The Importance of Method Selection in Determining Product Integrity for Nutrition Research 1 4

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1 REVIEW FROM ASN EB 2015 SYMPOSIUM The Importance of Method Selection in Determining Product Integrity for Nutrition Research 1 4 Elizabeth M Mudge, 5 Joseph M Betz, 6 Paula N Brown 5 * 5 Centre for Applied Research and Innovation, British Columbia Institute of Technology, Burnaby, Canada; and 6 Office of Dietary Supplements, NIH, Bethesda, MD ABSTRACT The American Herbal Products Association estimates that there as many as 3000 plant species in commerce. The FDA estimates that there are about 85,000 dietary supplement products in the marketplace. The pace of product innovation far exceeds that of analytical methods development and validation, with new ingredients, matrixes, and combinations resulting in an analytical community that has been unable to keep up. This has led to a lack of validated analytical methods for dietary supplements and to inappropriate method selection where methods do exist. Only after rigorous validation procedures to ensure that methods are fit for purpose should they be used in a routine setting to verify product authenticity and quality. By following systematic procedures and establishing performance requirements for analytical methods before method development and validation, methods can be developed that are both valid and fit for purpose. This review summarizes advances in method selection, development, and validation regarding herbal supplement analysis and provides several documented examples of inappropriate method selection and application. Adv Nutr 2016;7: Keywords: dietary supplements, herbal medicine, botanical identification, method selection, phytochemicals Introduction There is continuing demand for high-quality herbal products, but the rate of new product introduction far outpaces efforts to produce reliable analytical methods that verify botanical identity, quality, and strength. Further hindering research efforts, the physical forms in which herbal supplements are marketed have changed from tinctures, tablets, and capsules to gummies, softgels, and liquid emulsions. Most jurisdictions have rigorous requirements for product authenticity and quality that require methods for all incoming raw materials and finished products (1, 2). The scientific community has been unable to keep up with product innovation, leaving manufacturers to their own devices, which has resulted in improper method selection and use, dry-labbing, and concerns regarding product quality, safety, and authenticity (3). The function and use of many herbal supplements has been inferred through historical use, anecdotal information, 1 This article is a review from the symposium "What s New in Natural Products Analysis? Cutting-Edge Methods and Available Resources for Nutrition Research held 1 April 2015 at the ASN Scientific Sessions and Annual Meeting at Experimental Biology 2015 in Boston, MA. The symposium was sponsored by the ASN Nutritional Sciences Council and the Dietary Bioactive Components Research Interest Section. 2 The organizer has indicated that related reviews of this symposium will be submitted for publication in an upcoming issue of Advances of Nutrition. 3 Supported by Canada Research Chairs. 4 Author disclosures: EM Mudge, JM Betz, and PN Brown, no conflicts of interest. *To whom correspondence should be addressed. paula_brown@bcit.ca. and clinical studies. Each year, new products are introduced into the marketplace with minimal research on quality and safety. Herbal supplement quality is defined by ingredient authenticity, absence of impurities, content of desirable marker, and active compounds or profiles of these constituents. The selection of marker compounds for evaluation is typically based on historical information, uniqueness of nonubiquitous constituents, and identification of active compounds with use of bioassays related to the hypothesized clinical effect (4 6). The identity of compounds responsible for the therapeutic activity of many plants is unknown, but the community (including the regulatory and standard-setting community as reflected by the European Pharmacopoeia, USP, Hong Kong Chinese Materia Medica Standards, Pharmacopoeia of the People s Republic of China, and others) recognizes analysis of marker compounds as a surrogate means of assessing raw material and finished product integrity. In particular, measurement and adjustment of marker compounds through a process called standardization can ensure batch-to-batch consistency of formulations even if relevance to therapeutic activity has not yet been demonstrated. Examples of classes of phytochemicals measured as part of product integrity evaluation include anthocyanins in bilberry, caffeic acid derivatives in Echinacea, and isoquinoline alkaloids in goldenseal (7 9). 390 ã2016 American Society for Nutrition. Adv Nutr 2016;7:390 8; doi: /an

2 Product quality is compromised when undeclared active pharmaceuticals, dyes, or other substances are added to extracts with the intent of confounding nonspecific assays; inferior plants are substituted when desirable plants are scarce or expensive or when nontarget plants are unintentionally introduced into products (10, 11). The first 2 cases are examples of economically motivated adulteration and frequently occur where there is pressure to produce low-cost products (12). Plant identity and authenticity are classically determined by examining diagnostic anatomical features, which remain useful when raw materials are traded as minimally processed biomass (13). Modern-ingredient supply chains seldom deal in this type of material (14) because manufacturers may purchase highly pulverized plant powders or extracts from processors, distributers, or suppliers. Quality defined as authenticity depends on the competence and honesty of every player in a supply chain, which can be lengthy and in some cases untraceable. In addition to supply chain audits, manufacturers can confirm product authenticity through validated analytical methods for product identity and composition. This review summarizes advances in method development and validation for botanicals