Adulterated Dietary Supplements Research Articles

Adulterated dietary supplements commercialized in Brazil: development of a screening method and a preliminary study of cytotoxicity

The high consumption of dietary supplements was a fundamental driver for the creation of the regulatory framework by the Brazilian governmental authorities. However, the regulatory agencies lack official low-cost methodologies to evaluate the quality of food supplements. A preliminary screening method by HPLC-DAD was proposed and validated for screening and quantification of adulterants in dietary supplements. The limits of detection and quantification were <0.11 and 0.37 µg.g−1, respectively. The method was applied for the investigation of ten unauthorized substances (spironolactone, hydrochlorothiazide, furosemide, clenbuterol, testosterone, testosterone propionate, yohimbine, vardenafil, tadalafil, and sildenafil) with a time of analysis of <5 min. Sixteen percent of the 44 samples analyzed had at least one adulterant at or above therapeutic concentrations. Subsequently, in vitro evaluations were performed of the potential cytotoxicity to evaluate the cell viability, DNA damage, determination of nitric oxide levels, and quantification of reactive oxygen species. Despite the necessity of further studies, the results indicate a relationship between the presence of adulterants in food supplements and a potential cytotoxic effect.

Simultaneous Detection Method of 11 Respiratory Drug Substances Including Theobromine, Oxymetazoline, etc. in Adulterated Dietary Supplements Using Liquid Chromatography–Electrospray Ionization–Tandem Mass Spectrometry and Liquid Chromatography–Quadrupole Time-of-Flight Mass Spectrometry Analysis

Abstract Recently, the demand for respiratory disease-related products has surged due to the influence of coronavirus disease 2019, prompting warnings about illegal dietary supplements containing unauthorized substances. Additionally, adulterated dietary supplements are continuously detected in open markets, posing significant public health safety problem. In this study, we developed and validated an analytical method for 11 respiratory drug substances using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) and proposed optimal conditions for LC–quadrupole time-of-flight MS (LC–QTOF-MS) to determine the fragmentation patterns of each substance. This method underwent thorough validation considering specificity, linearity, limits of detection and quantification, accuracy, precision, matrix effect, stability, etc. All results met international guidelines. These validated methods were applied to 52 dietary supplements advertised for treating respiratory diseases and enhancing respiratory function, among which one sample was found to contain 313.7 mg/g of theobromine. This determination was made by comparing the product ion ratios with the standards and subsequent quantification. To re-confirm the detected substances, their fragmentation patterns were compared with those of the standards using LC–QTOF-MS. In conclusion, the mass-based information, coupled with the LC–ESI–MS/MS method development, can be successfully applied to rapidly identify 11 respiratory drug substances in illegal dietary supplements used for respiratory disease treatment. The developed simultaneous detection method contributes to public health and safety improvements.

Identification of appetite suppressants through Fourier transform infrared spectroscopy and filtered spectral feature extraction

Rapid inspection of adulterated dietary supplements containing appetite suppressants seized at the scene can provide clues and directions for case investigations. Meanwhile, applying filters and feature extraction techniques can effectively remove noise and interference from the original spectral data, thereby improving the recognition performance of the model. In this study, a total of 419 samples of 7 appetite suppressants confiscated from actual cases were collected. Fourier and Hilbert transform filters were used to preprocess the original spectral data of the samples. Feature extraction was performed using competitive adaptive reweighted sampling, and classification models were established using partial least squares discriminant analysis and random forest algorithms for identification purposes, aiming to select the optimal filter for noise removal. The results showed that the recognition rate and stability of the original spectral data significantly improved after filter processing, with the Hilbert transform filter demonstrating the best noise removal effect. The random forest model outperformed the partial least squares discriminant analysis model regarding recognition rate and stability. The optimized model achieved a recognition rate of 99.21%. This method effectively filters out noise from the spectral data using filters, enhancing the qualitative identification capability of the model and providing reference value for the rapid identification of appetite suppressants in forensic science.

