Compendial Perspectives on Botanical Identity Testing

Abstract

Quality control systems such as Good Agricultural and Collection Practices, current Good Manufacturing Practices, and regulations emphasize the importance of botanical identity verification for assuring safety and purported benefits. The inherent biological variability and chemical complexity of botanical raw materials, extracts, and fractions demand fit-for-purpose methods for the accurate identification and discrimination from confounding materials and adulterants. Compendial botanical identity testing comprises orthogonal procedures for morphological and chemical characterization; specifications and acceptance criteria versus reference standards; consistent nomenclature; and labeling. Pharmacopeial general chapters provide guidance on the system suitability and method validation tests needed for each matrix to demonstrate specificity and sensitivity.

Introduction

The term “botanicals” is used in the broad sense to include plants, algae, fungi, and lichens, as well as plant products such as exudates or oleo-gum-resins. Botanical identity as defined in the World Health Organization (WHO) Good Agricultural and Collection Practices (GACPs) is the verification/authentication of the genus, species, variety/cultivar/chemotype, and origin. The preamble to the current Good Manufacturing Practices (cGMPs) for dietary supplements in the United States highlights that the “identity” of a dietary supplement refers to its consistency with the master manufacturing record and/or that it is the same as described in the record. More specifically, the cGMPs require manufacturers to establish the identity, purity, strength, and composition specifications for ingredients and products and ensure that such specifications are met in the finished products. Simply put, consumers must be able to have confidence that what is on the label is in the bottle.

Regulatory requirements emphasize the importance of appropriate methods for determining identity. The preamble to the dietary supplement cGMPs states “Because of the critical importance of ensuring the proper identity of dietary ingredientsthey are the central defining ingredients of a dietary supplementwe are requiring each firm that uses a dietary ingredient to perform its own testing or examination for identity of each dietary ingredient prior to use”. Regulation 21 CFR 111.75­(h)(2) states that the tests and examinations must include at least one of the following: (i) Gross organoleptic analysis; (ii) Macroscopic analysis; (iii) Microscopic analysis; (iv) Chemical analysis; or (v) Other scientifically valid methods. To meet this requirement, manufacturers must employ analytical procedures to correctly identify the ingredients to be used. Specifications for identitythe scientifically valid analytical methods and acceptance criteriaplay an important role in defining and characterizing a dietary ingredient or finished product. While the basis of contractual agreements between the sellers and buyersthe Certificate of Analysisis an important tool, the cGMP preamble states that “Firms may not rely upon a certificate of analysis provided by suppliers to determine the identity of a dietary ingredient before use”. This reflects a universal tenet in pharmaceutical analysis, according to which the testing of identity is the minimal requirement when receiving materials for further use. The cGMP final rule also states, ″In the preamble to the 2003 cGMP Proposal (68 FR 12157 at 12209), we acknowledged that validated methods exist in official compendia for vitamins, minerals, and several botanicals, and we recommended you use validated methods whenever such methods are available. We explicitly stated that you may use validated methods that can be found in official references, such as AOAC International, USP, and others”.

Underscoring the importance of ensuring identity, the cGMP regulations require manufacturers to demonstrate evidence of “no material diminution of assurance, compared to the assurance provided by 100% identity testing, of the identity of the dietary ingredient before use,” when proposing alternative methods and processes and seeking a waiver from the need to conduct identity testing of all the dietary ingredients used in a formulation (commonly known as the requirement for 100% identity testing). We are not aware of any manufacturer who has availed themselves of the waiver from the responsibility to conduct 100% identity testing.

Note: Although the regulatory standards for the quality of botanicals used in drugs and dietary supplements differ, they share several common elementssuch as the use of scientific criteria to establish identity. This article primarily focuses on the botanical identity in the context of U.S. cGMP requirements for dietary supplements.

Why Identity Matters

Good Agricultural and Collection Practices

Verification of the botanical identity is a fundamental requirement of GACPs. GACP compliance is not internationally enforced; it is required in some countries for some classes of products (e.g., active ingredients of herbal medicinal products) but not required for other similar types of products (e.g., ingredients of herbal dietary supplement products). WHO GACPs require voucher specimens of wild collected botanicals to be submitted to a qualified herbarium for authentication. For cultivated botanicals, seeds, planting stock, or other propagation materials must be authenticated before planting. GACPs require agreement between producers and buyers to include written specifications based on recognized standards such as a pharmacopeial monograph. In both the United States and the European Union, GACP compliance is mandatory only for botanical drug active ingredients while it is voluntary for botanical dietary supplement ingredients. In Canada, GACP compliance is a requirement for both natural health products, which are a subset of drugs, and for foods, including supplemented foods containing added botanicals. Postharvest primary processing steps should also occur under GACP controls before transferring to secondary processing manufacturers that have implemented suitable cGMPs, whether for botanical medicines or supplements.

Reported Concerns About Identity

According to the Dietary Supplements Health and Education Act of 1994 (DSHEA), botanicals and derivatives in the form of a concentrate, metabolite, constituent, or extract are considered a category of dietary ingredients. cGMPs require the use of correctly identified botanical ingredients. Failure to establish specifications for identity is one of the most common reasons cited in cGMP warning letters to manufacturers of dietary supplement products in the United States. This may result from the failure to use appropriate analytical methods for establishing the identity of ingredients consistent with the raw material form, ingredient, or product matrix and target analytes. Adding to the challenges of analyzing botanicals are their inherent chemical and biological complexity, natural variability, and changes to their composition from the processing of the raw materials to the final product. Unintentional substitution with other species because of similarities in their botanical characteristics or because of similarities in common names poses a risk of misidentification. Substitution is also accepted in some systems of medicine when equivalent efficacy has been documented (e.g., Andrographis paniculata [Burm. f.] Nees, Acanthaceae, in place of the rarer and higher-cost Swertia chirayita [Roxb.] H.Karst., Gentianaceae, used interchangeably in the Ayurvedic system of medicine). , Similarly, multiple closely related species of certain genera are sometimes used, e.g., Salix, Crataegus, Bupleurum, and Epimedium. Suitable analytical methods are needed in order to identify the target species with specificity (the ability to identify nonauthentic samples as not authentic, i.e., true negatives) and sensitivity (the ability to identify authentic samples as authentic, i.e., true positives) to discriminate it from adulterants and substitutes. The increasing dependence on a global supply chain and competitive price pressures on the industry have created conditions where deliberate adulteration with cheaper or substandard ingredients of questionable quality is an unfortunate reality.

