LC-MS-based metabolomics for detecting adulteration in Tribulus terrestris-derived dietary supplements
Food Chemistry: X, Volume 27, April 2025, 102476: Fig. 3. a) Score plot of PCA model M4 containing: 1-Authentic samples of T. terrestris, 4- commercially obtained products; Fig. 4. b) Score plot of PCA model M5 containing: 1-Authentic samples of T. terrestris, 4a- commercially obtained products excluding outliers from model M4; c) Score plot of OPLS-DA model (M6) containing: 1-Authentic samples of T. terrestris, 4b- commercially obtained products appearing as a separate group in PCA model M5.
The goal of this study is to assess the authenticity and detect possible adulteration in Tribulus terrestris-derived dietary supplements. An untargeted liquid chromatography-high resolution mass spectrometry (LC-HRMS) metabolomics approach was employed to analyze authentic plant materials, simulated adulterated samples, and commercial products.
By combining LC-HRMS data with advanced statistical and machine learning tools, the study aimed to uncover undeclared substances and markers of product degradation. The findings highlight the power of metabolomics for quality control and the urgent need for stricter monitoring of supplement authenticity to ensure consumer safety.
The original article
LC-MS-based metabolomics for detecting adulteration in Tribulus terrestris-derived dietary supplements
Dejan Gođevac, Jovana Stanković Jeremić, Mirjana Cvetković, Katarina Simić, Ivana Sofrenić, Jovana Ljujić, Lazar Popović, Uroš Gašić, Yen-Nhi Hoang, Tao Huan, Stefan Ivanović
Food Chemistry: X, Volume 27, April 2025, 102476
https://doi.org/10.1016/j.fochx.2025.102476
licensed under CC-BY 4.0
Selected sections from the article follow. Formats and hyperlinks were adapted from the original.
Tribulus terrestris L. is a creeping, herbaceous annual plant from the Zygophyllaceae family, native to warm climates across Europe, Asia, Africa, America, and Australia. T. terrestris is an important medicinal plant with a long history of use in traditional medicine worldwide (Ghosh et al., 2012; Zahedi et al., 2024). It has been used to treat eye infections (Ghosh et al., 2012), abdominal bloating (Zahedi et al., 2024), swelling, pathological pains, edema (Ghosh et al., 2012), kidney disorders (Ghosh et al., 2012; Zahedi et al., 2024), cardiovascular system diseases (Zahedi et al., 2024), gastrointestinal liver diseases (Zahedi et al., 2024), itchy skin (Saeed et al., 2024), urinary tract infections, heart issues, and high blood pressure (Saeed et al., 2024). T. terrestris extracts have exhibited a range of pharmacological effects, including hypercholesteremic, antioxidant, antibacterial, anti-inflammatory, analgesic, and hepatoprotective properties (Saeed et al., 2024), free radical scavenging activity (Zahedi et al., 2024), as well as antihypertensive and antifungal effects (Ghosh et al., 2012; Zahedi et al., 2024). Additionally, the extracts demonstrated diuretic, anthelmintic, and anticancer effects (Ghosh et al., 2012; Saeed et al., 2024), provided protective effects against ischemic stroke, and demonstrated antispasmodic and immunomodulatory properties (Saeed et al., 2024). Globally, extracts of T. terrestris have been primarily used to enhance muscle strength and to treat impotence and sexual dysfunction (Saeed et al., 2024; Zahedi et al., 2024). The wide spectrum of biological activity of T. terrestris extracts comes from their rich and varied chemical composition, which includes steroidal saponins, flavonoids, alkaloids, tannins, and phenolic acids (Ghosh et al., 2012; Saeed et al., 2024; Zahedi et al., 2024).
The popularity of dietary supplements has increased dramatically in the last several years. Herbal supplements are particularly appealing to consumers because they are marketed as a natural alternative to treat nutrient deficiencies (Taghvimi et al., 2019), perceived as safer and healthier than synthetic drugs, and commonly used for weight loss, muscle building, and boosting energy levels (Taghvimi et al., 2019). T. terrestris dietary supplementation effectively reduces inflammation and oxidative stress, improves muscle tone, and supports sexual function in men (Saeed et al., 2024).
Our research introduces an advanced untargeted metabolomics approach employing Orbitrap HRMS for the rapid and comprehensive identification of adulterants in T. terrestris-based dietary supplements. This technology, in contrast to traditional approaches that typically focus on targeted screening of known contaminants, allows for the detection of both expected and unexpected adulterants with exceptional sensitivity and specificity. To establish and verify this methodology, three categories of samples were examined: (i) authentic plant material, (ii) simulated fraudulent products where authentic plant material was blended with known adulterants to mimic adulteration, and (iii) commercially available supplements of unknown authenticity.