pertaining to quantitation of marker compounds, contaminants, and botanical identification. The instrumental and chemical approach to botanical identification is an emerging field, and an array of techniques is being explored. Further development, validation, and adoption of these techniques by the botanical industry and researchers will ensure product integrity. Current Status of Knowledge Optimization and validation of methods for botanical products Most published methods for botanical analysis focus on the quantitation of target compounds in raw ingredients. These methods are often tedious and not suitable for routine ingredient analysis or finished product testing because of lowthroughput and matrix effects (excipients, confounding botanical ingredients). In addition, many commercial raw materials are dried extracts rather than biomass and bear little chemical resemblance to the plants from which they are derived (13). Because of product complexity, methods must undergo intensive optimization for extracting the active or marker compounds before method validation and use. For example, supplements made from plant seeds can contain high amounts of fat and thus interfere with the extraction of active ingredients in raw materials (15, 16). Microencapsulated ingredients may require additional sample preparation steps to break the shell material (17). Finished products are complex, and characteristics such as analyte solubility, excipient type, and coloring agents and combinations with several plant species, vitamins, and minerals can complicate sample preparation and analysis. Phytochemicals that are not quality control targets may interfere with the extraction or chromatographic separation of the marker compounds, requiring clean-up procedures or improved chromatography to meet the method performance requirements (18, 19). For the purposes of complying with current good manufacturing practices, manufacturers must develop product specifications that define their products, and this definition must include consideration of claims and testing methods with tolerances against which conformity to specifications are evaluated. The regulations regarding herbal products, claims, and quality vary depending on the country, but the same set of principles can be used as guidelines for setting product specifications. Claims may be related to a specific function and composition of an ingredient; for example, the dried aerial parts of feverfew [Tanacetum parthenium (L.) Sch. Bip.] are consumed for migraine prevention, and this appears to be related to a sesquiterpene lactone content of % parthenolide (20). A test method developed for feverfew would quantify these compounds at the expected concentrations in the types of matrixes used by the manufacturer. In addition to quantifying desirable constituents that are markers of identity and have been selected by standard setting bodies (e.g., USP) as indicators of high-quality materials, additional test methods capable of detecting adulteration should be included. Virtually all modern pharmacopoeias set limits for toxic elements, pesticides, mycotoxins, pathogenic and nonpathogenic microorganisms, nontarget plant material, and other extraneous contaminants. In some cases, a single method may be useful for quantifying desirable compounds and detecting adulterants (10, 21). More often, 2 or more methods are required. For routine testing, it is desirable to develop concise, simple methods with optimized extraction time and as few sample transfers as possible. This minimizes analyte losses and improves method precision. In addition to faster, simpler sample preparation, technological developments in rapid chromatographic separation and improved resolution should be investigated. The development of smaller particle-size columns, higher-pressure LC systems, and smaller-bore, shorter GC columns increase laboratory productivity and accelerate product release from current good manufacturing practicesmandated finished-product quarantine. There are several ways to optimize analytical methods. Planning before optimization ensures a higher probability of achieving methods that are precise and accurate. Method development with use of factorial designs can assist in identifying those factors that affect method performance, thereby directing the optimization process and reducing development time. Several optimization parameters should be considered to ensure optimal extract efficiency, including choice of extraction solvent, length of extraction time, number of extraction cycles, extraction temperature, and technique. Factorial designs have been used to optimize essential oil extraction parameters with microwave-assisted extraction and extraction of flavonoids from Bauhinia forticata (22, 23). Optimization must also be performed for chromatographic, spectroscopic, or enzymatic test methods. Raw The importance of method selection 391

3 botanicals can contain many classes of compounds such as flavonoids and anthocyanins in berries. Optimizing the chromatographic separation with use of a natural matrix rather than pure calibration standards will ensure peak resolution and permit the establishment of realistic system suitability requirements. A fully optimized method that includes extraction, separation, and detection must then be subjected to a validation to ensure the method is accurate and precise for proposed matrixes. Analytical method validation is the process by which a method s performance is assessed for its fitness for purpose, precision, and accuracy. Subjecting a method to a validation study ensures that it is suitable for its intended use with respect to detecting, identifying, and/or quantifying the components of interest. For botanical products, it is essential that validation take into account the entire analytical procedure, including sample preparation, because this may have the most important impact on the final result (5). Validated methods are essential for regulators, the industry, and basic and clinical researchers. By including accurate chemical characterization data in clinical trial reports, there is the potential for correlating material content with effectiveness, leading to more conclusive findings. In addition, regulators and the industry can ensure products meet specifications, leading to increased consumer trust. The degree of validation required for a method depends on the intended use of the final method. Methods to be used in a single laboratory require single-laboratory validation (SLV). 