Pharmacopeial Standards for the Quality Control of Botanical Dietary Supplements in the United States.

Botanicals are among the fastest growing segments of the dietary supplement industry in the U.S. The Dietary Supplement Health and Education Act (DSHEA; Public Law 103-417 [Oct. 25, 1994]) provided a regulatory classification for the trade of numerous botanicals and botanically-derived products as dietary supplements. The global supply chain, the adoption of many botanicals that are also recognized as traditional medicines around the world as dietary supplement ingredients, and the differing recognition of the national and international pharmacopeias as sources for voluntary or mandatory quality standards present challenges in ensuring the quality of the ingredients and products. The complexity of quality assurance by compliance with pharmacopeial standards is illustrated in this article with a brief history of pharmacopoeias including their official recognition in national laws, their approaches to the science behind the standards, the use of reference standards for quality assessment and regulatory compliance, the use of pharmacopeial standards by the industry and regulators within the DSHEA framework in the United States, and a discussion of the global supply chain. Pharmacopeial standards can help regulators and the industry adapt to the new technologies that present both opportunities and challenges.

Understanding plant to extract ratios in botanical extracts.

Dietary supplement current good manufacturing practice (cGMP) requires establishment of quality parameters for each component used in the manufacture of a dietary supplement to ensure that specifications for the identity, purity, strength, composition, and limits on contaminants are met. Compliance with botanical extract ingredient specifications is assured by using scientifically valid methods of analysis, the results of which are reported on certificates of analysis (CoAs). However, CoAs routinely include additional data that are not amenable to verification through methods of analysis. Such descriptive information may include Plant to Extract ratios, which are ratios of the quantity of botanical article used in the manufacture of the extract to the quantity of extract obtained. Plant to Extract ratios can be misleading when their meaning is not clearly understood. Plant to Extract ratios do not completely describe botanical extracts because other important factors influence the make-up of final extracts, such as the quality of the raw starting material (as can defined by pharmacopeial standards), extraction solvent(s) used, duration and temperature of extraction, and percentage and type of excipients present. Other important qualitative descriptions may include constituent "fingerprinting." Despite these issues, Plant to Extract ratios are often used as a measure of extract strength for dosage calculations. This article defines and clarifies the meaning of Plant to Extract ratios and their proper use in describing and labeling botanical extract ingredients and finished products containing them.

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.

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.

Development and Validation of a Species-specific PCR Method for the Identification of Ginseng Species Using Orthogonal Approaches.

When testing botanical ingredients of herbal medicines and dietary supplements, the complexity of botanical matrixes often requires the use of orthogonal methods to establish identification procedures suitable for quality control purposes. Genomic-based botanical identification methods are evolving and emerging as useful quality control tools to complement traditional morphological and chemical identification methods. Species-specific polymerase chain reaction methods are being evaluated for botanical quality control and as a cost-effective approach to identify and discriminate between closely related botanical species. This paper describes orthogonal identification of Panax ginseng, P. quinquefolius, and P. notoginseng materials in commerce as an example of the development and validation of a set of species-specific polymerase chain reaction methods to establish botanical identity in ginseng roots. This work also explored the possibility of extending the application of species-specific polymerase chain reaction methods to provide species identity information for processed materials, such as steamed roots and hydroalcoholic extracts, and showed success with this approach. Finally, the paper provides recommendations for an out-of-specification investigation of samples that may pass some of the orthogonal tests and fail others.

The qNMR Summit 5.0: Proceedings and Status of qNMR Technology.

The goal of the qNMR Summit is to take stock of the status quo and the recent developments in qNMR research and applications in a timely and accurate manner. It provides a platform for both advanced and novice qNMR practitioners to receive a well-rounded update and discuss potential qNMR-related applications and collaborations. For over a decade, scientists from academia, industry, nonprofit institutions, and governmental bodies have focused on the standardization of qNMR methodology, as well as its metrological and pharmacopeial utility. This paper reviews key content of qNMR Summits 1.0 to 4.0 and puts into perspective the outcomes and available transcripts of the October 2019 Summit 5.0, with attendees from the United States, Canada, Japan, Korea, and several European countries. Summit presentations focused on qNMR methodology in the pharmaceutical industry, advanced quantitation algorithms, and promising developments.

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.

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.

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.

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.

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)."

NMR reveals an undeclared constituent in custom synthetic peptides.

Whereas generic, LC-based pharmaceutical control quality procedures depend largely on the detection mode and can be particularly 'blind' to certain impurities, NMR is a more versatile and, thus, often more judicious detector. While adulteration presents ever-evolving challenges for the analysis of active pharmaceutical ingredients (APIs) and finished products sold in the worldwide (online) marketplace, research chemicals are usually trusted rather than being considered flawed or even adulterated. This report shows how NMR analysis uncovered the unanticipated presence of substantial amounts of mannitol (20 and 43% w/w) as undeclared constituent in two custom synthetic peptides, DR and DRVYI, that were sourced commercially. Quantitative 1H NMR (qHNMR) readily detected the contaminant, even on a 60 MHz benchtop instrument, and quantified the highly polar and UV-transparent adulterant. Quantum-mechanical 1H iterative Full Spin Analysis (HiFSA) not only achieved unambiguous identification of both the mannitol and the peptides, but also confirmed the quantitative results. The cases show that experimental verification supersedes trust in both pharmaceutical and research QC. They also highlight the promising utility of both established high-field and recently re-evolving low-field benchtop qHNMR. The unanticipated findings remind manufacturers and researchers alike about the advantages of including/performing NMR and qNMR with routine CofA documentation and/or verification of research grade chemicals. Especially when done jointly, this can greatly improve confidence in research and help streamline the pharmaceutical QC toolbox.