Hierarchical clustering of liquid chromatography-tandem mass spectrometry data for screening of phosphodiesterase type 5 inhibitors and their analogues in adulterated dietary supplements

Sexual enhancement dietary supplements have often been adulterated with phosphodiesterase type 5 (PDE-5) inhibitors used for treatment of erectile dysfunction, and widely distributed through online markets. As the illegal adulterants, the original PDE-5 inhibitor drugs and a numerous number of synthetized analogues, more than 80, have already been found. Therefore, analytical methods that detect various PDE-5 inhibitors and uncover newly synthesized analogues are needed. In this study, we have developed a rapid and reliable screening method for PDE-5 inhibitors and their structural analogues by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by hierarchical clustering based on similarity of MS/MS spectra. Forty reference standards of PDE-5 inhibitors/analogues were measured using a quadrupole-orbitrap mass spectrometer in data-dependent mode. The 60 most intense fragment ions were extracted from each MS/MS spectra, and the ions observed within 1.5 mDa mass tolerance were considered to be the same ion. Based on fragment ion tables representing detected ions for each compound, hierarchical clustering was performed. The resulting dendrogram showed that the reference standards were separated into seven clusters according to their characteristic structures. Subsequently, two additional standards spiked into a herbal sample were analyzed. While herbal components were clearly separated from the clusters of the reference standards, the spiked standards were clustered closely with the structurally similar standards. Furthermore, application of our method to dietary supplements allowed for detection of sildenafil and tadalafil as adulterants. These results suggest that our screening method facilitates discovery of adulterant PDE-5 inhibitors/analogues by illustrating their structural similarity.

Development of a detector tube for rapid detection of tadalafil in adulterated dietary supplements

ABSTRACT Introduction Products marketed as dietary supplements for improving sexual performance at times include phosphodiesterase type 5 (PDE5) inhibitors, such as sildenafil and analogues. The ingestion of these dietary supplements is extremely dangerous as these are not evaluated for their efficacy and safety as pharmaceuticals, and their manufacture is not controlled. For this reason, regulatory authorities conduct inspections on these products to prevent the occurrence and spread of health hazards. Tadalafil is one of the PDE5 inhibitors, and its analogues are found as the most major adulterants in sexual products after analogues of sildenafil. Tadalafil has two chiral carbons that are both R configuration, which is important for PDE5 inhibitory activity. To confirm the molecular configuration of the tested compound, it is subject to instrumental analysis such as circular dichroism detection or X-ray crystal structure analysis. Therefore, it takes longer for testing tadalafil than for other compounds that do not require these processes. Meanwhile, in 2020, the product containing 120 times more tadalafil than the normal dosage was detected in Tokyo, making it an urgent issue to remove these dangerous products from the market. This study proposes a simple and rapid method to detect tadalafil with a newly devised detector tube. Objective Development of a detector tube for the rapid screening of tadalafil with simple functioning. Methods Approximately 80 mg of silica gel powder impregnated with sulfuric acid was filled in a glass tube, having a 3-mm inner diameter, to prepare a detector tube. The measuring method of this detector tube is quite simple; first, both tips of the detector tube are cut, then the sample solution is aspirated, and a decision is made by the colour of the detector tube after 5 minutes. It was defined as a positive result when the detector tube turned violet and negative when it showed other colours. First, the reaction of tadalafil and analogues were confirmed using the prepared detector tube. Then, specificity was tested for active pharmaceutical ingredients and common additives. The tube was also tested for the applicability to some commercially available dietary supplements. Results All tested tadalafil and analogues showed positive results in the test. The limit of detection was 5 μg/mL for tadalafil. Except for levomepromazine, results for additives and any pharmaceutical ingredients tested were negative. This tube was able to detect all real samples containing tadalafil and analogues. None of the real samples that did not contain tadalafil and analogues gave false positives. Conclusions The developed detector tube could rapidly detect tadalafil and analogues with high sensitivity in a simple procedure. It was suggested that false-positive results could occur only if levomepromazine was included in the sample. Other than that, no significant interference that leads to false results was observed in tested common adulterants and additives of supplements. Furthermore, the tube was able to detect tadalafil and analogues in all tested real samples. Accordingly, the proposed test method has the potential for the use of preliminary screening of tadalafil.

