healthy Chinese subjects. Acta Pharmacol. Sin. 2012, 33, 1353–1358. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- National Center for Biotechnology Information. PubChem Substance Database; CID=7753. Available online: http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=7753 (accessed on 13 December 2021).
- Lisi, A.; Hasick, N.; Kazlauskas, R.; Goebel, C. Studies of methylhexaneamine in supplements and geranium oil. Drug Test Anal. 2011, 3, 873–876. [Google Scholar] [CrossRef] [PubMed]
- Gauthier, T.D. Evidence for the presence of 1,3-dimethylamylamine (1,3-DMAA) in geranium plant materials. Anal. Chem. Insights 2013, 8, 29–40. [Google Scholar] [CrossRef] [PubMed]
- Di Lorenzo, C.; Moro, E.; Dos Santos, A.; Uberti, F.; Restani, P. Could 1,3 dimethylamylamine (DMAA) in food supplements have a natural origin? Drug Test Anal. 2013, 5, 116–121. [Google Scholar] [CrossRef] [PubMed]
- Austin, K.G.; Travis, J.; Pace, G.; Lieberman, H.R. Analysis of 1,3 dimethylamylamine concentrations in Geraniaceae, geranium oil and dietary supplements. Drug Test Anal. 2014, 6, 797–804. [Google Scholar] [CrossRef] [PubMed]
- Fleming, H.L.; Ranaivo, P.L.; Simone, P.S. Analysis and Confirmation of 1,3-DMAA and 1,4-DMAAin Geranium Plants Using High Performance LiquidChromatography with Tandem Mass Spectrometryat ng/g Concentrations. Anal. Chem. Insights 2012, 7, 59–78. [Google Scholar] [CrossRef] [PubMed]
- Kwiatkowska, D.; Wójtowicz, M.; Jarek, A.; Goebel, C.; Chajewska, K.; Turek-Lepa, E.; Pokrywka, A.; Kazlauskas, R. N,N-dimethyl-2-phenylpropan-1-amine—New designer agent found in athlete urine and nutritional supplement. Drug Test. Anal. 2015, 7, 331–335. [Google Scholar] [CrossRef] [PubMed]
- Kazlauskas, R.; Hasick, N. ASDTL supplements project. In Recent Advances in Doping Analysis(19); Schänzer, W., Geyer, H., Gotzmann, A., Mareck, U., Eds.; Sport und Buch Strauß: Köln, Gremany, 2011; pp. 10–14. [Google Scholar]
- Van Thuyne, W.; van Eenoo, P.; Delbeke, F.T. Nutritional supplements: Preva-lence of use and contamination with doping agents. Nutr. Res. Rev. 2006, 19, 147–158. [Google Scholar]
- Geyer, H.; Braun, H.; Burke, L.M.; Stear, S.J.; Castel, L.M. A–Z of nutritional supple-ments. Br. J. Sports Med. 2011, 45, 752–754. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Wójtowicz, M.; Jarek, A.; Chajewska, K.; Turek-Lepa, E.; Kwiatkowska, D. Determination of designer doping agent2-ethylamino-1-phenylbutane—In dietary supplements andexcretion study following single oral supplement dose. J. Pharm. Biomed. 2015, 115, 523–533. [Google Scholar] [CrossRef] [PubMed]
- World Anti-Doping Agency (WADA). International Standard for Laboratories 2021. Available online: https://www.wadaama.org/sites/default/files/resources/files/isl2021.pdf (accessed on 25 October 2021).
No. | Manufacturer/ Product Name | Product Form/ EcdysteroneAmount | Estimated Concentration of Ecdysterone(mg/g) |
---|---|---|---|
1 | Magnum Nutraceuticals Thrust® Male Amplifier | Capsules 250 mg/portion | 5 |
2 | Universal Animal M. StakTM | Capsules 100 mg/portion | 69 |
3 | Activ LAB MACA | Capsules 2.5 mg/portion | 5 |
4 | VEM HERB Supplement DietyEcdysterone 95% Beta Ecdysterone | Capsules 490 mg/portion | 64 |
5 | PEAK Ecdysterone extract andL-leucine | Capsules 100 mg/portion | 25 |
6 | SyntraxSyntraTM Anabolic Agent | Capsules 200 mg/portion | 383 |
Compound | Precursor Ion | Product Ions | Cone Voltage (V) | Collision Energy (eV) |
---|---|---|---|---|
445.08 | 10 | |||
427.07 | 15 | |||
Ecdysterone | 481.32 | 371.06 | 10 | 10 |
165.07 | 10 | |||
Mefruside | 382.97 | 129.05 | 25 | 20 |
Ecdysterone Concentration (mg/g) | 1 | 5 | 10 |
---|---|---|---|
Coefficient of variation (CV) (%) | 8.3 | 6.6 | 6.6 |
Intra-day precision (N = 3) (CV) (%) | 2–12.8 | 3.9–4.7 | 0.6–6.1 |
Inter-day precision (N = 9) (CV) (%) | 8.1 | 4.4 | 3.9 |
Matrix effects (%) | 101 | 83 | 93 |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Grucza, K.; Wicka, M.; Drapała, A.; Kwiatkowska, D. Determination of Ecdysterone in Dietary Supplements and Spinach by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry. Separations 2022, 9, 8. https://doi.org/10.3390/separations9010008
Grucza K, Wicka M, Drapała A, Kwiatkowska D. Determination of Ecdysterone in Dietary Supplements and Spinach by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry. Separations. 2022; 9(1):8. https://doi.org/10.3390/separations9010008
Chicago/Turabian StyleGrucza, Krzysztof, Mariola Wicka, Aleksandra Drapała, and Dorota Kwiatkowska. 2022. "Determination of Ecdysterone in Dietary Supplements and Spinach by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry" Separations 9, no. 1: 8. https://doi.org/10.3390/separations9010008
APA StyleGrucza, K., Wicka, M., Drapała, A., & Kwiatkowska, D. (2022). Determination of Ecdysterone in Dietary Supplements and Spinach by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry. Separations, 9(1), 8. https://doi.org/10.3390/separations9010008