This method utilizes multivariate statistical techniques, including principal component analysis (PCA) and partial least squares (PLS), to efficiently distinguish between authentic and adulterated samples based on their metabolic profiles. Additionally, a convolutional neural network (CNN)-based filtering tool (Xing et al., 2021) was used to improve the detection of steroid-related compounds, given that steroidal saponins are the principal bioactive components of T. terrestris, while anabolic steroids are a common form of its adulteration. This workflow enhances quality control initiatives and offers a more efficient and high-throughput approach to ensure consumer safety and regulatory compliance in the dietary supplement sector.
2. Materials and methods
2.3. Sample preparation
Plant samples were first air-dried and then finely ground using an analytical mill (IKA A11 basic, Germany). For extraction, 2 g of aerial part samples, 1 g of fruit samples, and 2 g of supplement samples were utilized. The extraction was performed with 15 mL of 70 % EtOH in an ultrasonic bath (Elmasonic P 30H, Germany) for 30 min at 80 °C. The ethanolic extracts were filtered and evaporated under reduced pressure. The resulting extracts were further purified using solid-phase extraction (SPE) with 500 mg/6 mL C18-E cartridges (Strata, Phenomenex, USA). Conditioning was done with 5 mL of methanol, followed by equilibration with 5 mL of water. Subsequently, the dissolved extracts (20 mg in 10 mL of water) were loaded into the cartridges, washed with 5 mL of 5 % methanol in water, and analytes were eluted with 5 mL of methanol. The final extracts were filtered through a 0.45-μm nylon filter (Agilent, US) before LC-HRMS analysis.
2.4. LC-HRMS analysis
In total, 79 samples were prepared for LC-HRMS analysis, 38 authentic, 14 spiked samples, and 27 commercially available supplements. A QC solution was prepared by combining 100 μL of each tested sample, ensuring consistent and reliable comparison of results across all runs. The PCA score plot (Fig. S11) illustrates that all five QC sample runs are tightly clustered near the origin, indicating high reproducibility of the LC-HRMS analysis.
The LC-HRMS analysis was conducted using a Thermo Scientific™ Vanquish™ Core HPLC system coupled to the Orbitrap Exploris 120 mass spectrometer (San Jose, CA, USA).
The elution was performed at 25 °C on a Hypersil GOLD™ C18 analytical column (50 × 2.1 mm, 1.9 mm) from Thermo Fisher Scientific. The mobile phase consisted of (A) a 0.1 % aqueous formic acid solution and (B) acetonitrile MS grade containing 0.1 % formic acid, which were applied in the following gradient program: 5 % B in the first 1.12 min, 5–20 % B from 1.12 to 1.68 min, 20–80 % B from 1.68 to 7.26 min, 80–95 % B from 7.26 to 7.82 min, 95 % B from 7.82 to 9.94 min, 95 %–5 % B from 9.94 to 10.00 min, and 5 % B until the 13th min. The flow rate was set to 0.4 mL/min and the injection volume was 3 μL.
The Orbitrap Exploris 120 mass spectrometer was equipped with an ESI source operating in positive ionization mode. The capillary voltage, nebulizer gas pressure, drying gas flow rate, and source temperature were described by Xing et al. (2021). Full scan MS was monitored from 100 to 1500 m/z with the Orbitrap resolution set to 60,000 FWHM, RF Lens 70 %, and maximum injection time 100 ms, while data-dependent MS2 experiments were monitored from 50 m/z with an Orbitrap resolution of 15,000 FWHM and normalized collision energy set to 30 %, isolation window 1.5 m/z, and maximum injection time 22 ms. The dynamic exclusion time was set to 5 s, with exclusion applied after one occurrence of a specific scan. The intensity threshold was set to 5 × 103.
Data was acquired using the Xcalibur® data software (Thermo Finnigan, San Jose, CA, USA).
3. Results and discussion
3.1. LC-HRMS metabolomics profiling
To develop an untargeted metabolomics approach for detecting adulterants in T. terrestris-based dietary supplements, three categories of samples were obtained: i) authentic plant material, ii) simulated fraudulent plant material, and iii) commercially available supplements. To account for variations in metabolite composition due to ecological factors and differences in tissue composition, several biological replicates from different botanical populations were used in the selection of authentic plant material. Therefore, T. terrestris specimens were gathered from distinct places, across various years and months, and from diverse plant parts, including either fruits or the whole aerial parts (Table S1).
In recent years, numerous reports have documented the adulteration of aphrodisiac dietary supplements, primarily with active pharmaceutical ingredients from phosphodiesterase-5 (PDE5) inhibitors, such as sildenafil, vardenafil, and tadalafil – compounds intended for the treatment of erectile dysfunction (Jiru et al., 2019; Pujol et al., 2024). To mimic such adulteration, these PDE5 inhibitors were selected for blending with authentic plant material. Given that T. terrestris extracts are also marketed for muscle-strengthening purposes, adulteration with anabolic agents – known for reducing body fat and increasing muscle mass – poses a plausible concern (Roiffé et al., 2019). Therefore, testosterone propionate and 4-androstene-3,17-dione were incorporated into authentic plant material to simulate adulteration.
