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Determination of some sex hormones in health supplements by liquid chromatography with tandem mass spectrometry

Kieu Van Anh Nguyen Thi Hong Ngoc Phung Cong Ly Dang Thi Kim Hang Pham Thi Thanh Ha
Received: 23 Oct 2023
Revised: 24 Dec 2023
Accepted: 26 Dec 2023
Published: 29 Dec 2023

Article Details

How to Cite
Kieu Van Anh, Nguyen Thi Hong Ngoc, Phung Cong Ly, Dang Thi Kim Hang, Pham Thi Thanh Ha. "Determination of some sex hormones in health supplements by liquid chromatography with tandem mass spectrometry". Vietnam Journal of Food Control. vol. 6, no. 4 (en), pp. 445-457, 2023

Main Article Content


The trend of using dietary supplements in daily life is increasingly popular. However, sex hormones are illegally mixed to improve product effectiveness such as promoting hair growth, developing chest muscles, preventing skin aging, promoting growth, etc. In this study, a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed and applied to quantify sex hormones: progesterone (PG), testosterone (TS), and 17β-estradiol (E2) in health supplements. A Symmetry C18 reversed-phase chromatography column (150 mm x 3.0 mm x 3.5 μm) using a mobile phase system consisting of A: 10 mM HCOONH4/0.1% HCOOH, B: ACN was used.  Mass spectrometry conditions were set at positive ionization mode, precursor ion and two corresponding daughter ions were selected for each analyte. Quantification limits of PG and TS were 0.4 mg/kg and E2 was 4 mg/kg in liquid matrix, hard and soft capsules. The validated method was applied to detect sex hormones in 16 health supplement samples purchased at pharmacies in Hanoi, Vietnam and some online websites. Three samples were detected to be positive for sex hormones, including one sample containing a sex hormone (testosterone) banned from use in health supplements at a low content (about 0.5 mg/kg).