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.

Quality specifications for articles of botanical origin from the United States Pharmacopeia.

BACKGROUND: In order to define appropriate quality of botanical dietary supplements, botanical drugs, and herbal medicines, the United States Pharmacopeia (USP) and the Herbal Medicines Compendium (HMC) contain science-based quality standards that include multiple interrelated tests to provide a full quality characterization for each article in terms of its identity, purity, and content. PURPOSE: To provide a comprehensive description of the pharmacopeial tests and requirements for articles of botanical origin in the aforementioned compendia. Selective chromatographic procedures, such as high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC), are used as Identification tests in pharmacopeial monographs to detect species substitution or other confounders. HPLC quantitative tests are typically used to determine the content of key constituents, i.e., the total or individual amount of plant secondary metabolites that are considered bioactive constituents or analytical marker compounds. Purity specifications are typically set to limit the content of contaminants such as toxic elements, pesticides, and fungal toxins. Additional requirements highlight the importance of naming, definition, use of reference materials, and packaging/storage conditions. METHODS: Technical requirements for each section of the monographs were illustrated with specific examples. Tests were performed on authentic samples using pharmacopeial reference standards. The chromatographic analytical procedures were validated to provide characteristic profiles for the identity and/or accurate determination of the content of quality markers. RESULTS: The multiple tests included in each monograph complement each other to provide an appropriate pharmacopeial quality characterization for the botanicals used as herbal medicines and dietary supplements. The monographs provide detailed specifications for identity, content of bioactive constituents or quality markers, and limits of contaminants, adulterants, and potentially toxic substances. Additional requirements such as labeling and packaging further contribute to preserve the quality of these products. CONCLUSION: Compliance with pharmacopeial specifications should be required to ensure the reliability of botanical articles used for health care purposes.

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.

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.

Dietary supplements quality analysis tools from the United States Pharmacopeia.

The United States Food and Drug Administration (FDA) issued the dietary supplement (DS) current good manufacturing practice (GMP) regulations in compliance with the mandate from the Dietary Supplements Health and Education Act (DSHEA), with the intention of protecting public health by ensuring the quality of DS. The GMP regulations require manufacturers to establish their own quality specifications for identity, purity, strength, composition, and absence of contaminants. Numerous FDA-conducted GMP inspections found that the private specifications set by these manufacturers are often insufficient to ensure adequate quality of dietary ingredients and DS. Wider use of the public standards developed by the United States Pharmacopeial Convention (USP), in conjunction with GMP compliance, can help ensure quality and consistency of DS as they do for medicines. Public health protection could be enhanced by strengthening the GMP provisions to require conformance with relevant United States Pharmacopeia-National Formulary (USP-NF) standards, or in the absence of USP standards, other public compendial standards. Another serious concern is the presence of synthetic drugs and drug analogues in products marketed as DS. Use of the new USP General Chapter Adulteration of Dietary Supplements with Drugs and Drug Analogs <2251> may reduce the exposure of consumers to dangerous drugs disguised as DS. Copyright © 2016 John Wiley & Sons, Ltd.

Electrophoretic separation of alginic sodium diester and sodium hexametaphosphate in chondroitin sulfate that interfere with the cetylpyridinium chloride titration assay.

The most commonly used chondroitin sulfate (CS) assay method is cetylpyridinium chloride (CPC) titration. Cellulose acetate membrane electrophoresis (CAME) is the technique used for detection of impurities in the U.S. Pharmacopeia's CS monograph. Because CPC titration is a relatively nonspecific quantitative technique, the apparent amount of CS as determined by CPC titration alone may not reflect the true amount of CS due to possible interference with the CPC assay by impurities that contain CPC titratable functional groups. When CAME is used in conjunction with CPC titration, certain non-CS and adulterants can be visualized and estimated, and a true value for CS can be assigned once the presence of these non-CS impurities has been ruled out. This study examines conjunct application of CPC and CAME in ascertaining CS assay and purity in the presence of certain adulterants. These include propylene glycol alginate sulfate sodium, known in commerce as alginic sodium diester (ASD), and Zero One (Z1), a water-soluble agent newly reported in the CS marketplace and subsequently identified as sodium hexametaphosphate. ASD, Z1, and CS are similar in physical appearance and solubility in water and ethanol. They are also titratable anions and form ionic pairs with CPC, therefore interfering with the CPC titration assay for CS CAME separates these adulterants from each other and from CS by differences in their electrophoretic mobility. CAME is able to detect these impurities in CS at levels as low as 0.66% by weight. Although it is recommended that a method for detecting impurities (e.g., CAME) be used in cormbination with relatively nonspecific assay methods such as CPC titration, this is seldom done in practice. Assay results for CS derived fromn CPC titration may, therefore, be misleading, leaving the CS supply chain vulnerable to adulteration. In this study, the authors investigated ASD and Z1 adulteration of CS and developed an electrophoretic separation of these adulterants in CS and procedures to isolate ASD from CS matrixes containing these adulterants. The authors describe in this paper utilization of an orthogonal approach to establish the identity of Z1 as sodium hexametaphosphate and to confirm the identity of ASD, including ethanol fractionation, FTIR spectroscopy, differential scanning calorimetry, and NMR spectroscopy. The authors suggest that CAME is a cost-effective and easy to use methodfor detecting certain impurities in CS raw ingredients and recommend that CPC and CAME be used in combination by QC laboratories as a means of effectively deterring the practice of adulterating CS raw materials with the known adulterants ASD and Z1 and/or other non-chondroitin substances that can be separated from CSby CAME and that exhibit CPC titration behavior similar to CS.