Using an Orbitrap LC-HRMS-based untargeted methodology, all authentic and spiked samples, as well as commercially available supplements, were examined. A data-dependent acquisition (DDA) was used to preferentially fragment the most intense ions, generating high-quality MS/MS spectra to enhance metabolite identification (Guo et al., 2022). This methodology was appropriate, as the objective was not trace-level detection but rather the identification of adulteration involving significant or therapeutically relevant quantities of added adulterants.
Feature extraction and peak alignment in the positive mode LC-HRMS data were performed utilizing techniques integrated into MS-DIAL, an open-source LC-MS data processing software. To minimize false-positive metabolite feature detection, the extraction sensitivity was reduced by setting a high minimum peak height threshold. Furthermore, all extracted ion chromatograms were visually examined to confirm Gaussian-like peak shapes, thereby guaranteeing the reliability of the features found. Additionally, a five-fold sample average-to-blank average change filter was applied to further reduce false-positive detections. This resulted in a total of 1531 extracted features.
Compound annotation was performed using in silico fragmentation of all predicted molecular formulas, determined based on accurate mass, isotope ratios, and ion data obtained from databases integrated into the MS-FINDER software (Tsugawa et al., 2016). In cases where an equivalent feature in the negative MS mode was present, in silico analysis was conducted accordingly. The MS data of structural candidates generated by MS-FINDER were compared to those from the literature. Moreover, the botanical origin of the identified compounds was also verified through the literature. This approach enabled compound identification at confidence level 2, as outlined by the Compound Identification Work Group of the Metabolomics Society (Blaženović et al., 2018). The most important compounds identified in this study are listed in Table 1. The mass ppm error and MS/MS fragments for the adulterants identified are listed in Table S3.
3.3. Revealing adulteration and authenticity in commercial T. terrestris products
Next, a PCA model (M4) was created using authentic plant samples and commercially obtained products based on T. terrestris. In the score plot of the first two principal components of this model, two distinct groups of supplements deviated significantly from the central cluster, with samples S3, S10, S15, S19, and S20 identified as outliers (Table S4, Fig. 3a). The contribution plots of the PCA model, including samples S3, S10, and S15, were examined to identify the variables primarily responsible for the observed deviations. In all three instances, caffeine had the greatest contribution, followed by catechin tannins, i.e., gallocatechin gallate, gallocatechin, epicatechin gallate, and epicatechin, and the flavonoid vitexin-2′-rhamnoside (Figs. S3-S5). Since these metabolites are characteristic of green tea (Camellia sinensis) (Morikawa et al., 2013), their presence strongly indicates that supplements S3, S10, and S15 were adulterated with green tea extracts. It was reported that many herbal dietary supplements contain green tea, although their presence may not always be mentioned on the product label (Navarro et al., 2013).
Food Chemistry: X, Volume 27, April 2025, 102476: Fig. 3. a) Score plot of PCA model M4 containing: 1-Authentic samples of T. terrestris, 4- commercially obtained products; Fig. 4. b) Score plot of PCA model M5 containing: 1-Authentic samples of T. terrestris, 4a- commercially obtained products excluding outliers from model M4; c) Score plot of OPLS-DA model (M6) containing: 1-Authentic samples of T. terrestris, 4b- commercially obtained products appearing as a separate group in PCA model M5.
Producers may incorporate green tea to boost the stimulant properties of the supplement, appealing to consumers seeking increased energy, alertness, or improved athletic performance (Grgic et al., 2020). Compounds in green tea, particularly caffeine and catechin tannins, possess potent antioxidant effects and may enhance metabolism, fat oxidation, and endurance, making the supplement appear more efficacious (Meyer et al., 2023). Green tea extracts are widely available and cost-effective, allowing manufacturers to reduce production costs while retaining some properties of a T. terrestris supplement. Furthermore, it is crucial to recognize that the adulteration of supplements with green tea extracts may pose considerable safety risks. Numerous reported cases have documented significant hepatitis and liver damage, with symptoms improving upon discontinuation of the supplement (Navarro et al., 2013). This underscores the need for stringent analytical techniques, such as the untargeted LC-HRMS metabolomics method used in this study, to accurately identify and characterize adulterants, thereby ensuring product authenticity and customer safety (Navarro et al., 2013).
3.4. Conclusion remarks
In summary, our untargeted LC-HRMS metabolomics methodology offers considerable advantages over current techniques in detecting adulteration in T. terrestris-based products. Unlike chromatographic fingerprinting approaches, which primarily focus on plant identification, our method can detect a broader spectrum of adulterants. Similarly, while targeted LC-HRMS techniques offer exceptional sensitivity for known PDE5 inhibitors, they are intrinsically limited to predefined analyte lists. The comprehensive detection capability of our method, along with sophisticated chemometric techniques, ensures authenticity verification and effective adulteration screening, marking a significant improvement in the quality control of herbal dietary supplements.
These findings underscore the importance of comprehensive metabolomics analysis in evaluating the authenticity of herbal dietary supplements and identifying potentially fraudulent activities. The research emphasizes the need for stringent quality control and regulatory protocols to prevent the distribution of adulterated products and safeguard consumer safety.