Hierarchical Clustering of Lc-Ms/Ms Data for Screening of Phosphodiesterase Type 5 Inhibitors and Their Analogues in Adulterated Dietary Supplements

Hierarchical Clustering of Lc-Ms/Ms Data for Screening of Phosphodiesterase Type 5 Inhibitors and Their Analogues in Adulterated Dietary Supplements

Liquid chromatography-high-resolution mass spectrometry analysis of erectile dysfunction drugs and their analogues in food products

The presence of erectile dysfunction (ED) drugs in adulterated dietary supplements, mainly in pharmaceutical dosage forms, is frequently addressed in the literature. Little attention is given to food products despite their increasing adulteration trend. To address this knowledge gap targeted, suspected-target, and non-targeted strategies were utilised to analyse ED drugs and their analogues in powdered drink mix (PDM), honey, jelly, hard candy, and sugar-coated chewing gum using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). The method was optimised and validated using 23 target analytes, representing different ED drugs with structural similarities. The modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction exhibited insignificant matrix effect (ME) within − 9.2–8.8% and provided complete coverage of target analytes with acceptable extraction recovery (RE) within 75.5–123.9%, except for carbodenafil in the PDM matrix. Based on the ME and RE performance, the analytical method was validated to analyse 25 food samples that claimed to enhance male sexual performance. The method exhibited good specificity and linearity with a limit of detection within 10–70 ng/mL and limit of quantification of 80 ng/mL. Similarly, the accuracy and precision were satisfactory within 77.4–122.0% and< 16.7%RSD, respectively. The LC-HRMS targeted analysis, together with suspected-target and non-targeted screenings, identified and detected ten ED drugs from 24 food samples. The modified QuEChERS extraction with LC-HRMS-based method was demonstrated to be universally applicable to various food products, covering an extensive range of known and potentially novel ED drugs, which is valuable for routine casework.

Istotne problemy związane z bezpieczeństwem surowców roślinnych obecnych w wybranych grupach suplementów diety

Dietary supplements are a wide group of products belonging to the food category. Their sales are still growing. The composition, appearance and the intended use of these products make them resemble drugs. Dietary supplements are designed to meet the specific requirements of different consumer groups. Many dietary supplements contain herbal substances or their preparations. The safety of the herbal material is particularly affected by its proper identification, the part of the plant used as well as the standardization of the extract for a specific biologically active compound. Regardless of the group of consumers for which these products are intended, the risks associated with taking dietary supplements may relate to their contamination with heavy metals, pesticide residues or pathogenic microorganisms. There may also be adulterated dietary supplements on the market, the real composition of which differs from the composition presented on the original packing. Furthermore, the presence of restricted hazardous substances, or limited substances on the product is undoubtedly dangerous to health.

Screening of Phosphodiesterase-5 Inhibitors and Their Analogs in Dietary Supplements by Liquid Chromatography-Hybrid Ion Trap-Time of Flight Mass Spectrometry.

