Published May 21, 2025
| Version v1
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Development and validation of an LC-PDA method for detection of the natural β2 agonist higenamine
Authors/Creators
- 1. Medical University of Plovdiv, Plovdiv, Bulgaria
- 2. Medical University of Plovdiv, Sofia, Bulgaria
Description
Higenamine is a natural β2 agonist that can be found in the extracts of many plants, including Nelumbo nucifera Gaertn., Tinospora crispa L., Nandina domestica Thunb., Gnetum parvifolium Warb., Asarum sieboldii Miq., Asarum heterotropoides F. Schmidt, Aconitum carmichaelii Debeaux, and Aristolochia brasiliensis Mart. & Zucc. Since 2017, the compound has been included in WADA's prohibited list, and its detection in the biological samples of professional athletes is regarded as a violation of the anti-doping rules. Currently, many dietary supplements (DSs) contain plants that are rich in higenamine, posing a risk of inadvertent intake by athletes. In general, the presence of higenamine in DSs containing extracts from Nelumbo nucifera Gaertn. and other plant species that are a source of this β2 agonist is undisclosed. Most of these plants are involved in the treatment of various medical conditions in Eastern folk medicine, and their intake is associated with many benefits. The objective of this study was to establish an LC-PDA method for detecting higenamine in plant extracts and dietary supplements (DSs). The method development utilized the Shimadzu LCMS-2050 system (Shimadzu Co., Kyoto, Japan) along with a PDA detector. Detection was performed using gradient elution. The mobile phase consisted of formic acid in water and acetonitrile. The PDA detector was set at 282 nm. The total run time was 16 min. The method exhibited a good correlation coefficient of 0.999, a limit of detection (LOD) of 0.643 μg/mL, and a limit of quantification (LOQ) of 1.949 μg/mL. The method was validated in accordance with ICH guidelines, providing a fast and reliable detection process.
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References
- Austin KG, Travis J, Pace G, Lieberman HR (2014) Analysis of 1,3 dimethylamylamine concentrations in Geraniaceae , geranium oil and dietary supplements: 1,3 dimethylamylamine, dietary supplements, geranium oil and Geraniaceae. Drug Testing and Analysis 6: 797–804. https://doi.org/10.1002/dta.1491
- Cohen PA, Travis JC, Vanhee C, Ohana D, Venhuis BJ (2021) Nine prohibited stimulants found in sports and weight loss supplements: deterenol, phenpromethamine (Vonedrine), oxilofrine, octodrine, beta-methylphenylethylamine (BMPEA), 1,3-dimethylamylamine (1,3-DMAA), 1,4-dimethylamylamine (1,4-DMAA), 1,3-dimethylbutylamine (1,3-DMBA) and higenamine. Clinical Toxicology 59(11): 975–981. https://doi.org/10.1080/15563650.2021.1894333
- Cooper ER, McGrath KCY, Li X, Heather AK (2018) Androgen bioassay for the detection of nonlabeled androgenic compounds in nutritional supplements. International Journal of Sport Nutrition and Exercise Metabolism 28: 10–18. https://doi.org/10.1123/ijsnem.2017-0018
- Djaoudene O, Romano A, Bradai YD, Zebiri F, Ouchene A, Yousfi Y, Amrane-Abider M, Sahraoui-Remini Y, Madani K (2023) A global overview of dietary supplements: Regulation, market trends, usage during the COVID-19 pandemic, and health effects. Nutrients 15: 3320. https://doi.org/10.3390/nu15153320
- Garthe I, Maughan RJ (2018) Athletes and supplements: Prevalence and perspectives. International Journal of Sport Nutrition and Exercise Metabolism 28: 126–138. https://doi.org/10.1123/ijsnem.2017-0429
- Herbold NH, Visconti BK, Frates S, Bandini L (2004) Traditional and nontraditional supplement use by collegiate female varsity athletes. International Journal of Sport Nutrition and Exercise Metabolism 14: 586–593. https://doi.org/10.1123/ijsnem.14.5.586
- Kerpel dos Santos M, Gleco E, Davidson JT, Jackson GP, Pereira Limberger R, Arroyo LE (2018) DART-MS/MS screening for the determination of 1,3-dimethylamylamine and undeclared stimulants in seized dietary supplements from Brazil. Forensic Chemistry 8: 134–145. https://doi.org/10.1016/j.forc.2018.03.005
- Kosuge T, Yokota M (1976) Studies on cardiac principle of aconite root. Chemical and Pharmaceutical Bulletin 24: 176–178. https://doi.org/10.1248/cpb.24.176
- Lee S-R, Schriefer JM, Gunnels TA, Harvey IC, Bloomer RJ (2013) Acute oral intake of a higenamine-based dietary supplement increases circulating free fatty acids and energy expenditure in human subjects. Lipids in Health and Disease 12: 148. https://doi.org/10.1186/1476-511X-12-148
- Lin Y-A, Hsu M-C (2024) Determination of doping higenamine in Chinese herbal medicines and their concentrated preparations by LC-MS/MS. Journal of Pharmaceutical and Biomedical Analysis 246: 116188. https://doi.org/10.1016/j.jpba.2024.116188