7 Multilaboratory validation (MLV) is required when the expectation is that multiple locations within a company will use the method or if data produced by analyses will be compared across laboratories. Collaborative studies and other MLVs ensure consistency of results because the data acquired through these studies establish method reproducibility. There are several organizations with published validation guidelines, including the AOAC, International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, FDA, International Union of Pure and Applied Chemistry, and USP (24 28). Full collaborative studies require a minimum number of laboratories to assess method accuracy and precision. Several alternatives to MLV have been proposed to reduce the burden of collaborative studies with the goal of reducing overall time and costs (29). SLV guidelines evaluate the performance characteristics of a method. Most validation guidelines require a demonstration of method applicability, selectivity, linearity, repeatability (precision), accuracy, limit of detection, limit of quantitation, and stability. These parameters can vary depending on analyte concentrations. To demonstrate acceptable precision, the Horwitz ratio values described in the 7 Abbreviations used: CPC, cetylpyridinium chloride; CS, chondroitin sulfate; FIMS, flow-injection MS; MLV, multilaboratory validation; NIR, near-infrared; OMA, official method of analysis; PCA, principal component analysis; SIMCA, soft independent modeling of class analogy; SLV, single-laboratory validation; SMPR, standard method performance method requirement; SPDS, stakeholder panel for dietary supplements. AOAC guidelines evaluate method performance based on the ratios of actual precision to predicted precision. Acceptable Horwitz ratio ranges are for SLV and collaborative studies (24, 29). Details on validation parameters and procedures can be found in reviews and guidelines (5, 24 30). Several method optimization and validation studies have been successful for analyzing components. Those mentioned in this review have been highlighted in Table 1, whereas additional examples are listed on the NIH Office of Dietary Supplements Analytical Methods and Reference Materials website ( (38). AOAC International has published several official methods of analysis (OMAs) for dietary supplements. OMAs are analytical methods that have been subjected to expert scrutiny and the rigors of validation studies. These are also listed on the Analytical Methods and Reference Materials website (38). OMAs can be used in dispute resolution and quality control and are required for regulatory enforcement in many jurisdictions. In 2014, the AOAC initiated a stakeholder panel for dietary supplements (SPDS). The SPDS is composed of representatives from the industry, government agencies, and the academic research community who have an interest in dietary supplements. The goal of the SPDS is to determine areas where dietary supplement methods are needed, prioritize those needs, and provide guidance on method performance requirements (39). Within the SPDS, expert working groups recommend desirable performance characteristics by creating standard method performance requirements (SMPRs) as criteria by which methods will be evaluated. The AOAC then puts out a call for methods, and submitted methods are evaluated for conformity to the SMPRs by an expert review panel. Methods that meet SMPRs and have sufficient data demonstrating accuracy and precision may be adopted as firstaction OMAs. To reach final-action OMA status, expert review panels examine additional performance data accumulated over a 2-y period either through formal validation studies or use of certified reference materials to evaluate reproducibility of the methods. Depending on their performance, methods can be recommended as final-action OMAs or retired. The stage between first- and final-action OMAs requires considerable commitment from stakeholders because of the need to invest resources for the required studies. Curcuminoid analysis in turmeric (Curcuma longa L.) is traditionally performed with use of spectrophotometric absorbance, but because this is a nonspecific method there is a higher probability of product adulteration. Therefore, methods using chromatographic separation and quantitation of the 3 major curcuminoids curcumin, bisdemethoxycurcumin, and demethoxycurcumin are being implemented. An analytical method was recently developed for many different matrix types with use of the method optimization and validation protocols previously described (37). Sample preparation and chromatographic separation were optimized with use of 2-level factorial designs. The most important factor in the chromatographic separation was the 392 Symposium

4 TABLE 1 Several analytical methods for dietary supplements that successfully underwent rigorous optimization and validation protocols for a variety of product matrixes and raw materials 1 Material Constituents Method Matrixes Validation Refs. β-carotene β-carotene HPLC-VIS Multivitamin tablets, softgels, SLV, collaborative 31, 32 capsules, beadlets study, OMA Chondroitin sulfate Chondroitin sulfate Enzymatic Raw ingredients, capsules, chews, SLV 33 digestion: HPLC-UV tablets, softgels, liquids Goldenseal Isoquinoline alkaloids HPLC-UV Capsules, tinctures, caplets, and SLV, collaborative 8, 34 dried roots combined with Echinacea study, OMA Echinacea Phenolic constituents HPLC-UV Raw materials, softgels, tinctures, SLV 9, 35 capsules, single and multicomponent products Cranberry Anthocyanins HPLC-VIS Raw berries, juices, powdered SLV 36 extracts Turmeric Curcuminoids HPLC-UV Raw materials, capsules, tinctures, tablets, softgels SLV 37 1 OMA, official method of analysis; SLV, single-laboratory validation; VIS, visible. column temperature and sample preparation solvent composition. These were optimized to 55 8 C and methanol, respectively (37). The final method was subjected to a single-laboratory validation according to the AOAC and evaluated for linearity, repeatability, detection limits, and recovery. The findings confirmed that the method was suitable for quantitating curcuminoids in single- and multicomponent products in a variety of matrixes (37). Fitness for purpose A method of analysis can be shown to be accurate, precise, and reproducible through a validation study but may not be suitable for a particular matrix that was not evaluated in the validation even if the target analytes are the same. Methods must be shown to be suitable for their intended use (fit for purpose) in addition to being shown to be valid. The method user must properly understand the herb of interest, active/marker compounds and their expected concentrations, common excipients, additional botanical or nonbotanical ingredients, and potential contaminants to select a method that is valid and suitable for his or her own product. Analyzing a product with a method that has not been demonstrated to be valid and suitable for the material in hand can lead to incorrect or inconclusive results. As noted previously, methods must be developed based on manufacturer specifications, regulatory requirements, and the need to detect or quantify nutrients or contaminants or for identification purposes. As noted, method selection must take into account fitness for purpose to ensure the method chosen or developed meets the expectations of accuracy and precision in the matrix being evaluated. For example, a published study that reported the iodine content of several commercial multivitamin/mineral supplements did not provide details of the analytical method used (40). In reply to a letter to the editor (41) asking for methodological details, the authors noted that a method developed for determining iodine in human urine had been used for analyzing the products but failed to provide information on whether the urine method had been evaluated for its applicability to finished dietary supplement products (42). The need to understand the nature of the material to be analyzed and the importance of method selection can be illustrated by the example of cetylpyridinium chloride (CPC) titration for quantification of chondroitin sulfate (CS) in finished products. CPC titration is a popular method for determining the amount of CS in raw materials and finished products, but it cannot distinguish between CS and other polymeric glycosaminoglycans and will quantify any material having a high anionic charge, e.g., carrageenans, proteins, and surfactants (43). When assayed with CPC titration, finished products containing combinations of CS with glucosamine, methylsulfonylmethane, or emulsifiers such as carrageenan will produce erroneously high values for CS (33). This titration should not be used as a stand-alone method; a purity determination with use of electrophoresis must be performed before CPC titration (43). As such, this method is only suitable for quantitation under specificcircumstances (i.e., when the identity and lack of impurities in the tested material have been unequivocally established by other methods). Similarly, methods such as enzymatic hydrolysis of the glycosaminoglycan polymer followed by LC-UV separation and detection of the parent disaccharides can demonstrate that the tested material is CS, indicate the biological source of the material (bovine, porcine, ovine, marine), and quantify the amount of CS but cannot detect adulterants (33). Quality assurance of CS raw materials and finished products must be accomplished with use of a suite of analytical approaches and methods. Methods capable of establishing identity of the material include the aforementioned enzymatic digestion and HPLC disaccharide characterization, Fourier transform infrared, and proton NMR. Assay methods (designed to determine the amount of CS) include CPC titration, enzymatic treatment and HPLC disaccharide characterization, and proton NMR. Methods to detect impurities in CS include cellulose acetate membrane electrophoresis, agarose-gel electrophoresis, capillary electrophoresis, and size-exclusion chromatography. Methods for establishing finished-product composition include proton NMR and specific rotation (43). The importance of method selection 393

5 Chewable gummy supplements are increasing in popularity but provide a complex matrix not previously common to the industry. Gummies incorporate botanical extracts, vitamins, and minerals into a matrix that may contain gelatin, pectin, gum arabic, polysaccharides, and oligosaccharides alone or in combination with other ingredients. Fruit juices along with other natural and artificial flavors are often added to the finished product. The gummy matrix is complex, and extracting target compounds requires specialized techniques. One approach entails enzymatic digestion with protease followed by extraction with a mixture of ethanol and dichloromethane (31). Anthocyanins are often evaluated as the quality control target marker compounds in several gummy botanical products. Anthocyanins are also frequently present in the fruit juices used to flavor and color the gummies and can interfere with determining the target marker anthocyanins in the finished product. Finally, there are only a few validated methods for anthocyanin determination, and the validation studies of those methods lacked a gummy matrix and would not be fit for the purpose of analyzing anthocyanins in gummies (36). This is an example of the challenge faced by the highly innovative botanical supplement industry, where methods must be evaluated in new matrixes to ensure fitness for purpose. Prevention of adulteration Adulteration can be either intentional or accidental and can occur because of incorrect botanical identification or improper process control. Intentional adulteration can range in scope from the use of spent botanical materials in capsule manufacturing (leading to nonefficacious products lacking in phytochemical markers) to the addition or substitution of botanically or chemically similar (but cheaper) plant species (10). Several botanical products have been associated with negative health outcomes that