Economically motivated adulteration also occurs when someone adds an undeclared substance to a product to make it appear better or of greater value. It is not clear how extensive the problem of adulteration is but estimates suggest that up to 25% of dietary supplement products may be adulterated in some way. − Adulteration of food and medicines has been common throughout history. In his article A brief history of adulteration of herbs, spices, and botanical drugs, Steven Foster (1957–2022) notes that “Since the beginnings of civilization, once commerce develops, adulteration follows,” with documented cases of adulteration going back to the beginning of the common era.

Value of Identity for Reproducibility of Beneficial Effects and Detection of Adulteration

Given the complex nature of botanical materials, failure to assess the identity can impact the validity and reproducibility of research. Recognizing the value of completeness of information about a product, the National Center for Complementary and Integrative Health has established policies for ensuring natural product integrity for mechanistic and clinical research. The proper identity of botanical extracts, their nomenclature, and correlation to commonly used products is highlighted in relation to a study on ginkgo (Ginkgo biloba L.; Ginkgoaceae) leaf extract.

The sale of adulterated ingredients can have a number of impacts. For the consumer, an adulterated product may not provide the expected benefit or, in some instances, may even cause unwanted side effects. A lack of proper identification of botanical ingredients may also lead to irreproducible results and erroneous conclusions in scientific studies. The importance of proper characterization of botanical investigational materials has been discussed by Oketch-Rabah et al. using the example of cinnamon (Cinnamomum Schaeff, Lauraceae) (Figure ). The authors reviewed human clinical studies and tried to determine which species of Cinnamomum was used. They reported that identity was not verified in the majority of trials, and hence, the results may be called into question.

1.

Commercial samples of cinnamon bark from different countries, illustrating the variability of the material, which may all be marketed as cinnamon despite consisting of four different Cinnamomum species with differences in chemistry, pharmacology, and toxicology (images courtesy of T. Brendler).

It should be noted that the need for clinical trial investigational products to be characterized, manufactured, handled, and stored in compliance with applicable cGMPs is explicitly cited in the internationally recognized International Council for Harmonisation (ICH) Guideline for Good Clinical Practice E6­(R3), Principle 11 and Annex 1 paragraph 3.15.2­(a). A prominent investigation in 2002 into a supposedly herbal product, PC-SPES, revealed adulteration with pharmacologically active compounds such as diethylstilbestrol, leading to misleading efficacy signals and safety risks. This case highlighted that well-controlled studies using well-characterized materials are needed to yield reliable evidence of safety and therapeutic benefit. − The details needed for adequate characterization of botanical products investigated in clinical trials are itemized in the Consolidated Standards of Reporting Trials (CONSORT) standards for reporting of randomized, controlled trials of herbal interventions. ,

A lack of proper identity verification can also lead to erroneous conclusions about product safety and serious adverse effects in consumers. A series of case reports from 2003–2009 linked the consumption of products labeled to contain the North American herb black cohosh (Actaea racemosa L., Ranunculaceae) to liver injury. However, the identity of the botanical was not established in most cases, and analysis of some of the alleged black cohosh products linked to hepatotoxicity by scientists at Health Canada revealed that these products were adulterated with related Actaea species originating from Asia.

Similarly, a series of case reports suggested that the use of English lavender (Lavandula angustifolia Mill., Lamiaceae) essential oil-containing products caused premature breast development in young girls or prepubertal gynecomastia in boys. Again, the presence of lavender was not verified by the authors of the case reports orin one casebased on the detection of two marker constituents that are not suitable to identify lavender essential oil. One author proclaimed that lavender-containing products could be identified by the color of the bottle exteriors or caps, i.e., purple or dark blue often indicates lavender as an ingredient. Subsequent analysis of some of the implicated products found that these contained predominantly synthetic fragrance constituents. And lavenderif present at allwas detected only in trace amounts. , Despite evidence that at least some of these case reports were erroneously assigned to black cohosh and lavender, respectively, the medical literature continues to perpetuate these case reports as evidence for the risk of adverse events, hence limiting the use of potentially beneficial botanicals.

Botanical Identity Considerations in the USP

United States Pharmacopeial Convention (USP) is an independent, scientific, nonprofit public health organization devoted to improving global public health through the development of public standards and related programs that help ensure the quality, safety, and benefit of medicines, dietary supplements, and foods. USP publishes two legally recognized Official Compendia of the United States, combined into a single publication, the United States Pharmacopeia-National Formulary (USP-NF). One of USP’s areas of expertise and focus is the development of standards for articles of botanical origin, including analytical procedures and acceptance criteria to help ensure their identity, purity, and strength. USP monographs set forth the article’s name, definition, specifications, and other requirements related to packaging, storage, and labeling. Specifications consist of tests, procedures, and acceptance criteria for identity, composition or strength, purity, and limits for contaminants, which are aligned with the requirements of compliance with cGMPs. USP monograph standards for botanical ingredients include several requirements that are relevant for identity: monograph title, nomenclature, definition, identification tests, and labeling.

The Identification section of a monograph is intended to provide analytical tests that will help ensure that the tested article agrees with the definition and conforms to the description used on the label. USP botanical monographs typically include at least two complementary or orthogonal tests for identification. The Identification tests are a guide for assessing the plant’s inherent characteristics, including its macroscopic morphological features and its microscopic anatomical and histological features. Chemotaxonomy, or the classification of plants based on their chemical constituents, allows linking botanical and chemical identifications, which are intrinsic aspects of botanical identity verification. Compared with a reference standard (RS), characteristic chemical fingerprinting allows for the identification of plant materials and the distinction between closely related species.

Recognizing the critical need to ensure the identity of an ingredient, USP General Notices state that an ingredient that claims compliance with USP specifications “must comply with compendial identity standards or be deemed adulterated, misbranded, or both”. Accordingly, complying with the identification requirements in the compendia and claiming compliance determines whether the article must use the official compendial title as its nonproprietary name. When two or more substances share an identity, they must conform to the same comparative standard in the monograph.

