Analytical Challenges and Metrological Approaches to Ensuring Dietary Supplement Quality: International Perspectives.

The increased utilization of metrology resources and expanded application of its' approaches in the development of internationally agreed upon measurements can lay the basis for regulatory harmonization, support reproducible research, and advance scientific understanding, especially of dietary supplements and herbal medicines. Yet, metrology is often underappreciated and underutilized in dealing with the many challenges presented by these chemically complex preparations. This article discusses the utility of applying rigorous analytical techniques and adopting metrological principles more widely in studying dietary supplement products and ingredients, particularly medicinal plants and other botanicals. An assessment of current and emerging dietary supplement characterization methods is provided, including targeted and non-targeted techniques, as well as data analysis and evaluation approaches, with a focus on chemometrics, toxicity, dosage form performance, and data management. Quality assessment, statistical methods, and optimized methods for data management are also discussed. Case studies provide examples of applying metrological principles in thorough analytical characterization of supplement composition to clarify their health effects. A new frontier for metrology in dietary supplement science is described, including opportunities to improve methods for analysis and data management, development of relevant standards and good practices, and communication of these developments to researchers and analysts, as well as to regulatory and policy decision makers in the public and private sectors. The promotion of closer interactions between analytical, clinical, and pharmaceutical scientists who are involved in research and product development with metrologists who develop standards and methodological guidelines is critical to advance research on dietary supplement characterization and health effects.

Comparison of phytochemical composition of Ginkgo biloba extracts using a combination of non-targeted and targeted analytical approaches.

Ginkgo biloba extract (GbE) is a dietary supplement derived from an ethanolic extract of Ginkgo biloba leaves. Unfinished bulk GbE is used to make finished products that are sold as dietary supplements. The variable, complex composition of GbE makes it difficult to obtain consistent toxicological assessments of potential risk. The National Toxicology Program (NTP) observed hepatotoxicity in its rodent studies of a commercially available, unfinished GbE product, but the application of these results to the broader GbE supplement market is unclear. Here, we use a combination of non-targeted and targeted chromatographic and spectrophotometric methods to obtain profiles of 24 commercially available finished GbE products and unfinished standardized and unstandardized extracts with and without hydrolysis, then used principal component analysis to group unfinished products according to their similarity to each other and to National Institute of Standards and Technology (NIST) standard reference materials (SRM), and the finished products. Unfinished products were grouped into those that were characteristic and uncharacteristic of standardized GbE. Our work demonstrates that different analytical approaches produced similar classifications of characteristic and uncharacteristic products in unhydrolyzed samples, but the distinctions largely disappeared once the samples were hydrolyzed. Using our approach, the NTP GbE was most similar to two unfinished GbE products classified as characteristic, finished products, and the NIST GbE SRM. We propose that a simple analysis for the presence, absence, or amounts of compounds unique to GbE in unhydrolyzed samples could be sufficient to determine a sample's authenticity.Graphical abstract.

Identification of adulteration in botanical samples with untargeted metabolomics.

Adulteration remains an issue in the dietary supplement industry, including botanical supplements. While it is common to employ a targeted analysis to detect known adulterants, this is difficult when little is known about the sample set. With this study, untargeted metabolomics using liquid chromatography coupled to ultraviolet-visible spectroscopy (LC-UV) or high-resolution mass spectrometry (LC-MS) was employed to detect adulteration in botanical dietary supplements. A training set was prepared by combining Hydrastis canadensis L. with a known adulterant, Coptis chinensis Franch., in ratios ranging from 5 to 95% adulteration. The metabolomics datasets were analyzed using both unsupervised (principal component analysis and composite score) and supervised (SIMCA) techniques. Palmatine, a known H. canadensis metabolite, was quantified as a targeted analysis comparison. While the targeted analysis was the most sensitive method tested in detecting adulteration, statistical analyses of the untargeted metabolomics datasets detected adulteration of the goldenseal samples, with SIMCA providing the greatest discriminating potential. Graphical abstract.

Authentication of black cohosh (Actaea racemosa) dietary supplements based on chemometric evaluation of hydroxycinnamic acid esters and hydroxycinnamic acid amides.