An accurate and reliable method based on ion trap–time of flight mass spectrometry (IT–TOF MS) was developed for screening phosphodiesterase-5 inhibitors, including sildenafil, vardenafil, and tadalafil, and their analogs in dietary supplements. Various parameters affecting liquid chromatographic separation and IT–TOF detection were investigated, and the optimal conditions were determined. The separation was achieved on a reversed-phase column under gradient elution using acetonitrile and water containing 0.2% acetic acid at a flow rate of 0.2 mL/min. The chromatographic eluents were directly ionized in the IT–TOF system equipped with an electrospray ion source operating in the positive ion mode. The proposed screening method was validated by assessing its linearity, precision, and accuracy. Sequential tandem MS was conducted to obtain structural information of the references, and the fragmentation mechanism of each reference was proposed for providing spectral insight for newly synthesized analogs. Structural information, including accurate masses of both parent and fragment ions, was incorporated into the MSn spectral library. The developed method was successfully applied for screening adulterated dietary supplement samples.

Identification of Descarbonsildenafil in an Adulterated Dietary Supplement and Evaluation of Its Inhibitory Activity for Phosphodiesterase Type 5 (PDE5)

Some illegal dietary supplements contain phosphodiesterase type 5 (PDE5) inhibitors, such as sildenafil, for exerting "therapeutic" effects in erectile dysfunction. This is apparently dangerous, and thus, should be appropriately regulated. Identification of descarbonsildenafil was first reported in Singapore in a coffee sample labeled to exert male sexual performance enhancement effects. However, it is unclear whether the compound possesses PDE5 inhibitory activity. We encountered during our survey of dietary supplements, a sexual enhancement product commercially available in Tokyo, in which a peak presumed to be of descarbonsildenafil was detected by LC-UV and electrospray ionization-tandem MS (ESI-MS/MS). The compound was isolated and identified as descarbonsildenafil with liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS), NMR, and X-ray crystal structural analysis. In addition, descarbonsildenafil showed PDE5 inhibitory activity in PDE5 inhibition assay, and its IC50 value for PDE5A1 was found to be 30 nmol/L. The results of INADEQUATE NMR and X-ray crystal structural analysis in this study provide information for the identification of descarbonsildenafil. Since this study indicates that this compound is a PDE5 inhibitor having adequate activity, it is regulated as a drug component in Japan.

Development and validation of an LC-MS/MS method for the simultaneous analysis of 26 anti-diabetic drugs in adulterated dietary supplements and its application to a forensic sample

Development and validation of an LC-MS/MS method for the simultaneous analysis of 26 anti-diabetic drugs in adulterated dietary supplements and its application to a forensic sample

Unapproved Pharmaceutical Ingredients Included in Dietary Supplements Associated With US Food and Drug Administration Warnings

Over half of adults in the United States report consuming dietary supplements. The US Food and Drug Administration (FDA) has warned of numerous dietary supplements containing undeclared, unapproved pharmaceutical ingredients. These FDA warnings have not been comprehensively analyzed for recent years. To summarize trends across adulterated (containing unapproved ingredients) dietary supplements associated with a warning released by the FDA from 2007 through 2016. In this quality improvement study, data were extracted from the FDA's Center for Drug Evaluation and Research, Tainted Products Marketed as Dietary Supplements_CDER database from 2007 through 2016. Data from each warning were recorded unless multiple warnings were issued for the same product within a 6-month period. Date, product name, company, hidden ingredient(s), product category, source of sample, and warning document type were recorded for each included warning. Data analysis was conducted from February 2017 to June 2017. From 2007 through 2016, 776 adulterated dietary supplements were identified by the FDA and 146 different dietary supplement companies were implicated. Most of these products were marketed for sexual enhancement (353 [45.5%]), weight loss (317 [40.9%]), or muscle building (92 [11.9%]), with 157 adulterated products (20.2%) containing more than 1 unapproved ingredient. The most common adulterants were sildenafil for sexual enhancement supplements (166 of 353 [47.0%]), sibutramine for weight loss supplements (269 of 317 [84.9%]), and synthetic steroids or steroid-like ingredients for muscle building supplements (82 of 92 [89.1%]). There were 28 products named in 2 or 3 warnings more than 6 months apart. Of these products, 19 (67.9%) were reported to contain new unapproved ingredients in the second or third warning, consistent with the assumption that the FDA found the product to be adulterated more than once. In recent years (2014-2016), 117 of 303 adulterated samples (38.6%) were identified through online sampling and 104 of 303 (34.3%) were identified through the examination of international mail shipments. Active pharmaceuticals continue to be identified in dietary supplements, especially those marketed for sexual enhancement or weight loss, even after FDA warnings. The drug ingredients in these dietary supplements have the potential to cause serious adverse health effects owing to accidental misuse, overuse, or interaction with other medications, underlying health conditions, or other pharmaceuticals within the supplement.