The determination of whether two ingredients share an identity is based on whether they share the same quality attributes, as described in Definition and Labeling section. These sections include relevant information such as the part of the plant and season or growth stage of collection. In the case of botanical extracts, besides the name of the plant part used, other labeling recommendations include: the names of solvents, other than hydroalcoholic solvents, used in preparation; the content, in percentage Not Less Than (NLT) and/or Not More Than (NMT), of active principles or marker compounds identified in the individual monograph. Where the content of active principles is not specified, the ratio of starting material to the final product is stated (i.e., Plant to Extract ratio; also known as the drug-to-extract ratio in Europe), and the percentage of native extract when excipients are used may also be stated.

Compendial Identification tests use suitably specific analytical procedures in comparison with an RS and established acceptance criteria to assist in identifying the target species and discriminating it from closely related species, confounding materials, adulterants, and substitutes. While the term “identity” is often used interchangeably with “identification”, the Identification test is only an “aid” in verifying identity rather than itself defining identity.

The Identification tests for a monograph may consist of one or more analytical procedures. When a compendial test for Identification is undertaken, all requirements of all specified procedures in the test must be met to satisfy the requirements of the test. Failure of an article to meet all of the requirements of a prescribed Identification test (i.e., failure to meet the requirements of all of the specified procedures that are components of that test) indicates that the tested article is not the same as the article described in the monograph.

Nomenclature

The value of designating each substance by a nonproprietary name is important in terms of achieving simplicity and uniformity in nomenclature. For the purposes of establishing manufacturing specifications, Latin binomials should be used for specificity. For dietary supplement labeling in the U.S., 21 CFR 101.4­(h) states that “common or usual names of ingredients of dietary supplements that are botanicals (including fungi and algae) shall be consistent with the names standardized in Herbs of Commerce” or the Latin binomial if a standardized common name is not available. Unambiguous naming is critical to ensure that each unique ingredient is known by one name, and the identification tests and other requirements can establish its identity and discriminate against other substances.

The unique name is important in that if a closely related article, e.g., an ingredient obtained from the same plant but a different part or by different processing, does not meet an identity standard, it must use a distinct and differentiating name. For example, the USP–NF publishes separate monographs for Echinacea purpurea Aerial Parts (the aerial parts of Echinacea purpurea (L.) Moench (Asteraceae), harvested during the flowering stage) and for Echinacea purpurea Root (the dried rhizome and roots of Echinacea purpurea (L.) Moench (Asteraceae), harvested in the fall after 3 or more years of growth). An example based on manufacturing processing or preparation is the Cranberry (ripened fruits of Vaccinium macrocarpon Aiton (Ericaceae)) family of monographs, which consists of individual monographs for Cranberry Fruit Powder, Cranberry Fruit Juice, Cranberry Fruit Dry Juice, Cranberry Fruit Juice Concentrate, and Cranberry Fruit Juice Dry Extract. Figure illustrates the need for appropriate nomenclature and labeling to differentiate derivative products and the use of suitable identification test methods and acceptance criteria to characterize these articles of commerce. The identity of each of the articles derived from the cranberry fruit is linked to the specific name, definition, and labeling requirements, which describe the critical quality attributes of the derivative products.

2.

Universe of cranberry-derived ingredients as an example of a nomenclature exercise to identify, name, and define pharmacopeial from nonpharmacopeial ingredients.

USP has also created monographs for raw material articles of commerce from two different but closely related botanical species and one chemically enriched article of commerce that can be sourced from either. There are monographs for Garcinia gummi-gutta (L.) N.Robson (syn. G. cambogia Desr., Clusiaceae) dried pericarp of the fruits, Garcinia indica (Thouars) Choisy dried pericarp of the fruits, and Powdered Garcinia Hydroxycitrate Extract which may be prepared from either G. gummi-gutta or G. indica by extraction with water, alcohol, or mixtures of these solvents, followed by stabilization of the (−)-hydroxycitric acid content in the form of a calcium, potassium, magnesium, and/or sodium salt.

Note that the author of the botanical article’s Latin binomial is included as a key component of nomenclature for identity in each USP–NF monograph. The author’s citation becomes critical in tracing the source of the Latin binomial to prevent confusion over duplicate names or synonyms and confirm the correct nomenclature. A good example of this is the case of two different species that have been described in the literature with the same Latin binomial, Illicium anisatum (Schisandraceae). Only the author distinguishes the two names: Illicium anisatum L. is the correct Latin binomial for toxic Japanese star anise, and Illicium anisatum Lour. is an incorrect Latin binomial for edible Chinese star anise. The accepted Latin binomial in the NF monograph for edible Star Anise Oil is Illicium verum Hook.f. Synonyms of Latin binomials well-established in commerce are included in the Definition section of monographs for clarity, e.g., Aloe vera (L.) Burm.f. (syn. Aloe barbadensis Mill., Asphodelaceae).

The USP Guideline for Assigning Titles to USP Dietary Supplement Monographs and the Guideline for Assigning Titles to USP Herbal Medicines Compendium Monographs provide a systematic approach to the development of monograph titles for dietary ingredients and dietary supplement dosage forms and herbal medicine components admitted to the USP–NF and the Herbal Medicines Compendium (HMC), respectively. The importance of the nomenclature of plants and herbal substances is discussed in greater detail, with many examples and illustrations, by Allkin and Patmore (2025).

The unambiguous name by which an ingredient is recognized in commerce should be supported by appropriate tests of its identity. For example, a botanical extract rich in zeaxanthin and a specific purified isomeric form of zeaxanthin, meso-zeaxanthin, are differentiated with distinct names. USP–NF defines Aztec Marigold Zeaxanthin Extract as a purified extract, derived from the flowers of Tagetes erecta L. (Asteraceae), grown from seeds of varieties of the Scarletade cultivar rich in zeaxanthin.

Overview of Analytical Methods

Macroscopic and Microscopic Identification Methods

Diagnostic morphological and anatomical features determined by macroscopic and microscopic examinations are used to differentiate botanical articles from related species. The pharmacognostic features of the genuine article and those of the adulterants and substituents should be differentiated to indicate how to distinguish an adulterant from an authentic sample. USP–NF General Chapter <563> Identification of Articles of Botanical Origin defines the diagnostic features including organs, tissues, cell type and arrangement, type of secretory canal or ducts and their number surrounding epithelial cells, and the presence and type of ergastic substances occurring in the cytoplasm, organelles, vacuoles, cavities, or cell walls. To help account for the natural variability of botanical articles, the Macroscopic and Microscopic descriptions in the USP monographs and Supporting Information in the Dietary Supplements Compendium include size ranges and variations in color and texture for key diagnostic features, based on the examination of multiple specimens (See Figure as an example).