have been attributed to partial or complete substitution of 1 plant for another. An adverse event was associated with substituting purple foxglove (Digitals lanata) for plantain (Plantago sp.) in an herbal cleansing product (44). Hepatotoxicty associated with ingesting the herb skullcap (Scutellaria lateriflora) was most likely the result of substitution with germander (Teucrium spp.) (45, 46). Other types of economically motivated adulteration also occur. Adulteration of bilberry extract (Vaccinium myrtilus) involves adding amaranth dye (FD&C Red No. 2, a synthetic napthylazo food dye banned by the FDA in 1976 because it is a suspected carcinogen) for the purpose of inflating the apparent anthocyanin content when measured with use of a USP assay (47). The method measures spectrophotometric absorbance at 528 nm and assumes that the products are extracts that contain only bilberry. Because amaranth dye absorbs strongly at 528 nm, it can report high amounts of anthocyanins in the extract. Several approaches have been proposed to eliminate this issue, including use of a method such as HPTLC to confirm the identity of extracts in parallel with the spectrophotometric method for the quantitation of anthocyanins (48) or HPLC quantification of the individual anthocyanins, where profiling also confirms the authenticity of the product (49 51). Chemical techniques used to establish ingredient identity and to detect adulteration include HPLC, HPLC-MS, infrared, NMR, HPTLC, and MS. Data collected may be evaluated by visual inspection or by chemometric-modeling techniques to compare the phytochemical profiles or entire metabolomes of the different species. AOAC International has published recommendations for the validation of identity methods (24, 52). The utility of this approach was illustrated by differentiating between ginseng species by applying ANOVA to spectral fingerprints [UV, near-infrared (NIR), and MS] acquired from 3 Panax species and soft independent modeling of class analogy (SIMCA) modeling of flow-injection MS (FIMS) data acquired from 2 Panax species, respectively (53, 54). These methods and the robustness of the models generated require a large number of authenticated test samples, including both biological and technical replicates, to ensure the methods are valid. Acquiring authentic specimens can be a daunting task, so validation of botanical identity protocols can be a lengthy and expensive proposition (13). Chemometric modeling of spectral data has been used to affirm botanical identity and differentiate a target botanical from common adulterants or closely related species. Statistical approaches to data analysis include principal component analysis (PCA), partial least squares discriminant analysis, SIMCA, and linear discriminant analysis. PCA and discriminant analysis are powerful tools that evaluate large data sets or specific portions of large data sets derived from chromatograms or spectra and differentiate the samples into classes or groups based on similarities and differences across the data. Several pretreatment techniques can be applied to the data sets, including alignment, scaling, binning, and normalization, to account for data variations (55). The success of these models depends largely on material authenticity, the number and diversity of authenticated materials, sampling techniques, and data acquisition parameters. HPLC, ultra-performance LC, and MS coupled with chemometric modeling or detection of specific marker compounds or contaminants are common chemical approaches to botanical identification. The overall quality of goldenseal (Hydrastis canadensis L.) has classically been established by measuring the isoquinoline alkaloids hydrastine and berberine in root and rhizome. However, common adulterants of goldenseal include Chinese goldthread (Coptis chinensis), Oregon grape root (Berberis aquifolium), barberry (Berberis vulgaris), yellow dock root (Rumex crispus), and synthetic or purified berberine (8). These plants contain berberine, whereas hydrastine and canadine are unique to goldenseal; therefore, HPLC profiling and quantitation of all 3 alkaloids is 1 way to ensure product authenticity (8). SIMCA analysis of HPLC-UV data distinguished Gingko biloba raw materials and supplements from adulterated products. The adulterants were identified as rutin, quercetin, and an unknown flavonol glycoside (56). PCA analysis of HPLC-UV profiles differentiated between P. notoginseng root extract, other 394 Symposium

6 ginseng species, and nonroot extracts (57). None of these was possible by visually inspecting the chromatographic profiles alone. FIMS coupled with discriminant analysis can differentiate the 3 ginseng species without a lengthy chromatographic separation step (58). HPTLC delivers excellent band resolution and can be used to obtain precise radiofrequency values and permit effective direct visual comparison of chromatograms. The technique requires sample extraction, planer separation, and visual inspection of the resolved bands. HPTLC can also be coupled to densitometric detectors to generate quantitative and qualitative data for use in chemometric modeling, although visual inspection is generally used for differentiating between species. HPTLC has been shown to be capable of differentiating between various Actaea, Echinacea, and Panax species (59 61). Information regarding the validation of HPTLC identity methods has been summarized (62). A considerable amount of experience and skill is required to properly interpret the data, but as new detection and recording techniques are developed, visual interpretation may be replaced by more objective instrumental and statistical interpretation techniques. NIR is a form of infrared spectroscopy that utilizes a different part of the electromagnetic spectrum and has been applied to botanical identification. For application as a tool to differentiate species, NIR data must be processed with use of discriminant analysis. E. purpurea root powder was differentiated from E. angustifolia, E. pallida, and Parthenium integrifolium root powders with the addition of 1 to the other detected at concentrations as low as 10% with use partial least squares and data pretreatment (63). One technical quirk of this approach noted by the authors was the need for each material to be milled identically because particle-size distribution can confound the results. Using the AOAC International validation guidelines for the probability of identifying botanicals (24), calibration and validation materials were acquired to detect the adulteration of E. purpurea root powder with E. angustifolia root powder (64). Inferior and superior test materials composed of 90% and 98% E. purpurea roots with E. angustifolia roots, respectively, were utilized. In this case, the method specifications considered material that contained 2% nontarget species as acceptable material that the method should positively identify as E. purpurea root powder (64). Based on the high variability of biomass found during method development, the authors recommended that authentic samples of each botanical be used to ensure a robust calibration set. Validation of the model found that 100% E. purpurea was differentiated from 100% E. angustifolia roots, but both the superior and inferior test materials were not distinguishable. The final method was therefore not deemed fit for purpose (64). Several factors affected NIR method development, including population variation, particle size, moisture content, and vial rotation; thus, the method is not robust enough to account for unidentified sources of variation. The USP purity standard for E. purpurea root is not >3.0% content of foreign matter (65); the method for that determination is physical separation of E. purpurea root from non E. purpurea root and gravimetric determination that non E. purpurea material does not exceed 3.0%. Because of the nature of the material there is no specification for foreign matter in the powdered E. purpurea root monograph. Although not as sensitive as visual inspection and gravimetric analysis, NIR may become a useful identification tool for powdered Echinacea. Other investigators have differentiated P. notoginseng, P. quinquefolius, and P. ginseng with use of NIR, whereas mid-infrared and Fourier transformation infrared could distinguish Pelargonium sidoides from a similar contaminant, P. reniforme (53, 66). NIR and mid-infrared spectroscopy can also detect adulterants such as sildenafil, sibutramine, and phenolphthalein in herbal products (67, 68). Infrared is a rapid and noninvasive technique that requires minimal sample preparation, and once methods have been designed and validated for specific plant materials and adulterants, it will allow rapid screening of raw materials. Unfortunately, a considerable amount of development is required to set up the methods, chemometric modeling, and data analysis. NIR has primarily been successful on raw materials because extracts and/or excipients present in finished products will change the spectral profiles, leading to complex and significantly different spectra from the raw materials, as shown with Ginkgo biloba (56). NMR spectroscopy has traditionally been used for structurally elucidating pure compounds, determining purity determination, and assigning stereochemistry to isolated or synthesized compounds. More recently it has become a favored data acquisition technique for metabolomic studies. It has several advantages over other techniques, including simple sample preparation, universal detection, and reproducibility across laboratories. Detection by NMR is much less sensitive than by MS and can be more costly with respect to instrument purchase and maintenance, but NMR is capable of quantifying phytochemicals in botanical products without a separation step. Furthermore, when coupled with discriminate data analysis it has been demonstrated to be a potentially useful tool for differentiating botanical species. Blueberry (Vaccinium angustifolium) leaf was differentiated from V. ovalifolium and V. macrocarpon leaves with use of NMR acquisition followed by PCA analysis (69), demonstrating the ability of this technique to differentiate dried powders. Black cohosh (Actaea racemosa L.) roots are used for their purported utility in treating menopausal symptoms. In the past several years, severe adverse reaction reports have associated black cohosh products with liver damage. It is indigenous to North America and is primarily obtained from wild collections. As demand (and therefore price) has increased, substitution of other Actaea species for authentic A. racemosa has been reported (70). At the moment, it is not possible to establish causality between black cohosh and liver toxicity in the reported cases, but it is a major concern, making black cohosh a consistently high-priority herb for both quality and identity testing (71, 72). HPLC with UV and The importance of method selection 395

7 MS detection can distinguish A. racemosa from other species based on the detection of phenolic acids and triterpene glycosides not present in A. racemosa (70, 73, 74). The presence of cimifugin, cimiracemoside F, and cimiracemoside C distinguish Asian Actaea species from A. racemosa in raw materials and commercial products. HPTLC demonstrates that the presence of cimifugin in biomass or finished product is an indicator of adulteration with A. foetida. Visualization with a boric acid-oxalic acid derivatization reagent permits detection of adulteration as low as 5% (68). FIMS coupled with visual interpretation and PCA differentiated methanol extracts of A. racemosa from A. dahurica and A. podocarpa based on the major molecular ions (75). DNA testing has gained widespread acceptance as a universal technique for taxonomic identification, although it has many limitations when applied to plant and herbal supplement identification. Its utility in botanical identification is still emerging as techniques and approaches evolve, and inappropriate method selection and data interpretation have led to controversy. For suitable fitness for purpose, botanical authentication methods that target DNA loci need to use genetic regions that are unique and specific to the plant species of interest. This can be challenging because the taxonomy of many plant species is confusing and in flux, whereas a genetic region selected for species differentiation may vary from closely