Ester and amide derivatives of hydroxycinnamic acids are found in black cohosh (Actaea racemosa) and other Actaea plants. These two compound groups were evaluated for authentication of black cohosh dietary supplements. The hydroxycinnamic acid esters (HCAE) were profiled by ultra-performance liquid chromatography-photodiode array detection (UPLC-PDA). The hydroxycinnamic acid amides (HCAA) were acquired simultaneously by mass spectrometry-multiple reaction monitoring (UPLC-MRM) mode. In contrast with the traditional HCAE method using 8 compounds, profiles of HCAA using only 4 feruloyl dopamine-O-hexosides was more convenient for peak by peak comparison. Partial least square discriminant analysis (PLS-DA) was applied to both HCAE and HCAA datasets. Authenticated plant samples of five Actaea species were randomly divided into training and test sets to build and validate the two PLS-DA models. Both models provided reasonable estimates for the classification of A. racemosa and other Actaea plant samples. However, HCAA model performs better in sensitivity, specificity, and accuracy. Assessment of supplement samples provided quite different results for the solid and liquid dietary supplement samples, indicating the dosage form could affect the composition of marker compounds. Graphical abstract.

How similar is similar enough? A sufficient similarity case study with Ginkgo biloba extract.

Botanical dietary supplements are complex mixtures that can be highly variable in composition and quality, making safety evaluation difficult. A key challenge is determining how diverse products in the marketplace relate to chemically and toxicologically characterized reference samples (i.e., how similar must a product be in order to be well-represented by the tested reference sample?). Ginkgo biloba extract (GBE) was used as a case study to develop and evaluate approaches for determining sufficient similarity. Multiple GBE extracts were evaluated for chemical and biological-response similarity. Chemical similarity was assessed using untargeted and targeted chemistry approaches. Biological similarity was evaluated using in vitro liver models and short-term rodent studies. Statistical and data visualization methods were then used to make decisions about the similarity of products to the reference sample. A majority of the 26 GBE samples tested (62%) were consistently determined to be sufficiently similar to the reference sample, while 27% were different from the reference GBE, and 12% were either similar or different depending on the method used. This case study demonstrated that approaches to evaluate sufficient similarity allow for critical evaluation of complex mixtures so that safety data from the tested reference can be applied to untested materials.

The Dietary Supplement Label Database: Recent Developments and Applications.

Objective: To describe the history, key features, recent enhancements, and common applications of the Dietary Supplement Label Database (DSLD). Background and History: Although many Americans use dietary supplements, databases of dietary supplements sold in the United States have not been widely available. The DSLD, an easily accessible public-use database was created in 2008 to provide information on dietary supplement composition for use by researchers and consumers. Rationale: Accessing current information easily and quickly is crucial for documenting exposures to dietary supplements because they contain nutrients and other bioactive ingredients that may have beneficial or adverse effects on human health. This manuscript details recent developments with the DSLD to achieve this goal and provides examples of how the DSLD has been used. Recent Developments: With periodic updates to track changes in product composition and capture new products entering the market, the DSLD currently contains more than 71,000 dietary supplement labels. Following usability testing with consumer and researcher user groups completed in 2016, improvements to the DSLD interface were made. As of 2017, both a desktop and mobile device version are now available. Since its inception in 2008, the use of the DSLD has included research, exposure monitoring, and other purposes by users in the public and private sectors. Future Directions: Further refinement of the user interface and search features to facilitate ease of use for stakeholders is planned. Conclusions: The DSLD can be used to track changes in product composition and capture new products entering the market. With over 71,000 DS labels it is a unique resource that policymakers, researchers, clinicians, and consumers may find valuable for multiple applications.

Feruloyl dopamine-O-hexosides are efficient marker compounds as orthogonal validation for authentication of black cohosh (Actaea racemosa)-an UHPLC-HRAM-MS chemometrics study.

Due to the complexity and variation of the chemical constituents in authentic black cohosh (Actaea racemosa) and its potential adulterant species, an accurate and feasible method for black cohosh authentication is not easy. A high-resolution accurate mass (HRAM) LC-MS fingerprinting method combined with chemometric approach was employed to discover new marker compounds. Seven hydroxycinnamic acid amide (HCAA) glycosides are proposed as potential marker compounds for differentiation of black cohosh from related species, including two Asian species (A. foetida, A. dahurica) and two American species (A. pachypoda, A. podocarpa). These markers were putatively identified by comparing their mass spectral fragmentation behavior with those of their authentic aglycone compounds and phytochemistry reports. Two isomers of feruloyl methyldopamine 4-O-hexoside ([M + H]+ 506) and one feruloyl tyramine 4-O-hexoside ([M + H]+ 476) contributed significantly to the separation of Asian species in principle component analysis (PCA) score plot. The efficacy of the models built on four reasonable combinations of these markers in differentiating black cohosh and its adulterants were evaluated and validated by partial least-square discriminant analysis (PLS-DA). Two models based on these reduced dataset achieved 100% accuracy based on the current sample collection, including the model that used only three feruloyl dopamine-O-hexoside isomers ([M + H]+ 492) and one feruloyl dopamine-O-dihexoside ([M + H-hexosyl]+ at m/z 492). Graphical abstract Hydroxycinnamic acid amide glycosides are proposed as potential marker compounds for authentication of black cohosh.