Determination of 26 anti-diabetic compounds in dietary supplements using a validated UPLC method

ABSTRACTThe purpose of this study was to validate a rapid, simple and accurate method using ultra-performance liquid chromatography (UPLC) for the simultaneous determination of 26 anti-diabetic compounds in illegally adulterated dietary supplements. The method was validated for specificity, linearity, limit of detection, limit of quantitation, precision, accuracy, recovery and stability. All compounds were separated with a resolution of over 1.5. The limits of detection and quantitation were 0.10–1.70 and 0.30–5.10 µg g–1 in a solid sample, respectively; the corresponding values were 0.10–1.25 and 0.30–3.75 µg ml–1 in a liquid sample. The correlation coefficient was > 0.99, precisions were 0.11–3.30% (intra-day) and 0.05–6.15% (inter-day), and accuracies were 83–108% (intra-day) and 85–109% (inter-day). The recoveries were measured with six dosage forms, and the results were acceptable as 87–117% with relative standard deviations ≤ 6.44%. The relative standard deviations of stability were ≤ 3.40% and the standard solution was stable for 48 h. Ninety-six samples were obtained from on/off-line markets and were analysed using the developed method. Among these samples, pioglitazone and glibenclamide were found in seven samples and the concentrations of each compound were 0.15% and 0.26–0.51%, respectively. With the increasing adulteration of dietary supplements with anti-diabetic drugs, this method may be helpful to protect public health and safety.

Collision-induced dissociation pathways of H1-antihistamines by electrospray ionization quadrupole time-of-flight mass spectrometry.

Over the past decades, mass spectrometry technologies have been developed to obtain mass accuracies of one ppm or less. Of the newly developed technologies, quadrupole time-of-flight mass spectrometry (Q-TOF-MS) has emerged as being well suited to routine and high-throughput analyses of pharmaceuticals. Dietary supplements and functional foods have frequently been found to be contaminated with pharmaceuticals. In our continuous efforts to develop methodologies to protect public health against adulterated dietary supplements, we have constructed a mass spectral database for 21 H1-antihistamines encountered as adulterants by using liquid chromatography-electrospray ionization (LC-ESI)/Q-TOF-MS, and have proposed their possible collision-induced dissociation pathways. This database will be very useful for the rapid and accurate detection of H1-antihistamines (known) and their analogues (unknown) illegally added to dietary supplements as well as in other sample matrices.

Adulterated dietary supplements threaten the health and sporting career of up-and-coming young athletes.

The authors provide the perspective of the United States Anti-Doping Agency on the actions youth sport organizations should take to protect the health of developing young athletes.

Comparison of three development approaches for Stationary Phase Optimised Selectivity Liquid Chromatography based screening methods Part II: A group of structural analogues (PDE-5 inhibitors in food supplements)

Three approaches for the development of a screening method to detect adulterated dietary supplements, based on Stationary Phase Optimised Selectivity Liquid Chromatography were compared for their easiness/speed of development and the performance of the optimal method obtained. This comparison was performed for a heterogeneous group of molecules, i.e. slimming agents (Part I) and a group of structural analogues, i.e. PDE-5 inhibitors (Part II).The first approach makes use of primary runs at one isocratic level, the second of primary runs in gradient mode and the third of primary runs at three isocratic levels to calculate the optimal combination of segments of stationary phases. In each approach the selection of the stationary phase was followed by a gradient optimisation.For the PDE-5 inhibitors, the group of structural analogues, only the method obtained with the third approach was able to differentiate between all the molecules in the development set. Although not all molecules are baseline separated, the method allows the identification of the selected adulterants in dietary supplements using only diode array detection. Though, due to the mobile phases used, the method could also be coupled to mass spectrometry.The method was validated for its selectivity following the guidelines as described for the screening of pesticide residues and residues of veterinary medicines in food.

