NMR-Based Quantum Mechanical Analysis Builds Trust and Orthogonality in Structural Analysis: The Case of a Bisdesmosidic Triglycoside as Withania somnifera Aerial Parts Marker.

The present study demonstrates the relationship between conventional and quantum mechanical (QM) NMR spectroscopic analyses, shown here to assist in building a convincingly orthogonal platform for the solution and documentation of demanding structures. Kaempferol-3-O-robinoside-7-O-glucoside, a bisdesmosidic flavonol triglycoside and botanical marker for the aerial parts of Withania somnifera, served as an exemplary case. As demonstrated, QM-based 1H iterative full spin analysis (HiFSA) advances the understanding of both individual nuclear resonance spin patterns and the entire 1H NMR spectrum of a molecule and establishes structurally determinant, numerical HiFSA profiles. The combination of HiFSA with regular 1D 1H NMR spectra allows for simplified yet specific identification tests via comparison of high-quality experimental with QM-calculated spectra. HiFSA accounts for all features encountered in 1H NMR spectra: nonlinear high-order effects, complex multiplets, and their usually overlapped signals. As HiFSA replicates spectrum patterns from field-independent parameters with high accuracy, this methodology can be ported to low-field NMR instruments (40-100 MHz). With its reliance on experimental NMR evidence, the QM approach builds up confidence in structural characterization and potentially reduces identity analyses to simple 1D 1H NMR experiments. This approach may lead to efficient implementation of conclusive identification tests in pharmacopeial and regulatory analyses: from simple organics to complex natural products.

Cannabis Inflorescence for Medical Purposes: USP Considerations for Quality Attributes.

There is an active and growing interest in cannabis female inflorescence (Cannabis sativa) for medical purposes. Therefore, a definition of its quality attributes can help mitigate public health risks associated with contaminated, substandard, or adulterated products and support sound and reproducible basic and clinical research. As cannabis is a heterogeneous matrix that can contain a complex secondary metabolome with an uneven distribution of constituents, ensuring its quality requires appropriate sampling procedures and a suite of tests, analytical procedures, and acceptance criteria to define the identity, content of constituents (e.g., cannabinoids), and limits on contaminants. As an independent science-based public health organization, United States Pharmacopeia (USP) has formed a Cannabis Expert Panel, which has evaluated specifications necessary to define key cannabis quality attributes. The consensus within the expert panel was that these specifications should differentiate between cannabis chemotypes. Based on the secondary metabolite profiles, the expert panel has suggested adoption of three broad categories of cannabis. These three main chemotypes have been identified as useful for labeling based on the following cannabinoid constituents: (1) tetrahydrocannabinol (THC)-dominant chemotype; (2) intermediate chemotype with both THC and cannabidiol (CBD); and (3) CBD-dominant chemotype. Cannabis plants in each of these chemotypes may be further subcategorized based on the content of other cannabinoids and/or mono- and sesquiterpene profiles. Morphological and chromatographic tests are presented for the identification and quantitative determination of critical constituents. Limits for contaminants including pesticide residues, microbial levels, mycotoxins, and elemental contaminants are presented based on toxicological considerations and aligned with the existing USP procedures for general tests and assays. The principles outlined in this review should be able to be used as the basis of public quality specifications for cannabis inflorescence, which are needed for public health protection and to facilitate scientific research on cannabis safety and therapeutic potential.

The Importance of Quality Specifications in Safety Assessments of Amino Acids: The Cases of l-Tryptophan and l-Citrulline.

The increasing consumption of amino acids from a wide variety of sources, including dietary supplements, natural health products, medical foods, infant formulas, athletic and work-out products, herbal medicines, and other national and international categories of nutritional and functional food products, increases the exposure to amino acids to amounts far beyond those normally obtained from the diet, thereby necessitating appropriate and robust safety assessments of these ingredients. Safety assessments of amino acids, similar to all food constituents, largely rely on the establishment of an upper limit [Tolerable Upper Intake Level (UL)] considered to be a guide for avoiding high intake, above which adverse or toxic effects might occur. However, reliable ULs have been difficult or impossible to define for amino acids because of inadequate toxicity studies in animals and scarce or missing clinical data, as well as a paucity or absence of adverse event reporting data. This review examines 2 amino acids that have been associated with in-market adverse events to show how quality specifications might have helped prevent the adverse clinical outcomes. We further highlight the importance of various factors that should be incorporated into an overall safety assessment of these and other amino acids. In addition to the traditional reliance on the established UL, well-defined quality specifications, review of synthesis and production strategies, potential interactions with drugs, contraindications with certain disease states, and cautionary use within certain age groups should all be taken into consideration.