3.

Macroscopic characteristics of rhizomes and roots of three species of ginseng.

When establishing unambiguous morphological and anatomical identification characteristics, reference to authenticated herbarium specimens of the plant in the appropriate growth stage (e.g., with flowers and fruits) is crucial to set specifications that capture the necessary range of size, shape, and other variable features. These characteristics can then be reliably applied, with comparison to an authenticated botanical reference standard, to the identification of the material of commerce, which typically consists of missing plant parts lost during postharvest processing. The characteristic shape and color of the inflorescence may be removed due to trimming when the root is the article of commerce, and processing may change a botanical article’s color and texture, resulting in it potentially closely resembling other unrelated species. For example, the freshly harvested root of wild quinine, also known as Missouri snakeroot (Parthenium integrifolium L., Asteraceae), is not similar in appearance to the root of Echinacea angustifolia DC. or E. pallida (Nutt.) Nutt. (Asteraceae). However, once the root is dried, cut, and sifted, it has an uncanny resemblance to E. angustifolia or E. pallida roots, although it possesses its own characteristic flavor and fragrance. For preliminary visual identification, by comparing the morphology of a voucher specimen of the plant collected at the source with specimens in a herbarium, an innovative tool is the virtual herbarium. One example is the C. V. Starr Virtual Herbarium, which is the gateway to the digitized specimens of the New York Botanical Garden’s William and Lynda Steere Herbarium, with over four million specimens digitized. Figure illustrates the differentiation of the three species of ginseng based on morphological characteristics of the material of commerce, i.e., the rhizome and root.

Chromatography Identification Methods

The fingerprint (also termed profiling) approach is one of the widely used identification methods.

High-Performance Thin-Layer Chromatography

For high-performance thin-layer chromatography (HPTLC) fingerprinting, the relative position, color, and intensity of the bands under white light or ultraviolet (UV) light, before and after derivatization, in comparison to an RS, help identify some of the diagnostic constituents. In a cGMP-compliant environment, the reproducibility and integrity of the results are of great importance. USP–NF General Chapters <203> High-Performance Thin-Layer Chromatography Procedure for Identification of Articles of Botanical Origin provides guidance and standard parameters to ensure reproducibility of the results across laboratories. Individual RS (or their mixtures) and Botanical Extract RS are typically employed for system suitability monitoring to help locate the position and resolution of the bands. The resolution, position, and colors of the key bands of the RS chromatogram should match the description in the monograph within a specified tolerance. See Figure as an example for differentiation of the three species of ginseng (tracks 4–6) based on their ginsenoside profile.

4.

HPTLC profile of the ginsenosides and differentiation of the three species of ginseng under long-wave UV light. Developing solvent: methylene chloride, anhydrous ethanol, and water (60:45:6.5). Derivatization reagent: sulfuric acid and ice-cold alcohol (1:9). Track assignment: 1) USP Escin RS (0.5 mg/mL), arbutin (0.5 mg/mL) with increasing RF; 2) ginsenoside Rb1 (0.5 mg/mL), ginsenoside Rb3 (0.5 mg/mL), notoginsenoside R1 (0.5 mg/mL), ginsenoside Rf (0.5 mg/mL), pseudoginsenoside F11 (0.5 mg/mL), with increasing RF; 3) USP Ginsenoside Rg1 RS (0.5 mg/mL); 4) USP Powdered American Ginseng Extract RS (10 mg/mL); 5) USP Powdered Asian Ginseng Extract RS (10 mg/mL); 6) USP Panax notoginseng Root and Rhizome Dry Extract RS (10 mg/mL).

Liquid Chromatography

For HPLC or UHPLC fingerprinting, the constituents’ peak relative retention time and intensity are compared to an RS, usually as a Botanical Extract. The area or content ratio between characteristic peaks can also be defined as another parameter or requirement for the identification tests. USP–NF General Chapter <621> Chromatography provides the SST requirements for chromatographic methods. For HPLC, these system suitability tests typically specify the following: (1) a resolution requirement to ensure that the peaks to be quantitated achieve baseline separation (NLT 1.5); (2) a maximum tailing factor, typically NMT 2.0; (3) a maximum value of relative standard deviation (%RSD) of NMT 2.0%. In addition to these SST, botanical monographs also include the test for Chromatography Similarity, in which the profile of a USP botanical extract RS needs to match the profile provided in the Certificate of Analysis regarding peak identification and relative retention times.

When using chemical fingerprinting by HPTLC, HPLC, or UHPLC, it is important not to directly extend the identification test used for botanical raw materials to botanical extracts without previous method validation since the fitness for purpose of a method needs to be confirmed for each matrix independently. The extraction process may result in the loss of chemical components due to the use of different solvents and/or different solvent concentrations. In addition, a mismatch of the polarity of the solvent and individual components can reduce the extraction efficiency, even if repeated several times. Comparison of a raw material with a finished product can be particularly problematic, as other constituents are frequently added, i.e., a mixed supplement. The change in composition negates the use of chemometric methods to establish similarity, as discussed in the next section. See Figure as an example of a typical UHPLC chromatogram of USP Panax notoginseng Root and Rhizome Dry Extract RS.

5.

Typical UHPLC chromatogram of USP Panax notoginseng Root and Rhizome Dry Extract RS. Column: L1, 5 cm × 2.1 mm, 1.7 μm, Kinetex C18, Phenomenex; Mobile phase: 0.03% phosphoric acid in water (Solution A) and acetonitrile (Solution B); Elution: gradient program; Flow rate: 0.8 mL/min; Column temperature: 30 °C; Detection: UV 203 nm. Legend: A, Notoginsenoside R1; B, Ginsenoside Rg1; C, Ginsenoside Re; D, Ginsenoside Rb1; and E, Ginsenoside Re.