Comparison of three development approaches for Stationary Phase Optimised Selectivity Liquid Chromatography based screening methods Part I: A heterogeneous group of molecules (slimming agents in food supplements)

Three approaches for the development of a screening method to detect adulterated dietary supplement, based on Stationary Phase Optimised Selectivity Liquid Chromatography were compared for their easiness/speed of development and the performance of the optimal method obtained. This comparison was performed for a heterogeneous group of molecules, i.e. slimming agents (Part I) and a group of structural analogues, i.e. PDE-5 inhibitors (Part II).The first approach makes use of primary runs at one isocratic level, the second of primary runs in gradient mode and the third of primary runs at three isocratic levels to calculate the optimal combination of segments of stationary phases. In each approach the selection of the stationary phase was followed by a gradient optimisation.For the slimming agents, the heterogeneous group of molecules, the method obtained with the first approach was selected as optimal, based on the speed of development and the performance of the method. The method shows a good separation of the compounds, allowing the screening to be performed with diode array detection, and is fully compatible with mass spectrometry. The method was validated for its selectivity following the guidelines as described for the screening of pesticide residues and residues of veterinary medicines in food.

Application of Orbitrap-mass spectrometry to differentiate isomeric sildenafil- and thiosildenafil-like analogues used for the adulteration of dietary supplements

Two groups of isomeric phosphodiestrase-type 5 inhibitors (PDE-5), consisting of four sildenafil- and three thiosildenafil-like analogues, have been successfully differentiated using high-resolution MS/MS. The optimised MS/MS data obtained from each compound were used to build a database with the aid of mass processing software. Isomeric compounds with very close chromatographic separation like dimethylsildenafil and homosildenafil could be distinguished by their unique fingerprint fragment ions in the MS/MS database. All fragment ions were within the mass tolerance of 5 ppm. One case study using an adulterated dietary supplement is included to provide more insights into this application.

Identification of a new tadalafil analogue in an adulterated dietary supplement: Trans-Bisprehomotadalafil

A new tadalafil analogue was identified along with homotadalafil during routine screening of an adulterated dietary supplement using HPLC-DAD. The UV spectrum of this analogue was almost identical with that of tadalafil. This compound was isolated from the supplement by using semi-preparative HPLC and its structure was subsequently elucidated by performing Q-TOF/MS/MS and NMR spectroscopic experiments. The spectral data indicate that this tadalafil analogue is a dimeric compound that consists of an ethylamino group and two pretadalafil moieties. NOE experiments and comparison with 1H NMR spectra of tadalafil and trans-tadalafil suggested the trans-relationship between the substituents on piperidine rings in the pretadalafil moieties.

Detection of adulteration in Maca dietary supplements using NMR spectroscopy

Maca, Lepidium meyenii Walpers (Brassicaceae), is a perennial, herbaceous plant grown on the high Andean plateaus. Its small, turnip-like root has been traditionally used as food. In recent years, maca has been used as an aphrodisiac, energizer, fertility enhancer, and is a so-called natural Viagra or Peruvian ginseng. As maca products have gained increasing popularity throughout the world, both the demand and price have increased dramatically. A global shortage of maca plant material has resulted in the commercial sale of seriously adulterated dietary supplements. In the present study, a nuclear magnetic resonance (NMR) spectroscopic method was developed to assess the quality of maca products sold on the market. The method can be used as a rapid tool for detection of maca adulteration.