Use of qNMR to determine HPLC relative response factors for botanical reference standards used in pharmacopeial monographs.

In modern botanical pharmacopeial monographs, one measurement of content is the quantitation of relevant constituents as marker compounds. The use of suitable reference standards (RSs) to quantify multiple compounds by HPLC is recommended in the U.S. Pharmacopeial (USP) botanical monographs. However, these substances may be expensive and difficult to develop into an RS. Surrogate RSs could be used instead of the actual constituents, provided that the relative response factors (RRFs) of each analyte to the selected surrogate RS are known. USP monographs of both Sichuan Lovage Rhizome and Dong Quai Root recognize Z-ligustilide as a major characteristic marker compound, making quantitation of Z-ligustilide and its analog(s) relevant for quality control. However, because Z-ligustilide is unstable, it is difficult to develop it into a quantitative RS. Instead, oxybenzone was selected as a surrogate external quantitative RS because of its similar chromatographic behavior to Z-ligustilide, its stability, and affordable cost. The RRF determination of Z-ligustilide to oxybenzone by the conventional HPLC procedure is challenging due to both the instability of the purified Z-ligustilide at ambient temperature and the difficulty of determining its purity. Therefore, a qNMR method was used to overcome these challenges as it enables to directly measure the mass ratio of Z-ligustilide to oxybenzone in the stock solution without the need for weighing and purity information. In the present study, RRF values of 1.01, 0.46, and 0.89 for Z-ligustilide, senkyunolide A, and ferulic acid relative to oxybenzone, respectively, were determined using the qNMR-based methodology.

What Should Clinicians Know About Dietary Supplement Quality?

Increase in dietary supplement use in the United States suggests a great need for clinicians to be aware of the range of supplements' quality parameters. Regulatory requirements exist, but specific quality parameters for each ingredient are not set by regulators. This article considers how clinicians can evaluate dietary supplement product quality, assess manufacturers' adherence to public quality standards, and encourage use of verification and certification programs.

Pharmaceutical analysis by NMR can accommodate strict impurity thresholds: The case of choline.

The ICH guidelines recommend reporting thresholds for regular impurities in drug substances at the level of 0.05% or 0.03% (w/w) depending on the maximum daily intake. Therefore, any instrumental method of analysis applicable to the impurity analysis should be able to detect and quantify the analytes at those levels. This investigation was designed to verify the suitability of 1H NMR spectroscopy for the detection of impurities, as a first step in the process before attempting quantification. In order to minimize demand on equipment, this study employed a 400 MHz instrument for structural confirmation and signal assignments of choline (1) and O-(2-hydroxyethyl)choline (2), a known impurity. The limit of detection (LOD) of 2 in 10 mg of 1 was established as 0.01% on a 400 MHz instrument and 2% on a 60 MHz (benchtop) NMR spectrometer. Thus, impurities for which quantification is required are readily detected at 400 MHz or above. These results are in contrast to the widespread belief that 1H NMR sensitivity is insufficient for pharmaceutical impurity analysis. The choice of solvent was recognized as a critical parameter for 1H NMR LOD analysis. Furthermore, publicly available NMR raw data (HMDB) proved to be valuable for unveiling the otherwise cryptic information hidden in complex signal patterns via 1H NMR iterative Full Spin Analysis. Finally, the study uncovered the less noticed, yet characteristic, 14N-1H coupling in the -N+(CH3)3 groups, adding strong arguments for the Raw NMR Data Initiative. Collectively, the data prove that the analytical capabilities of high-field NMR easily fulfill the ICH requirements for detection of impurity in the presence of an actual substance of interest which makes it a step closer to achieving regulatory standards.

United States pharmacopeia safety evaluation of spirulina.