sex hormones, health supplement, LC-MS/MS


[1]. X. He, C. Xi, B. Tang, G. Wang, D. Chen, T. Peng and Z. Mua, “Simultaneous determination of 30 hormones illegally added to anti-ageing functional foods using UPLC-MS/MS coupled with SPE clean-up”, Food Additives & Contaminants: Part A, vol. 31, no. 10, pp. 1625-1638, 2014.
[2]. Ministry of Health, 10/2021/TT-BYT circular: Regulations on the list of substances banned from use in the production and trading of health supplements, 2021. (In Vietnamese)
[3]. U.S. Department of Health and Human Services, National Toxicology Program, “Fourth Annual Report on Carcinogens: Summary, 1985”, U.S. Department of Health and Human Services, 1985.
[4]. M. Farre, R. Brix, M. Kuster, F. Rubio, Y. Goda, M. J. L. Alda and D. Barcelo, “Evaluation of commercial immunoassays for the detection of estrogens in water by comparison with highperformance liquid chromatography tandem mass spectrometry HPLC-MS/MS (QqQ)”, Analytical and Bioanalytical Chemistry, vol. 385, no. 6, pp. 1001-1011, 2006.
[5]. L. Zhang, J. Chen, Y. He, Y. Chi and G. Chen, “A new mixed micellar electrokinetic chromatography method for analysis of natural and synthetic anabolic steroids”, Talanta, vol. 77, no. 3, pp. 1002-1008, 2009.
[6]. C. Almeida and J. M. F. Nogueira, “Determination of steroid sex hormones in water and urine matrices by stir bar sorptive extraction and liquid chromatography with diode array detection”, Journal of Pharmaceutical and Biomedical Analysis, vol. 41, no. 4, pp. 1303-1311, 2006.
[7]. R. Barreiro, P. Regal, M. Diaz-Bao, C. A. Fente and A. Cepeda, “Analysis of Naturally Occurring Steroid Hormones in Infant Formulas by HPLC-MS/MS and Contribution to Dietary Intake”, Foods, vol. 4, no. 4, pp. 605-621, 2015.
[8]. S. S. Alqahtani, D. M. B. Humaid, S. H. Alshail, D. T. AlShammari, H. Al-Showiman, N. Z. Alzoman and H. M. Maher, “Development and validation of a highperformance liquid chromatography/diode array detection method for estrogen determination: Application to residual analysis in meat products”, Open Chemistry, vol. 18, no. 1, pp. 995-1010, 2020.
[9]. R. Simerský, O. Novák, D. A. Morris, V. Pouzar and M. Strnad, “Identification and Quantification of Several Mammalian Steroid Hormones in Plants by UPLC-MS/MS”, Journal of Plant Growth Regulation, vol. 28, no. 2, pp. 125-136, 2009.
[10]. A. Goyon, J. Z. Cai, K. Kraehenbuehl, C. Hartmann, B. Shao and P. Mottier, “Determination of steroid hormones in bovine milk by LC-MS/MS and their levels in Swiss Holstein cow milk”, Food Additives & Contaminants: Part A, vol. 33, no. 5, pp. 804-816, 2016.
[11]. E. Vulliet, J. B. Baugros, M. M. Flament-Waton and M. F. Grenier-Loustalo, “Analytical methods for the determination of selected steroid sex hormones and corticosteriods in wastewater”, Analytical and Bioanalytical Chemmistry, vol. 387, no. 6, pp. 2143-2151, 2007.
[12]. S. He, R. Wang, W. Wei, H. Liu and Y. Ma, “Simultaneous determination of 22 residual steroid hormones in milk by liquid chromatography–tandem mass spectrometry”, International Journal of Dairy Technology, vol. 73, no. 2, pp. 357-365, 2019.
[13]. H. Noppe, K. D. Wasch, S. Poelmans, N. V. Hoof, T. Verslycke, C. R. Janssen and H. F. D. Brabander, “Development and validation of an analytical method for detection of estrogens in water”, Analytical and Bioanalytical Chemistry, vol. 382, no. 1, pp. 91-98, 2005.
[14]. M. Hansen, N. W. Jacobsen, F. K. Nielsen, E. Bjorklund, B. Styrishave and B. Halling-Sorensen, “Determination of steroid hormones in blood by GC-MS/MS”, Analytical and Bioanalytical Chemistry, vol. 400, no. 10, pp. 3409- 3417, 2011.
[15]. T. Trinh, N. B. Harden, H. M. Coleman and S. J. Khan, “Simultaneous determination of estrogenic and androgenic hormones in water by isotope dilution gas chromatography-tandem mass spectrometry”, Journal of Chromatography A, vol. 1218, no. 12, pp. 1668-1676, 2011.
[16]. R. Simerský, O. Novák, D. A. Morris, V. Pouzar, M. Strnad, “Identification and Quantification of Several Mammalian Steroid Hormones in Plants by UPLC-MS/MS”, Journal of Plant Growth Regulation, vol. 28, no. 2, pp. 125-136, 2009.
[17]. W. Meiling, Y. Hongfei, F. Shanliang, Z. Fan, Y. Jinting, D. Hua and L, “Rapid screening and confirmation of hormones in health foods by high performance liquid chromatography-ion trap-time of flight tandem mass spectrometry[J]”, Chinese Journal of Chromatography, vol. 30, no .10, pp. 980-985, 2012.
[18]. AOAC International, AOAC Official Methods of Analysis, Guidelines for Standard Method Performance Requirements, Appendix F, 2016.
[19]. International Conference on Harmonisation, Validation of Analytical Procedures: Text and Methodology, ICH harmonised tripartite guideline, pp. 1-13, 2005.
[20]. Tran Cao Son, Method validation in chemical analysis, Science and Technics Publishing House, Hanoi, 2021. (In Vietnamese)
[21] H. S. Yoo and J. L. Napoli, “Quantification of Dehydroepiandrosterone, 17β-Estradiol, Testosterone, and Their Sulfates in Mouse Tissues by LC-MS/MS”, Anal Chem, vol. 91, no. 22, pp. 14624-14630, 2019.
[22]. Commission Implementing Regulation (EU) 2021/808 on the performance of analytical methods for residues of pharmacologically active substances used in food-producing animals and on the interpretation of results as well as on the methods to be used for sampling and repealing Decisions 2002/657/EC and 98/179/EC.