related species by only a single bp substitution (76, 77). Several DNA methods have been used to identify herbal products, including Echinacea, Ginkgo, and black cohosh (78 80). Validation of identity methods is complex and requires a considerable number of authentic materials in all forms that will be evaluated in the final assay. For DNA barcoding, the material to be examined must contain intact DNA. Although DNA has successfully been extracted from herbal extracts, a number of processes for manufacturing botanical products degrades DNA to the point where it is no longer a viable tool for identification (78, 79). Such processes can include drying, extraction with very hot water, extraction with organic solvents, exposure to sunlight, etc. The utility of DNA for dietary ingredient and supplement authentication is complex and outside the scope of this review. Regardless of the technology used for botanical identification, the user must ensure that it is valid and fit for purpose before routine use. Conclusions Because of the increasing use of herbal supplements, demand for safe, high-quality products, and regulatory requirements, manufacturers must rely on the use of analytical methods to verify the quality and authenticity of their ingredients and products. Knowing as much as possible about the raw materials and finished products they will be using either to manufacture a product or that will be studied in a clinical trial beforehand will allow manufacturers and investigators to make rational decisions about analytical needs and approaches that they can use to design products or studies (81). By applying standard procedures to develop and validate analytical methods, including establishing desired performance characteristics, methods will be fit for purpose when used routinely in laboratories. Chemical identification methods are being designed to help ward off adulteration of herbal supplements. There are a variety of techniques that can be used and when coupled with discriminant analysis allow for the detection of known adulterants. It is critical to ensure that botanical identification methods are established and validated for each plant species separately because there are still issues surrounding finishedproduct identity as a result of the complexity of the different matrixes and excipients. Because of the prevalence of new herbal supplements, new adulteration techniques, and identity and safety concerns regarding herbal supplements, there is a continuing need for validated analytical methods for purity and identity. Acknowledgments We thank the ASN and Barbara Sorkin (NIH Office of Dietary Supplements) for organizing and supporting the symposium at the ASN Scientific Sessions and Annual Meeting at Experimental Biology All authors read and approved the final manuscript. References 1. Current good manufacturing practice in manufacturing, packaging, labeling, or holding operations for dietary supplements, 21 CFR Sect 111 (2009). 2. Natural Health Product Regulations, SOR/ (2003). 3. Brown PN, Betz JM, Jaksch FL. How to qualify an analytical laboratory for analysis of herbal dietary ingredients and avoid using a dry lab : a review of issues related to using a contract analytical laboratory by industry, academia and regulatory agencies. HerbalGram. 2013;99: Li S, Han Q, Qiao C, Song J, Cheng C, Xu H. Chemical markers for the quality control of herbal medicines: an overview. Chin Med 2008;3: Betz JM, Brown PN, Roman MC. Accuracy, precision, and reliability of chemical measurements in natural products research. Fitoterapia 2011; 82: Inui T, Wang Y, Pro SM, Fransblau SG, Pauli GF. Unbiased evaluation of bioactive secondary metabolites in complex matrices. Fitoterapia 2012;83: Zhang Z, Kuo X, Fugal K, McLaughlin J. Comparison of HPLC methods for determination of anthocyanins and anthocyanidins in bilberry extracts. J Agric Food Chem 2004;52: Brown PN, Roman M. Determination of hydrastine and berberine in goldenseal raw materials, extracts and dietary supplements by highperformance liquid chromatography with UV: collaborative study. J AOAC Int 2008;91: Brown PN, Chan M, Betz JM. Optimization and single-laboratory validation of a high-performance liquid chromatography (HPLC) method for the determination of phenolic Echinacea constituents. Anal Bioanal Chem 2010;397: Smillie TJ, Khan IA. A comprehensive approach to identifying and authenticating botanical products. Clin Pharmacol Ther 2010;87: Foster S. A brief history of adulteration of herbs, spices and botanical drugs. HerbalGram. 2011;92: Cumberford G. EMI vs EMA: economically motivated integrity vs. economically motivated adulteration in the natural products supply chain. HerbalGram. 2012;94: Applequist WL, Miller JS. Selection and authentication of botanical materials for the development of analytical methods. Anal Bioanal Chem 2013;405: Symposium

8 14. Betz JM, Fisher KD, Saldanha LG, Coates PM. The NIH analytical methods and reference materials program for dietary supplements. Anal Bioanal Chem 2007;389: Wallace SN, Carrier DJ, Clausen EC. Extraction of nutraceuticals from milk thistle. Part II. Extraction with organic solvents. Appl Biochem Biotechnol 2003;105: Soong Y, Barlow PJ. Antioxidant activity and phenolic content of selected fruit seeds. Food Chem 2004;88: Curtis JM, Berrigan N, Dauphinee P. The determination of n-3 fatty acid levels in food products containing microencapsulated fish oil using the one-step extraction method. Part 1: measurements in the raw ingredient and in dry powdered foods. J Am Oil Chem Soc 2008;85: Ma J, Ma YC, Cai C, Wang D, Hou FF, Luo M, Lu S, Gorecki DC, Patel AV, Chen A, et al. Simultaneous quantification of Echinacea species, Flos Loniverae, Radix Scutellaria and Fructus Forsythiae combinations by rapid resolution liquid chromatography. Nat Prod Commun 2011;6: Ma YC, Chen SL, Thibault ME, Ma J. Enhancing quality control of botanical medicine in the 21st century from the perspective of industry: the use of chemical profiling and DNA barcoding to ensure accurate identity. HerbalGram. 