The Dietary Supplements Information Expert Committee (DSI-EC) of the United States Pharmacopeial Convention (USP) reviews the safety of dietary supplements and dietary supplement ingredients for the purpose of determining whether they should be admitted as quality monographs into the United States Pharmacopeia and National Formulary (USP-NF). The United States Food and Drug Administration (FDA) has enforcement authority to pursue a misbranding action in those instances where a dietary supplement product indicates that it conforms to USP standards but fails to so conform. Recently DSI-EC undertook a safety evaluation of spirulina, a widely used dietary ingredient. DSI-EC reviewed information from human clinical trials, animal studies, and regulatory and pharmacopeial sources and analyzed 31 adverse event reports regarding spirulina to assess potential health concerns. At the conclusion of this review, DSI-EC assigned a Class A safety rating for Spirulina maxima and S. platensis, thereby permitting the admission of quality monographs for these dietary supplement ingredients in USP-NF. DSI-EC continually monitors reports concerning the safety of dietary supplements and dietary supplement ingredients for which USP dietary supplement monographs are developed. The DSI-EC may revisit the safety classification of spirulina as new information on this dietary ingredient becomes available.

An Effective Workflow for Differentiating the Same Genus Herbs of Chrysanthemum morifolium Flower and Chrysanthemum Indicum Flower.

C. morifolium flower and C. indicum flower are two closely related herbal species with similar morphological and microscopic characteristics but are discriminated in edible and medicinal purpose. However, there is no effective approach to distinguish the two herbs. A novel workflow for quickly differentiating C. morifolium flower and C. indicum flower was developed. Firstly, the difference in anti-inflammatory effects for C. morifolium flower and C. indicum flower was characterized using lipopolysaccharide-treated rats. Then HPLC fingerprint analysis for 53 batches of C. morifolium flowers and 33 batches of C. indicum flower was carried out to deep profile the chemical components. The preliminary markers were screened out by OPLS-DA, identified by HPLC-ESI-QTOF-MS, and quantified by the improved SSDMC (single reference standard to determine multiple compounds) approach. Finally, multiple statistical data mining was performed to confirm the markers and a binary logistic regression equation was built to differentiate C. morifolium flower and C. indicum flower successfully. In general, the established workflow was rapid, effective and highly feasible, which would provide a powerful tool for herb identification.

Multi-Criteria Decision Analysis Model for Assessing the Risk from Multi-Ingredient Dietary Supplements (MIDS).

Military personnel use dietary supplements (DS) for performance enhancement, bodybuilding, weight loss, and to maintain health. Adverse events, including cardiovascular (CV) effects, have been reported in military personnel taking supplements. Previous research determined that ingestion of multi-ingredient dietary supplements (MIDS), can lead to signals of safety concerns. Therefore, to assess the safety of MIDS, the Department of Defense via a contractor explored the development of a model-based risk assessment tool. We present a strategy and preliminary novel multi-criteria decision analysis (MCDA)-based tool for assessing the risk of adverse CV effects from MIDS. The tool integrates toxicology and other relevant data available on MIDS; likelihood of exposure, and biologic plausibility that could contribute to specific aspects of risk.Inputs for the model are values of four measures assigned based on the available evidence supplemented with the opinion of experts in toxicology, modeling, risk assessment etc. Measures were weighted based on the experts' assessment of measures' relative importance. Finally, all data for the four measures were integrated to provide a risk potential of 0 (low risk) to 100 (high risk) that defines the relative risk of a MIDS to cause adverse reactions.We conclude that the best available evidence must be supplemented with the opinion of experts in medicine, toxicology and pharmacology. Model-based approaches are useful to inform risk assessment in the absence of data. This MCDA model provides a foundation for refinement and validation of accuracy of the model predictions as new evidence becomes available.

United States Pharmacopeia (USP) comprehensive review of the hepatotoxicity of green tea extracts.

As part of the United States Pharmacopeia's ongoing review of dietary supplement safety data, a new comprehensive systematic review on green tea extracts (GTE) has been completed. GTEs may contain hepatotoxic solvent residues, pesticide residues, pyrrolizidine alkaloids and elemental impurities, but no evidence of their involvement in GTE-induced liver injury was found during this review. GTE catechin profiles vary significantly with manufacturing processes. Animal and human data indicate that repeated oral administration of bolus doses of GTE during fasting significantly increases bioavailability of catechins, specifically EGCG, possibly involving saturation of first-pass elimination mechanisms. Toxicological studies show a hepatocellular pattern of liver injury. Published adverse event case reports associate hepatotoxicity with EGCG intake amounts from 140 mg to ∼1000 mg/day and substantial inter-individual variability in susceptibility, possibly due to genetic factors. Based on these findings, USP included a cautionary labeling requirement in its Powdered Decaffeinated Green Tea Extract monograph that reads as follows: "Do not take on an empty stomach. Take with food. Do not use if you have a liver problem and discontinue use and consult a healthcare practitioner if you develop symptoms of liver trouble, such as abdominal pain, dark urine, or jaundice (yellowing of the skin or eyes)."