Interlaboratory Trial for Measurement of Vitamin D and 25-Hydroxyvitamin D [25(OH)D] in Foods and a Dietary Supplement Using Liquid Chromatography-Mass Spectrometry.

Assessment of total vitamin D intake from foods and dietary supplements (DSs) may be incomplete if 25-hydroxyvitamin D [25(OH)D] intake is not included. However, 25(OH)D data for such intake assessments are lacking, no food or DS reference materials (RMs) are available, and comparison of laboratory performance has been needed. The primary goal of this study was to evaluate whether vitamin D3 and 25(OH)D3 concentrations in food and DS materials could be measured with acceptable reproducibility. Five experienced laboratories from the United States and other countries participated, all using liquid chromatography tandem-mass spectrometry but no common analytical protocol; however, various methods were used for determining vitamin D3 in the DS. Five animal-based materials (including three commercially available RMs) and one DS were analyzed. Reproducibility results for the materials were acceptable. Thus, it is possible to obtain consistent results among experienced laboratories for vitamin D3 and 25(OH)D3 in foods and a DS.

Computer-aided method for identification of major flavone/flavonol glycosides by high-performance liquid chromatography-diode array detection-tandem mass spectrometry (HPLC-DAD-MS/MS).

A new computational tool is proposed here for tentatively identifying major (UV quantifiable) flavone/flavonol glycoside peaks of high performance liquid chromatogram (HPLC)-diode array detection (DAD)-tandem mass spectrometry (MS/MS) profiles based on a MATLAB-based script implementing an in-house algorithm. The HPLC-DAD-MS/MS profiles of red onion, Chinese lettuce, carrot leaf, and celery seed extracts were analyzed by the proposed computer-aided screening method for identifying possible flavone/flavonol glycoside peaks from the HPLC-UV and MS total ion current (TIC) chromatograms. The number of identified flavone/flavonol glycoside peaks of the HPLC-UV chromatograms is four, four, six, and nine for red onion, Chinese lettuce, carrot leaf, and celery seed, respectively. These results have been validated by human(s) experts. For the batch processing of nine HPLC-DAD-MS/MS profiles of celery seed extract, the entire script execution time was within 15 s while manual calculation of only one HPLC-DAD-MS/MS profile by a flavonoid expert could take hours. Therefore, this MATLAB-based screening method is able to facilitate the HPLC-DAD-MS/MS analysis of flavone/flavonol glycosides in plants to a large extent.

UHPLC-PDA-ESI/HRMSn profiling method to identify and quantify oligomeric proanthocyanidins in plant products.

Oligomeric proanthocyanidins were successfully identified by UHPLC-PDA-HRMS(n) in a selection of plant-derived materials (jujube fruit, Fuji apple, fruit pericarps of litchi and mangosteen, dark chocolate, and grape seed and cranberry extracts). The identities of 247 proanthocyanidins were theoretically predicted by computing high-accuracy masses based on the degree of polymerization, flavan-3-ol components, and the number of A type linkages and galloyls. MS(n) fragments allowed characterization on flavan-3-ol based on the monomer, connectivity, and location of A-type bonds. Identification of doubly or triply charged ions of 50 PAs was made on the basis of theoretical calculations. A single catechin standard and molar relative response factors (MRRFs) were used to quantify the well-separated PAs. The ratios of the SIM peak counts were used to quantify each of the unseparated isomers. This is the first report of direct determination of each of the proanthocyanidins in plant-derived foods and proanthocyanidins containing an epifisetinidol unit in grape seeds.

Profiling of glucosinolates and flavonoids in Rorippa indica (Linn.) Hiern. (Cruciferae) by UHPLC-PDA-ESI/HRMS(n).

An UHPLC-PDA-ESI/HRMS(n) profiling method was used to identify the glucosinolates and flavonoids of Rorippa indica (Cruciferae), a wild vegetable and Chinese herb used to treat cough, diarrhea, and rheumatoid arthritis. Thirty-three glucosinolates, more than 40 flavonol glycosides, and 18 other phenolic and common organic compounds were identified. The glucosinolates and polyphenols were separated by UHPLC. High-resolution deprotonated molecules provided high accuracy mass values that were used to determine formulas and provide putative identification of the glucosinolates and flavonoids. The fragments from multistage mass spectrometry were used to elucidate the structures. The concentrations of the main components were based on UV peak areas and molar relative response factors with a single calibration standard. This study found this plant to be a rich source for glucosinolates, containing 24 new glucosinolates, including 14 glucosylated glucosinolates that were previously unidentified.