- Mehta NR, Patel EP, Patani PV, Shah B (2013) Nelumbo nucifera (Lotus): A review on ethanobotany, phytochemistry and pharmacology. Indian Journal of Pharmaceutical and Biological Research 1: 152–167. https://doi.org/10.30750/ijpbr.1.4.26
- Moro CF, Yonekura M, Kouzuma Y, Agrawal GK, Rakwal R (2013) Lotus – A Source of Food and Medicine: Current Status and Future Perspectives in Context of the Seed Proteomics. International Journal of Life Sciences 7: 1–5. https://doi.org/10.3126/ijls.v7i1.6394
- Parr M, Pokrywka A, Kwiatkowska D, Schänzer W (2011) Ingestion of designer supplements produced positive doping cases unexpected by the athletes. Biology of sport / Institute of Sport 28: 153–157. https://doi.org/10.5604/959289
- Paudel KR, Panth N (2015) Phytochemical profile and biological activity of Nelumbo nucifera. Evidence-Based Complementary and Alternative Medicine 2015: 1–16. https://doi.org/10.1155/2015/789124
- Qiu J, Zhang R, Liu W, Lu Y, Liu J (2025) Immunomagnetic bead-based sample preparation method and immunoassay for detecting higenamine in herbal products. Journal of Pharmaceutical and Biomedical Analysis 257: 116712. https://doi.org/10.1016/j.jpba.2025.116712
- Rangelov Kozhuharov V, Ivanov K, Ivanova S (2022) Higenamine in plants as a source of unintentional doping. Plants 11: 354. https://doi.org/10.3390/plants11030354
- Reeuwijk NM, Venhuis BJ, de Kaste D, Hoogenboom RLAP, Rietjens IMCM, Martena MJ (2014) Active pharmaceutical ingredients detected in herbal food supplements for weight loss sampled on the Dutch market. Food Additives & Contaminants: Part A 31: 1783–1793. https://doi.org/10.1080/19440049.2014.958574
- Russo G, Barbato F, Grumetto L (2016) A validated LC/UV method for the determination of four adulterating drugs in herbal slimming capsules. Journal of Pharmaceutical and Biomedical Analysis 117: 436–445. https://doi.org/10.1016/j.jpba.2015.09.027
- Singhuber J, Zhu M, Prinz S, Kopp B (2009) Aconitum in traditional chinese medicine—A valuable drug or an unpredictable risk? Journal of Ethnopharmacology 126: 18–30. https://doi.org/10.1016/j.jep.2009.07.031
- Stajić A, Anđelković M, Dikić N, Rašić J, Vukašinović-Vesić M, Ivanović D, Jančić-Stojanović B (2017) Determination of higenamine in dietary supplements by UHPLC/MS/MS method. Journal of Pharmaceutical and Biomedical Analysis 146: 48–52. https://doi.org/10.1016/j.jpba.2017.08.017
- Stojanovic B, Rasic J, Andjelkovic M, Dikic N, Dragicevic N, Djordjevic B, Forsdahl G, Gmeiner G (2024) Urinary excretion profile of higenamine in females after oral administration of supplements – Doping scenario. Journal of Chromatography B 1235: 124047. https://doi.org/10.1016/j.jchromb.2024.124047
- Tietje C, Brouder A [Eds] (2010) International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. In: Handbook of Transnational Economic Governance Regimes. Brill | Nijhoff, 1041–1053. https://doi.org/10.1163/ej.9789004163300.i-1081.897
- Venhuis B, Keizers P, van Riel A, de Kaste D (2014) A cocktail of synthetic stimulants found in a dietary supplement associated with serious adverse events: A cocktail of synthetic stimulants found in a dietary supplement associated with serious adverse events. Drug Testing and Analysis 6: 578–581. https://doi.org/10.1002/dta.1664
- Williams M (2006) Dietary Supplements and Sports Performance: Herbals. Journal of the International Society of Sports Nutrition 3: 1. https://doi.org/10.1186/1550-2783-3-1-1
- Woo H, Kim JW, Han KM, Lee JH, Hwang IS, Lee JH, Kim J, Kweon SJ, Cho S, Chae KR, Han SY, Kim J (2013) Simultaneous analysis of 17 diuretics in dietary supplements by HPLC and LC-MS/MS. Food Additives & Contaminants: Part A 30: 209–217. https://doi.org/10.1080/19440049.2012.738939
- World Anti Doping Agency (2024) WADA The Prohibited List. World Anti Doping Agency. https://www.wada-ama.org/en/prohibited-list [October 25, 2024]
- World Anti-Doping Agency [WADA] (2019) WADA technical document TD2019MRPL—minimum required performance levels for detection and identification of non-threshold substances. https://www.wada-ama.org/sites/default/files/resources/files/td2019mrpl_eng.pdf [November 17, 2021]
- Yan K, Wang X, Wang Z, Wang Y, Luan Z, Gao X, Wang R (2019) The risk of higenamine adverse analytical findings following oral administration of Plumula nelumbinis capsules. Drug Testing and Analysis 11: 1731–1736. https://doi.org/10.1002/dta.2701
- Yen C-C, Tung C-W, Chang C-W, Tsai C-C, Hsu M-C, Wu Y-T (2020) Potential Risk of Higenamine Misuse in Sports: Evaluation of Lotus Plumule Extract Products and a Human Study. Nutrients 12: 285. https://doi.org/10.3390/nu12020285
- Zhang N, Lian Z, Peng X, Li Z, Zhu H (2017) Applications of Higenamine in pharmacology and medicine. Journal of Ethnopharmacology 196: 242–252. https://doi.org/10.1016/j.jep.2016.12.033
- Zlatanova M (2025) Administrative regulation of food supplements – coercive administrative measures and administrative penalties. In: Medical Law and Healthcare. Ciela Norma AD, 15–41.