United States Pharmacopeia review of the black cohosh case reports of hepatotoxicity.

OBJECTIVE: Black cohosh [Actaea racemosa L., formerly Cimicifuga racemosa (L.) Nutt.] is a botanical used mainly for the management of menopausal symptoms. Recently, regulatory agencies in Australia, Canada, and the European Union have released statements regarding the "potential association" between black cohosh and hepatotoxicity. In response, the Dietary Supplement Information Expert Committee of the US Pharmacopeia's Council of Experts reviewed safety information for black cohosh products. DESIGN: The Expert Committee analyzed information from human clinical case reports, adverse event reports, animal pharmacological and toxicological data, historical use, regulatory status, and contemporaneous extent of use. Reports were obtained from diverse sources, including the European Medicines Agency, Health Canada, the Australian Therapeutic Goods Administration, and the US Food and Drug Administration. Case reports pertaining to liver damage were evaluated according to the Naranjo causality algorithm scale. RESULTS: Thirty nonduplicate reports on use of black cohosh products concerning liver damage were analyzed. All the reports of liver damage were assigned possible causality, and none were probable or certain causality. The clinical pharmacokinetic and animal toxicological information did not reveal unfavorable information about black cohosh. CONCLUSIONS: Based on this safety review, the Dietary Supplement Information Expert Committee determined that black cohosh products should be labeled to include a cautionary statement. This is a change from the Expert Committee's decision of 2002, which required no such statement. With this decision, the US Pharmacopeia's Botanical Expert Committee may develop monographs for black cohosh, and the US Pharmacopeia may offer its verification programs to dietary supplement ingredient and product manufacturers.

Safety of green tea extracts : a systematic review by the US Pharmacopeia.

Green tea [Camellia sinensis (L.) Kuntze] is the fourth most commonly used dietary supplement in the US. Recently, regulatory agencies in France and Spain suspended market authorization of a weight-loss product containing green tea extract because of hepatotoxicity concerns. This was followed by publication of adverse event case reports involving green tea products. In response, the US Pharmacopeia (USP) Dietary Supplement Information Expert Committee (DSI EC) systematically reviewed the safety information for green tea products in order to re-evaluate the current safety class to which these products are assigned. DSI EC searched PubMed (January 1966-June 2007) and EMBASE (January 1988-June 2007) for clinical case reports and animal pharmacological or toxicological information. Reports were also obtained from a diverse range of other sources, including published reviews, the US FDA MedWatch programme, USP's MEDMARX adverse event reporting system, the Australian Therapeutic Goods Administration, the UK Medicines and Healthcare products Regulatory Agency, and Health Canada's Canadian Adverse Drug Reaction Monitoring Program. Case reports pertaining to liver damage were evaluated according to the Naranjo causality algorithm scale. In addition, the Committee analysed information concerning historical use, regulatory status, and current extent of use of green tea products. A total of 216 case reports on green tea products were analysed, including 34 reports concerning liver damage. Twenty-seven reports pertaining to liver damage were categorized as possible causality and seven as probable causality. Clinical pharmacokinetic and animal toxicological information indicated that consumption of green tea concentrated extracts on an empty stomach is more likely to lead to adverse effects than consumption in the fed state. Based on this safety review, the DSI EC determined that when dietary supplement products containing green tea extracts are used and formulated appropriately the Committee is unaware of significant safety issues that would prohibit monograph development, provided a caution statement is included in the labelling section. Following this decision, USP's DSI ECs may develop monographs for green tea extracts, and USP may offer its verification programmes related to that dietary ingredient.

The state of dietary supplement adverse event reporting in the United States.