Chemometrics

The use of multivariate statistical methods of analysis can provide an objective means of determining the similarity of samples based on their chromatographic or spectroscopic profiles. USP–NF General Chapter <1039> Chemometrics provides guidelines regarding scientifically sound practices for the chemometric analysis and interpretation of typical multivariate data. It is possible to discriminate between botanical articles, with respect to their taxonomic identity, geographic origin, season of collection, and other variants.

Chemometric methods can be applied to targeted (a matrix of digitized components such as a food database) or nontargeted (raw chromatograms or spectra) data sets and can range from fundamental principal component analysis (PCA) for initial data exploratory analyses to highly sophisticated deep learning methods using artificial neural networks for image analyses. For botanical materials, chemometric methods are generally used to establish whether a test material is the same as a reference material. Statistically different botanical composition patterns will result in separate groupings in a PCA scores plot. With appropriate experimental design, chemometric methods are generally applied to quantitative data sets and can be used to analyze many varieties of spectroscopic and chromatographic data. AOAC Appendix K describes a Guideline for Validation of Botanical Identification Methods. In this guideline, the generic validation method is illustrated using PCA to discriminate between American (Panax quinquefolius L., Araliaceae) and Asian ginseng (Panax ginseng C.A.Mey.), but chemometric methods have been applied to a wide variety of other botanicals. Most recently, it has been used to discriminate between cranberry supplements based on HPTLC analyses after appropriate digitization and preprocessing. Another excellent example of PCA applied to a botanical material was the analysis of maca (Lepidium meyenii Walp., Brassicaceae; syn. L. peruvianum G.Chacón), a tuber grown as a food source in its native range in Peru at altitudes between 3,500 and 4,500 m and now also grown in China. ,

The simplest chemometric approach uses one-class modeling to determine whether a test sample matches a set of reference samples. This approach produces a binary result telling the analyst whether the test sample is similar (YES) or not (NO) to the reference samples. The analyst can specify the statistical level of confidence for the test. Using a set of reference samples can account for the biological variation of the material if samples are collected for a variety of growing locations, years, climates, and processing methods. One-class modeling can be regarded as a survey or preliminary test to identify samples that need further testing using targeted methods.

Genomic Methods

Traditionally, species determination relied on the observation and comparison of morphological characteristics. However, with advancements in molecular biology, the genetic information underlying those features has increasingly become a powerful tool for species differentiation. For example, the internal transcribed spacer region 2 (ITS2), a widely used DNA barcode, has been analyzed in over 4,800 medicinal plant species and achieved an overall identification success rate of 92.7% at the species level. These studies highlight the robustness of genomic methods for species identification, especially when the plant material has lost its diagnostic features through postharvest processing.

A broad spectrum of genomic information can be used to test for botanical identity, ranging from entire genomes and complete plastid sequences to short barcode regions. − Correspondingly, a variety of laboratory techniques enable researchers to extract and interrogate these data, including various sequencing approaches and Polymerase Chain Reaction (PCR)-based methods for generating species-specific amplicon profiles. , Figure illustrates a species-specific amplification example for Panax ginseng.

6.

Specific amplification of Panax ginseng DNA using a targeted primer pair (Forward: 5′-TGTACCCTCCCCCTAGGTGT-3′; Reverse: 5′-CGATAAAATCCCTTTCCAGCTTAAAAAATA-3′) that amplifies a 166 bp region within the psbM–trnD intergenic spacer. Amplification is observed only in P. ginseng (PG) botanical reference materials, with no amplification in P. quinquefolius (PQ), P. notoginseng (PN), or the nontemplate control (NTC).

Genomic data, much like voucher specimens in herbaria, are preserved and made accessible in public databases such as GenBank and the Barcode of Life Data Systems. While these repositories serve as valuable references for the scientific community and industry, a curated database specifically dedicated to botanical identity testing in quality control has yet to be established.

Despite their utility, genomics methods have important limitations. Highly processed plant materials may contain degraded DNA, significantly reducing the effectiveness of DNA-based identification and requiring careful test design and cautious interpretation to avoid incorrect conclusions. , Moreover, once morphological context is lost, genetic information alone cannot reveal from which specific plant part (e.g., root, stem, or leaf) the DNA originated from. Therefore, DNA data must be interpreted with caution to avoid erroneous conclusions. Cross-validation of emerging DNA-based methods using established chromatographic techniques is essential to build confidence in their utility. See the section on Need for Orthogonal Tests for further discussion of DNA-based methods.

Fitness for Purpose: Applying Methods across Different Matrices

Fitness for the purpose of an identity method, defined by the specificity and sensitivity of the method, must be established for the specific matrices of the analyte to avoid false-positive or false-negative outcomes. The matrix of the analyte has a significant effect on the ability of the analytical test procedure to accomplish the objective of testing for identity. Several forms of botanical raw materials existcrude fresh or dried plant parts in whole or rough chopped forms, as well as comminuted materials of varying particle sizes and extracts that are processed by different manufacturing processes. Excipients may be intentionally used to standardize or enhance functionality, adding another layer of complexity to the botanical matrix and, thus, to the method validation task. Furthermore, it is common practice in the industry to subject botanical raw materials to microbial reduction processes that impact composition, results of some identity tests, and quality parameters including alteration of chemical composition, macroscopic or sensory changes (e.g., discoloration of leaves and flowers, discernible changes in both odor and taste), and microscopic changes (e.g., microscopic profiles are discernibly different between raw and steamed samples).

Some commonly used methods (not an exhaustive list) and the rationale for their use in testing different matrices are captured in Table .