2013;97: Natural Health Products Ingredients Database: feverfew [Internet]. Ottawa (ON): Health Canada; 2010 [cited 2015 Jul 24]. Available from: Wohlmuth H, Savage K, Dowell A, Mouatt P. Adulteration of Gingko biloba products and a simple method to improve its detection. Phytomedicine 2014;21: Souza CRF, Bott RF, Oliveira WP. Optimization of the extraction of flavonoids compounds from herbal material using experimental design and multi-response analysis. Lat Am J Pharm. 2007;26: Cardoso-Ugarte GA, Juarez-Becerra GP, Sosa-Morales ME, Lopez-Malo A. Microwave-assisted extraction of essential oils from herbs. J Microw Power Electromagn Energy 2013;47: AOAC International. Appendix K: guidelines for dietary supplements and botanicals. AOAC Off Methods Anal 2013: International Conference on Harmonisation Expert Working Group. ICH harmonised tripartite guideline. Validation of analytical procedures: text and methodology [Internet]. [cited 2015 Jul 24]. Available from: Guidelines/Quality/Q2_R1/Step4/Q2_R1 Guideline.pdf. 26. U.S. FDA. Guidelines for validation of chemical methods for the FDA foods program [Internet]. [cited 2015 Jul 24]. Available from: http: // pdf. 27. International Union of Pure and Applied Chemistry. Harmonized guidelines for single-laboratory validation of methods of analysis. Pure Appl Chem 2002;74: Magnusson B, Örnemark U, editors. Eurochem guide. The fitness for purpose of analytical methods a laboratory guide to method validation and related topics [Internet]. [cited 2015 Jul 24]. Available from: https: // pdf. 29. AOAC International. Appendix D: Guidelines for collaborative study procedures to validation characteristics of a method of analysis. AOAC Off Methods Anal 2013: Brown PN, Lister P. Current initiatives for the validation of analytical methods for botanicals. Curr Opin Biotechnol 2014;25: Schierle J, Pietsch B, Ceresa A, Fizet C, Waysek EH. Method for the determination of b-carotene in supplements and raw materials by reversed-phase liquid chromatography: single laboratory validation. J AOAC Int 2004;87: Szpylka J, DeVries JW, Bhandari S, Bui MH, Ji D, Konings E, Lewis R, Maas P, Parish H, Post B, et al. Determination of b-carotene in supplements and raw materials by reversed-phase high pressure liquid chromatography: collaborative study. J AOAC Int 2005;88: Ji D, Roman M, Zhou J, Hildreth J. Determination of chondroitin sulfate content in raw materials and dietary supplements by high-performance liquid chromatography with ultraviolet detection after enzymatic hydrolysis: single-laboratory validation. J AOAC Int 2007;90: Brown PN, Paley LA, Roman MC, Chan M. Single-laboratory validation of a method for the detection and/or quantification of select alkaloids in goldenseal supplements and raw materials by reversed-phase high-performance liquid chromatography. Pharm Biol 2008;46: Brown PN, Chan M, Paley L, Bezt JM. Determination of major phenolic compounds in Echinacea spp. Raw materials and finished products by high-performance liquid chromatography with ultraviolet detection: single-laboratory validation matrix extension. J AOAC Int 2011;94: Brown PN, Shipley PR. Determination of anthocyanins in cranberry fruit and cranberry fruit products by high-performance liquid chromatography with ultraviolet detection: single-laboratory validation. J AOAC Int 2011;94: Mudge E, Chan M, Venkataraman S, Brown PN. Curcuminoids in turmeric roots and supplements: method optimization and validation. Food Anal Methods 2015: National Institute of Health Office of Dietary Supplements. Publications directory [Internet]. [cited 2015 Nov 25]. Available from: https: //ods.od.nih.gov/amrmpubsdirectory.aspx 39. AOAC International. Stakeholder panel on dietary supplements (SPDS) [Internet]. [cited 2015 Jul 28]. Available from: imis15_prod/aoac/sd/spds/aoac_member/sh/spdscf/spdsm. aspx?hkey=b8cbd524 33d1 4e51 8cc0 4e2028c367f Leung AM, Pearce EN, Braverman LE. Iodine content of prenatal multivitamins in the United States. N Engl J Med 2009;360: Betz JM, Wise SA. More on iodine content of prenatal vitamins. N Engl J Med 2009;360: Leung AM, Pearce EN, Braverman LE. More on iodine content of prenatal vitamins: authors reply. N Engl J Med 2009;360: Weiguo Z, Zhang W, Giancaspro G, Adams KM, Neal-Kababick J, Hildreth J, Li A, Roman M, Betz JM. Electrophoretic separation of alginic sodium diester and sodium hexametaphosphate in chondroitin sulfate that interfere with the cetylpyridinium chloride titration assay. J AOAC Int 2014;97: Slifman NR, Obermeryer WR, Aloi BK, Musser SM, Correll WA, Cichowicz SM, Betz JM, Love LA. Contamination of botanical dietary supplements by Digitalis lanata. N Engl J Med 1998;339: Lin LZ, Harnly JM, Upton R. Comparison of the phenolic component profiles of skullcap (Scutellaria lateriflora) and germander (Teucrium canadense and T. chamaedrys), a potentially hepatotoxic adulterant. Phytochem Anal 2009;20: Chen P, Lin LZ, Harnly JM. Mass spectroscopic fingerprinting method for differentiation between Scutellaria lateriflora and the germander (Teucrium canadense and T. chamaedrys) species. J AOAC Int 2010; 93: Penman KG, Halstead CW, Matthias A, De Voss JJ, Stuthe JMU, Bone KM, Lehmann RP. Bilberry adulteration using the food dye amaranth. J Agric Food Chem 2006;54: Gardana C, Ciappellano S, Marinoni L, Fachechi C, Simonetti P. Bilberry adulteration: identification and chemical profiling of anthocyanins by different analytical methods. J Agric Food Chem 2014;62: Giacomelli L, Appendino G, Franceschi F, Togni S, Pace R. Omne Ignotim pro Magnifico: characterization of commercial Bilberry extracts to fight adulteration. Eur Rev Med Pharmacol Sci 2014;18: Primetta AK, Jaakola L, Ayaz FA, Inceer H, Riihinen KR. Anthocyanin fingerprinting for authenticity studies of bilberry (Vaccinium myrtillus L.). Food Contr 2013;30: Govindaraghavan S. Pharmacopeial HPLC identification methods are not sufficient to detect adulterations in commercial bilberry (Vaccinium myrtillus) extracts.anthocyaninprofile provides additional clues. Fitoterapia 2014;99: LaBudde RA, Harnly J. Probability of identification: a statistical model for the validation of qualitative botanical identification methods. J AOAC Int 2012;95: Chen P, Luthria D, Harrington PB, Harnly JM. Discrimination among Panax species using spectral fingerprinting. J AOAC Int 2011;94: The importance of method selection 397