PURPOSE: The Dietary Supplements Information Expert Committee (DSI-EC; the Committee) of the United States Pharmacopeial Convention (USP) reviews safety profiles of dietary supplements before development of USP-National Formulary (USP-NF) quality monographs. Because the veracity of dietary supplement adverse event reports (DS AERs) directly affects DSI-EC safety reviews, the Committee reviewed the current status of DS AER reporting in the US. METHODS: DSI-EC reviewed PubMed searches, information from the US Food and Drug Administration's (FDA) MedWatch program, the Toxic Exposure Surveillance System (TESS) of the American Association of Poison Control Centers (AAPCC), and reports from US and other agencies. DSI-EC analyzed this information to identify key factors that affect the quality of DS AERs. RESULTS: The overall incidence of DS AERs appears generally to be low. However, the primary reporting portal (FDA MedWatch) receives fewer AERs than do poison control centers (PCCs), and limited coordination exists among national and international surveillance programs for evaluating signals that may indicate potential public health risks. Both inadequate and poor-quality reporting of DS AERs are major limitations of DS safety monitoring in the US. CONCLUSIONS: Based on its assessments, the Committee advances recommendations to improve the quality of reporting, monitoring, and assessing DS AERs. These include (1) enhanced data collection approaches, (2) improved coordination of AER surveillance programs, (3) strengthening of educational programs for public and health care sectors, and (4) conduct of research concerning the safety of DS. If taken, these approaches are expected to improve the health and well-being of DS users.

Challenges and Opportunities for Improving the Safety Assessment of Botanical Dietary Supplements: A United States Pharmacopeia Perspective.

The United States Pharmacopeia (USP) is an independent, nonprofit, science-based organization whose mission is to improve global health through public quality standards for dietary supplements, medicines, and food ingredients.1 Before developing standards for dietary supplement ingredients, the USP performs an "Admission Evaluation" (Figure 1), which includes an assessment to ascertain that an ingredient does not present a serious health risk.2 This article discusses the challenges encountered during the evaluation of botanicals and proposes possible solutions.

Safety and performance benefits of arginine supplements for military personnel: a systematic review.

CONTEXT: Dietary supplements are widely used by military personnel and civilians for promotion of health. OBJECTIVE: The objective of this evidence-based review was to examine whether supplementation with l-arginine, in combination with caffeine and/or creatine, is safe and whether it enhances athletic performance or improves recovery from exhaustion for military personnel. DATA SOURCES: Information from clinical trials and adverse event reports were collected from 17 databases and 5 adverse event report portals. STUDY SELECTION: Studies and reports were included if they evaluated the safety and the putative outcomes of enhanced performance or improved recovery from exhaustion associated with the intake of arginine alone or in combination with caffeine and/or creatine in healthy adults aged 19 to 50 years. DATA EXTRACTION: Information related to population, intervention, comparator, and outcomes was abstracted. Of the 2687 articles screened, 62 articles meeting the inclusion criteria were analyzed. Strength of evidence was assessed in terms of risk of bias, consistency, directness, and precision. RESULTS: Most studies had few participants and suggested risk of bias that could negatively affect the results. l-Arginine supplementation provided little enhancement of athletic performance or improvements in recovery. Short-term supplementation with arginine may result in adverse gastrointestinal and cardiovascular effects. No information about the effects of arginine on the performance of military personnel was available. CONCLUSIONS: The available information does not support the use of l-arginine, either alone or in combination with caffeine, creatine, or both, to enhance athletic performance or improve recovery from exhaustion. Given the information gaps, an evidence-based review to assess the safety or effectiveness of multi-ingredient dietary supplements was not feasible, and therefore the development of a computational model-based approach to predict the safety of multi-ingredient dietary supplements is recommended.

Evidence-based evaluation of potential benefits and safety of beta-alanine supplementation for military personnel.

This Department of Defense-sponsored evidence-based review evaluates the safety and putative outcomes of enhancement of athletic performance or improved recovery from exhaustion in studies involving beta-alanine alone or in combination with other ingredients. Beta-alanine intervention studies and review articles were collected from 13 databases, and safety information was collected from adverse event reporting portals. Due to the lack of systematic studies involving military populations, all the available literature was assessed with a subgroup analysis of studies on athletes to determine if beta-alanine would be suitable for the military. Available literature provided only limited evidence concerning the benefits of beta-alanine use, and a majority of the studies were not designed to address safety. Overall, the strength of evidence in terms of the potential for risk of bias in the quality of the available literature, consistency, directness, and precision did not support the use of beta-alanine by military personnel. The strength of evidence for a causal relation between beta-alanine and paresthesia was moderate.