1. Identification Methods for Botanical Materials.

Nature of the Analyte	Method for Identification	Rationale
Whole or semiwhole botanical material	• Macroscopic and microscopic methods	Morphological and anatomical, and histological identifying features can be seen; phytochemical characterization is possible; and DNA may be suitable for amplification and downstream analysis
	• Chromatography methods: HPTLC-UV/vis; HPLC with various detectors (UV/vis, DAD, MS, ELSD, and possible hyphenations)
	• DNA-based methods
Comminuted botanical material	• Microscopic methods	Macroscopic morphological identifying features are lost when a plant material is processed but characteristic anatomical details may be identified by microscopy; phytochemistry remains characteristic; DNA may remain suitable for amplification and downstream analysis
	• Chromatography methods: HPTLC-UV/vis; HPLC with various detectors (UV/vis, DAD, MS, ELSD, and possible hyphenations)
	• DNA-based methods
Botanical extract	• Chromatography methods: HPTLC-UV/vis; HPLC with various detectors (UV/vis, DAD, MS, ELSD, and possible hyphenations)	Morphological and anatomical identifying features are lost when a plant material is processed; phytochemical characterization is possible; DNA may be damaged
	• DNA-based methods limited application (depending on the extraction processes, species, and plant parts)

Guidelines for Validation

With regard to the analytical methods used to set quality specifications, cGMP regulations require manufacturers to verify that the laboratory examination and testing methodologies are appropriate for their intended use and to identify and use appropriate scientifically valid method(s) for each established specification for which testing or examination is required (21 CFR 111.320).

The application and interpretation of technical guidelines and requirements for identification tests are defined in several resources including USP–NF General Chapter <1225> Validation of Compendial Procedures, <1226> Verification of Compendial Procedures, ICH Q2 guidelines, and by the WHO. These guidelines require the use of identification methods that possess the attribute of specificity, the ability to assess unequivocally the analyte(s) in the presence of components that may be expected to be present, such as impurities, degradation products, and matrix components. At the same time, a specific method should allow the identification of the target species from the nontarget species in the concomitant use of an RS and should be able to identify the target species within the variability expected in commercial samples (i.e., demonstrate sensitivity). Usually, the attribute of specificity needs to be cross-validated using an orthogonal method.

AOAC Guidelines for Dietary Supplements and Botanicals, Appendix K, Part II, AOAC Guidelines for Validation of Botanical Identification Methods, defines a botanical identification method as “a method that establishes identity specifications for a botanical material and determines, within a specified statistical limit, a binary test result: YES, the test material is a true example of the target botanical material and meets the identity specifications, or NO, it is not the target botanical...In most cases, the method will achieve this goal by comparison of the test material with materials from the inclusivity panel and will return a YES/NO (or, in some cases, a consistent/non-consistent) answer”. ,

The validation of the specificity attribute of qualitative identification methods for botanicals is challenging because it largely depends on the availability of the target species (representing the variability expected from botanical commercial samples) and the nontarget species (i.e., closely related species, confounding materials, adulterants, and substitutes). The lack of specificity of an individual analytical procedure may be compensated for by other supporting analytical procedures. In some cases, the specificity of a particular method can be improved by proposing a semiquantitative parameter to the same analytical method (i.e., adding intensity markers by HPTLC or the area or content ratio between relevant peaks by HPLC). In other cases, a second complementary or orthogonal test must be proposed.

The recent U.S. Food and Drug Administration (FDA) Botanical Drug Guidance also emphasized the need to evaluate the current and emerging technologies and develop orthogonal analytical methods to provide adequate identification and quantification of the active or chemical constituents in a botanical drug. The Guidance notes that “When the active constituents are not known and the botanical mixture cannot be fully characterized, the applicant may then select a characteristic profile of chemical constituents (which shows sensitivity to changes in the quality of the raw material(s) and/or manufacturing conditions for drug substance and product) for identity testing”. This again reinforces that specificity and sensitivity are important validation attributes of robust identification methods for botanicals, as they define the ability of a method to capture the expected variability of the target samples.

Identification Tests in USP Botanical Monographs

USP monographs provide analytical methods for identity relative to a standard common name as well as specifications for purity, strength, and composition and limits on contaminants. For example, USP monographs for the three commonly used species of ginseng, American Ginseng Root and Rhizome, Asian Ginseng Root and Rhizome, and Tienchi Ginseng Root and Rhizome (Panax notoginseng (Burkill) F.H. Chen ex C.Y. Wu & K.M. Feng), include macroscopic and microscopic characterization, as well as HPTLC and HPLC methods based on the differential presence and relative abundance of the ginsenosides.

The USP–NF General Chapter <563> Identification of Articles of Botanical Origin describes the different approaches that can be used to define the identity of botanical articles: (1) Botanical identification, including Diagnostic Plant Morphology and Anatomy, and Histology, (2) Chemical identification, and (3) DNA identification. Macroscopic and microscopic analyses are considered orthogonal to chemical and DNA analysis. In addition, the combination of several chromatographic techniques for chemical fingerprinting provides complementary identity information. As an example, the application of macroscopic, HPTLC, and UHPLC identification tests for ginseng according to the corresponding monographs is illustrated in Figures –. Botanical drug, dietary supplement, and herbal medicine monographs for raw materials in the USP–NF typically include identification tests for botanical characteristics (i.e., macroscopic and microscopic analysis) and chemical fingerprinting. In the case of monographs for botanical extracts, chemical fingerprinting using both HPLC and HPTLC techniques is usually proposed. The combination of chromatographic analysis allows the detection of characteristic profiles of different families of secondary metabolites, which may serve to distinguish potential confounding materials. The constituents to be tested may be plant-specific markers, or the pattern of constituents may be plant-specific. Bioactive marker constituents are those suspected to have pharmacological activity contributing to some extent to efficacy such as anthraquinone glucosides (calculated as sennosides) in Senna Leaf and Senna Pods (Senna alexandrina Mill., Fabaceae). However, when the constituents responsible for clinical efficacy are not known, the characteristic marker constituents are used to characterize the materials. Plant-specific analytical marker compounds are those that may not have a relevant pharmacological activity but aid in the positive identification due to their presence as secondary metabolites characteristic of the article, such as parthenolide in Feverfew (dried leaves of Tanacetum parthenium (L.) Sch. Bip. (Asteraceae), collected when the plant is in flower).