Profiling polyphenols in five Brassica species microgreens by UHPLC-PDA-ESI/HRMS(n.).

Brassica vegetables are known to contain relatively high concentrations of bioactive compounds associated with human health. A comprehensive profiling of polyphenols from five Brassica species microgreens was conducted using ultrahigh-performance liquid chromatography photodiode array high-resolution multistage mass spectrometry (UHPLC-PDA-ESI/HRMS(n)). A total of 164 polyphenols including 30 anthocyanins, 105 flavonol glycosides, and 29 hydroxycinnamic acid and hydroxybenzoic acid derivatives were putatively identified.The putative identifications were based on UHPLC-HRMS(n) analysis using retention times, elution orders, UV-vis and high-resolution mass spectra, and an in-house polyphenol database as well as literature comparisons. This study showed that these five Brassica species microgreens could be considered as good sources of food polyphenols.

LC-PDA-ESI/MS identification of the phenolic components of three compositae spices: chamomile, tarragon, and Mexican arnica.

Chamomile (Matricaria chamomilla L.), tarragon (Artemisia dracunculus L.) and Mexican arnica (Heterotheca inuoides) are common compositae spices and herbs found in the US market. They contain flavonoids and hydroxycinnamates that are potentially beneficial to human health. A standardized LC-PDA-ESI/MS profiling method was used to identify 51 flavonoids and 17 hydroxycinnamates. Many of the identifications were confirmed with authentic standards or through references in the literature or the laboratory's database. More than half of the phenol compounds for each spice had not been previously reported. The phenolic profile can be used for plant authentication and to correlate with biological activities.

Quantitation of flavanols, proanthocyanidins, isoflavones, flavanones, dihydrochalcones, stilbenes, benzoic acid derivatives using ultraviolet absorbance after identification by liquid chromatography-mass spectrometry.

A general method was developed for the systematic quantitation of flavanols, proanthocyanidins, isoflavones, flavanones, dihydrochalcones, stilbenes, and hydroxybenzoic acid derivatives (mainly hydrolyzable tannins) based on UV band II absorbance arising from the benzoyl structure. The compound structures and the wavelength maximum were well correlated and were divided into four groups: the flavanols and proanthocyanidins at 278 nm, hydrolyzable tannins at 274 nm, flavanones at 288 nm, and isoflavones at 260 nm. Within each group, molar relative response factors (MRRFs) were computed for each compound based on the absorbance ratio of the compound and the group reference standard. Response factors were computed for the compounds as purchased (MRRF), after drying (MRRFD), and as the best predicted value (MRRFP). Concentrations for each compound were computed based on calibration with the group reference standard and the MRRFP. The quantitation of catechins, proanthocyanidins, and gallic acid derivatives in white tea was used as an example.

Probability of identification: a statistical model for the validation of qualitative botanical identification methods.

A qualitative botanical identification method (BIM) is an analytical procedure that returns a binary result (1 = Identified, 0 = Not Identified). A BIM may be used by a buyer, manufacturer, or regulator to determine whether a botanical material being tested is the same as the target (desired) material, or whether it contains excessive nontarget (undesirable) material. The report describes the development and validation of studies for a BIM based on the proportion of replicates identified, or probability of identification (POI), as the basic observed statistic. The statistical procedures proposed for data analysis follow closely those of the probability of detection, and harmonize the statistical concepts and parameters between quantitative and qualitative method validation. Use of POI statistics also harmonizes statistical concepts for botanical, microbiological, toxin, and other analyte identification methods that produce binary results. The POI statistical model provides a tool for graphical representation of response curves for qualitative methods, reporting of descriptive statistics, and application of performance requirements. Single collaborator and multicollaborative study examples are given.

Classification of cultivation locations of Panax quinquefolius L samples using high performance liquid chromatography-electrospray ionization mass spectrometry and chemometric analysis.