HPLC and HPTLC procedures used in identification tests must have sufficient specificity individually or in combination to distinguish potential confounding materials. In the case of HPTLC, using different derivatization conditions and detection wavelengths enables multiprofiling identification patterns covering a wide range of analytes. Peak profiles from images and densitometry also allow the quantitative estimation of specific bands. In the case of HPLC, the relative abundance or intensity of characteristic peaks proposed as area ratios or content ratios is another important parameter in describing the acceptance criteria. Ginseng (Panax) species is a typical example in which many of the constituents are the same, but the ratios of the constituents are significantly different. For example, the peak intensities of ginsenosides Rd and Rb₂ are used in the Asian Ginseng Root and Rhizome monograph to distinguish Panax ginseng root and rhizome from the aerial parts, while the presence/absence and ratios of other ginsenosides are used to distinguish P. ginseng root and rhizome from those of P. notoginseng and P. quinquefolius. Identification tests proposed in USP monographs are validated by using a sufficient number of authentic samples from different sources (e.g., representative of the impact of genetics, environment, management and processing, geographical regions, harvest years, management practices, or ingredients elaborated using different manufacturing processes in the case of derived botanical ingredients) of the plant material or article represented by the monograph specifications as well as samples of the potential confounding materials and adulterants. Suitable reference standard materials are used to check the system suitability and confirm the identification of marker compounds (please see section: Use of Reference Standards for Establishing Identity).

Need for Orthogonal Tests

cGMPs require dietary supplement manufacturers to “conduct at least one appropriate test or examination to verify the identity of any component that is a dietary ingredient”. Regulations note that a single test may be adequate in some cases, but in other cases, additional tests may be necessary: “It is the responsibility of the manufacturer to determine the appropriate test(s) or examination(s) necessary to verify the identity of a dietary ingredient”.

In many cases, choosing only one of the tests or examinations outlined in the regulation will be insufficient to verify the identity, especially of highly processed ingredients. For example, a ginkgo leaf may be identified using morphological examination, while identification of a highly processed ginkgo extract would necessitate multiple tests for markers that are unique for ginkgo.

The use of additional orthogonal test methods can provide greater assurance than a single test about the identity of an article if a single test lacks sufficient specificity. While orthogonal tests offer greater combined accuracy, each method must be accurately characterized with respect to its sensitivity. Given the complex composition of botanical ingredients, a test that measures one set of parameters may lack the specificity to discriminate and not be sufficient to exclude false positive outcomes in establishing the identity. This measurement uncertainty with one test may be compensated by including another test that measures a different characteristic (orthogonal) of the analyte. For example, if two tests that measure different parameters each have only 70% certainty of establishing the identity of an ingredient (i.e., failing 3 out of 10 times in discriminating closely related species), the probability of uncertainty with each test is 0.3. However, when tested using both methods, the confidence level rises by reducing the uncertainty of the combined methods to 0.09 (0.3 × 0.3), or 91% certainty, with the combined tests since both tests would need to fail to provide a false result. In other words, the degree to which the result of a measurement, calculation, or specification can be depended on to be accurate increases by using orthogonal testing. The use of a third orthogonal method with similar uncertainty will further reduce the uncertainty of the combined methods to 0.027 (0.3 × 0.3 × 0.3) or 97% certainty of a correct identification, with the combined tests.

Orthogonal methods target the quantitative evaluation of the true value of a product attribute to address an unknown bias or interference. Complementary measurements include a broader scope of methods that reinforce each other to support a common decision. Methods with low specificity [defined as the ability to identify nonauthentic samples as not authentic (true negative rate)] or low sensitivity [defined as the ability to identify authentic samples as authentic (true positive rate)] benefit from orthogonal designs by ensuring a higher reliability of correctly identifying the target sample.

As an example for the limitation of a single test, adulteration of ginkgo leaf extract is known to occur with less expensive flavonol glycosides and/or aglycones to achieve the desired 24% flavonol glycoside content or by using other parts of the plant (root bark) to achieve the standard of 6% of ginkgo terpene lactones. To identify the ingredient and discriminate from adulterants, USP RS for Powdered Ginkgo Extract includes orthogonal test methods for identification by two complementary methods (HPTLC and HPLC). These methods compare the chromatographic patterns of the sample with those of the RS and define the acceptance criteria in terms of the ratio and relative abundance of flavonol glycosides and ginkgo terpene lactones.

Use of orthogonal methods becomes important if the identifying characteristics of a botanical are lost due to processing or if the test methods have limitations in identifying the target sample with specificity. For example, organoleptic testing using the combined sensory perceptions of characteristic smell, taste, color, and feel may be sufficient and provide a rapid and inexpensive option in some cases if the identifying morphological features are intact, for example, in the case of ginger or garlic, versus when they are powdered and extracted. As is well-established by tasting panels in the wine and coffee industries, the variability and subjectivity of identifying the appropriate grade of the ingredient by sensory methods can be challenging, and being part of an expert sensory panel requires rigorous training of the assessors and well-established processes to avoid inaccurate outcomes. The FDA noted in one warning letter that color, particle size, pH, and comparison of the certificate of analysis supplied by the ingredient manufacturer are not a sufficient basis for identification because these tests were not established as scientifically valid methods to identify an ingredient nor discriminate it from other ingredients.

The use of DNA-based methods as the sole approach for detecting adulteration in botanical dietary supplements, including products labeled as containing ginkgo leaf, St. John’s wort (Hypericum perforatum L., Hypericaceae) flowering tops, echinacea (E. purpurea L., Asteraceae) aerial parts and root, and other medicinal species, has recently reignited the debate over the need for complementary analytical techniques in botanical identification. Although DNA-based methods are highly sensitive, they also present important limitations. , False negatives may occur when DNA is degraded or removed during processing steps, such as solvent extraction, heat treatment, or purification processes intended to concentrate bioactive compounds. In these cases, the DNA may no longer be recoverable, even though the formulation still contains the intended levels of bioactive constituents. Negative results may also be caused by inefficient DNA extraction, flawed method design, or from the sample matrix that interferes the recovery of amplifiable DNA. On the other hand, false positives can result from trace amounts of organic matter naturally present in plant materials at acceptable levels (NMT 2%), laboratory contamination, or unintended detection of closely related species due to poorly developed genomic assays. The high sensitivity of DNA methods may also lead to misleading results when small quantities of DNA-rich material from the labeled plant are intentionally added, even in the absence or low level of the desired bioactive components. Furthermore, DNA analysis does not typically provide information about the plant part(s) used.

Therefore, it is essential to use appropriate reference materials to confirm system suitability, include robust negative controls to eliminate the risk of contamination, and apply validated analytical methods. While DNA-based techniques are valuable for identifying plant materials during the early stages of processing, they should not be relied upon exclusively to assess the identity of processed botanicals, such as extracts or finished dietary supplements. These assessments require the integration of additional orthogonal techniques to ensure accuracy and reliability.

Use of Reference Standards for Establishing Identity

The use of RSs is an integral part of assuring the identity, purity, quality, strength, and composition of a dietary supplement or dietary ingredient. For establishing the identity of a botanical ingredient, qualitative RSs may be used in identification tests, finding applications in system suitability tests, and chromatographic fingerprinting for the detection of marker compounds. RSs with established nominal properties help in determining the pass/fail criteria. However, it must be remembered that some of the RSs from metrological institutes are established for method validation or verification of analytical accuracy and not as identity standards.

The preamble to the dietary supplement cGMPs reiterated the distinction between “two general types of reference materials: (1) Compendial RSs that do not require characterization and (2) noncompendial standards that should be of the highest purity that can be obtained by reasonable effort and that should be thoroughly characterized to ensure their identity, purity, quality, and strength”. The preamble recommended the use of compendial RSs whenever possible and to establish appropriately characterized in-house materials prepared from representative lots if no compendial reference standard exists. §111.315­(d) requires the laboratory control processes to be established and followed to include the use of criteria for selecting standard reference materials used in performing tests and examinations. The FDA had warned manufacturers about the comparison of test results with a previous lot received from a supplier without written acceptance criteria established for the passing identification test and about the use of noncompendial reference materials that are not thoroughly characterized to be acceptable as an RS using appropriate testing methodologies. ,

USP RSs are highly characterized specimens reflective of specified ingredients, including dietary supplements and herbal medicines. USP RSs for botanical monographs are offered in the form of powdered plant material, extracts, fractions, and purified individual compounds. They are fit for purpose for the applications described in the tests and procedures included in the corresponding documentary standards. Characterization of USP RSs is performed using multiple methods, including high-resolution mass spectrometry and nuclear magnetic resonance techniques, by three independent laboratories.

USP Botanical Extract RSs represent all phytochemicals or marker compounds required for the identification tests of the article of commerce described in both the plant material and botanical extract monographs. The stability of the phytochemicals in these extracts is confirmed through stability studies to ensure suitability for continued use across the different compendial applications. The selection of candidate materials for developing USP Botanical Extract RSs is based on important considerations, including sourcing from authentic USP-grade botanical raw material, extraction conditions, manufacturing technology, and further purification steps. The fitness for purpose of the candidate material is demonstrated during the method validation phase by allowing confirmation of both the specificity and sensitivity of the identity method under consideration for each of the ingredients derived from the same plant across a family of monographs. Also essential to developing fit-for-purpose USP Botanical Extract RSs is the demonstration of its dual application in system suitability tests to ensure the appropriate resolution between critical pairs of compounds and to confirm chromatographic similarity in comparison with the chromatogram provided in the certificate of analysis of the USP RS. Considering that many phytochemical compounds are ubiquitous in nature, the use of marker compounds alone might only provide partial identification of the botanical ingredient under consideration. In this way, the use of USP Botanical Extract RSs provides a more complete assessment by identifying the loci of the marker compounds and defining the chromatographic fingerprints for both area/intensity and retention time. Sections of the chromatogram with no significant signals are also important and can be described as displaying no peaks or peaks with less intensity compared with one of the lowest intensity markers consistently observed in the recognizable pattern. This is especially important when working with botanicals with complex chromatographic fingerprints. Another way to assess complex materials is to develop multiprofiling RS, which provides multiple dimensions that complement the identity verification test. The USP Cranberry Fruit Juice Dry Extract RS is a recent example that includes chromatographic fingerprinting at three different wavelengths for the detection of anthocyanins, flavonol glycosides, and phenolic compounds. This RS is fit for the identification of characteristic cranberry polyphenols and the detection of potential adulterants. Similar development was recently completed for the USP European Elder berry Dry Extract RS (Sambucus nigra L.), which includes chromatographic fingerprinting at two different wavelengths (535 and 365 nm) for the identification of anthocyanins, and for flavonol glycosides and hydroxycinnamic acids, respectively.

The use of USP Botanical Extract RS in Identification tests is also supported by USP RS in the form of purified compounds. These USP purified compound RS could be developed for qualitative use or both qualitative and quantitative use. These RS are important not only to confirm the presence of characteristic marker compounds but also to ensure system suitability requirements, in particular chromatography peak tailing factor and system precision of chromatographic systems.

Conclusions

Appropriate tests and RS for identity help ensure that consumers receive the ingredients that they expect. Science-based public standards are available for most commonly used botanicals; these standards help establish specifications for the identity.

Manufacturers of dietary supplement products may develop appropriate analytical methods for identity testing of an incoming ingredient or choose to use the validated methods and RS provided by public standard-setting bodies, such as the USP.

Ensuring the proper identity of botanical materials is fundamental to safeguarding product quality, consumer trust, regulatory compliance, and reproducibility of clinical research. As emphasized in GACPs and cGMPs, identity verification is a minimal yet critical requirement that underpins broader quality assurance measures. The inherent complexity of botanical ingredientsdriven by factors such as natural variability, processing methods into different types of ingredients, and supply chain globalizationnecessitates the use of fit-for-purpose and specific analytical methods and RS, tailored to the unique characteristics of each matrix. Building on that, employing orthogonal and complementary methods mitigates the limitations of any single test and increases the confidence in identity determinations.

Beyond analytical methods, clear and consistent nomenclature and labeling practices are also essential components of botanical identity, supporting transparency across commercial and regulatory landscapes. This work further highlights the importance of using validated methods and appropriately characterized RS to address challenges in testing outcomes with scientific justification. Despite the existence of diverse advanced analytical methods for chemical fingerprinting, method validation along with reference standard selection, which are critical aspects to determine the acceptance criteria and scope of identity methods, remains a difficult task for the industry.

Ultimately, achieving botanical quality and integrity goes beyond identityencompassing composition, purity, potency, and safetyand requires a holistic, science-driven approach. The alignment of compendial standards, regulatory expectations, and scientific innovation offers a path forward to ensuring botanical products consistently meet their intended specifications and fulfill their roles in healthcare and commerce. Ongoing development of robust, validated, and complementary analytical techniques, alongside transparent and standardized nomenclature systems, will be critical to strengthening the reliability and reproducibility of botanical ingredients and products worldwide.

The authors declare no competing financial interest.