Panax quinquefolius L ( P. quinquefolius L) samples grown in the United States and China were analyzed with high performance liquid chromatography-mass spectrometry (HPLC-MS). Prior to classification, the two-way data sets were subjected to pretreatment including baseline correction and retention time (RT) alignment. Principal component analysis (PCA) and projected difference resolution (PDR) metrics were used to evaluate the data quality and the pretreatment effects. A fuzzy rule-building expert system (FuRES) classifier was used to classify the P. quinquefolius L samples grown in the United States and China with the optimized partial least-squares (o-PLS) classifier as the positively biased control method. A classification rate as high as 98 ± 3% with FuRES was obtained after baseline correction and RT alignment, which is equivalent to the result obtained by using the positively biased o-PLS control method (98 ± 3%). RT alignment improved the classification rates for both FuRES and o-PLS classifiers (18% improvement for the FuRES classification rate and 10% improvement for the o-PLS classification rate with baseline correction). From the rule obtained to classify the P. quinquefolius L samples grown in the United States and China, peaks were identified that can be prospective biomarkers for differentiating samples from different growth regions. HPLC-MS with chemometric analysis has the potential to be used as an authentication method for P. quinquefolius L grown in China and the United States.

Detection of adulterated Ginkgo biloba supplements using chromatographic and spectral fingerprints.

The fingerprints of 18 commercially available Ginkgo biloba supplements, 12 samples of raw G. biloba leaves, and three G. biloba standard reference materials from the National Institute of Standards and Technology were acquired directly (no chromatography) by UV spectrometry and after separation using HPLC with a diode array detector. The fingerprints consisted of the UV spectral images, the chromatographic images, and the areas of the 21 most prominent chromatographic peaks. Data were analyzed by principal component analysis and one-class soft independent modeling of class analogy (SIMCA). It was determined that three of the commercial products were adulterated with rutin, four with quercetin, and one with an unidentified flavonol glycoside. One-class SIMCA of the authentic products allowed the adulterated products to be easily distinguished using Q-residuals. Authentic supplements and raw leaf materials were easily distinguished. The finely powdered samples were also analyzed by near-IR (NIR) spectrometry. The authentic and adulterated products could not be distinguished by NIR spectrometry because of the excipients.

Discrimination among Panax species using spectral fingerprinting.

Spectral fingerprints of samples of three Panax species (P. quinquefolius L., P. ginseng, and P. notoginseng) were acquired using UV, near-infrared (NIR), and MS. With principal component analysis, all three methods allowed visual discrimination among the three species. All three methods were able to discriminate between white and red ginseng, and showed distinctive subgroupings of red ginseng related to root quality (age/size). Analysis of variance was used to evaluate the relative variance arising from the species, run, and analytical uncertainty, and was used to identify the most information-rich portions of the spectrum for NIR and UV. Accurate classification of the three species was obtained by using partial least squares-discriminant analysis and a fuzzy rule-building expert system. Relatively poor accuracy was obtained using soft independent modeling of class analogy when a single component was used.

A non-targeted approach to chemical discrimination between green tea dietary supplements and green tea leaves by HPLC/MS.

Green tea-based dietary supplements (GTDSs) have gained popularity in the U.S. market in recent years. This study evaluated the phytochemical composition difference of GTDS in comparison with green tea leaves using an HPLC/MS fingerprinting technique coupled with chemometric analysis. Five components that are most responsible for class separation among samples were identified as (-) epicatechin gallate, strictinin, trigalloylglucose, quercetin-3-O-glucosyl-rhamnosylglucoside, and kaempferol-3-O-galactosyl-rhamnosylglucoside, according to the accurate mass measurements and MS/MS data. The similarity coefficients between the GTDSs in solid form with green tea were 0.55 to 0.91, while for the GTDSs in liquid form they were 0.12 to 0.89, which suggested that chemical composition variance across the GTDSs was significant. Flavonol aglycone concentrations were higher in GTDSs than in tea leaves, indicating the degradation of flavonol glycosides or the oxidation of catechin during the manufacturing and storage processes. In some GTDS samples, compounds were identified that were on the label. The results demonstrate the urgency of QC for GTDS products.

A comparison of analytical and data preprocessing methods for spectral fingerprinting.

Spectral fingerprinting, as a method of discriminating between plant cultivars and growing treatments for a common set of broccoli samples, was compared for six analytical instruments. Spectra were acquired for finely powdered solid samples using Fourier transform infrared (FT-IR) and Fourier transform near-infrared (NIR) spectrometry. Spectra were also acquired for unfractionated aqueous methanol extracts of the powders using molecular absorption in the ultraviolet (UV) and visible (VIS) regions and mass spectrometry with negative (MS-) and positive (MS+) ionization. The spectra were analyzed using nested one-way analysis of variance (ANOVA) and principal component analysis (PCA) to statistically evaluate the quality of discrimination. All six methods showed statistically significant differences between the cultivars and treatments. The significance of the statistical tests was improved by the judicious selection of spectral regions (IR and NIR), masses (MS+ and MS-), and derivatives (IR, NIR, UV, and VIS).