CMC Process testing

The development of a pharmaceutical product requires a broad spectrum of scientific expertise to lead it through the complex pathway from discovery through characterization of quality, efficacy and safety, which are the hallmarks of a successful drug product. A company must be highly proactive in setting targets for appraising and selecting a compound that has the highest probability of success. In addition, the compound and its therapeutic use must be consistent with the research and marketing goals of the company in order to leverage existing resources and experience. To ensure scientific and commercial success, it is critical to understand the drug development process (Figure 1) and the myriad tasks and milestones that are vital to a comprehensive development plan.

Although the primary purpose of a well-designed pathway is to assure an efficient process for providing new, high quality and effective drugs for patients, it is also essential to effectively maximize the return on investment. In this context, some primary drivers contributing to maximizing return on investment include the cost of development, market price, product life cycle and competition (Table 1). Each step along the path from discovery to commercialization is important. However, if material cannot be manufactured, the drug development process cannot proceed. As a result, an effective chemistry, manufacturing and controls (CMC) process plays an integral role in the success of a therapeutic compound.

CMC Process
The ability to assure, over time, the physical and chemical properties of an active pharmaceutical ingredient, drug product or nutraceutical is critical for regulatory approval and therapeutic success. The CMC process is necessary for an efficient and comprehensive development strategy. The major challenges for the manufacturing and control component of drug development is to assure the chemical and physical properties of the compound and product are monitored at all critical phases of the pathway. This process matches the scientific and analytical tasks to the manufacturing and commercialization strategy (Table 2).

In recent years, the International Conference on Harmon-isation of Technical Requirements for Registration of Pharma-ceuticals for Human Use (ICH) has adopted scientific standards for quality control monitoring. These standards are the basis of most regulatory guidelines, including those published by the FDA. Key steps on the path include pharmaceutical analysis and stability studies that are required to determine and assure the identity, potency and purity of ingredients, as well as those formulated products. Stability testing facilitates the establishment of recommended storage conditions, determination of retest periods and definition of acceptable shelf life. These data play a key role in determining labeling requirements, as well as in the development and monitoring process.

A Continuous Process
Stability testing is performed on drug substances and products at various stages of product development (Table 3). In early stages, accelerated stability testing (at relatively high temperatures and/or humidities) is used as a "worst case" evaluation to determine what types of degradation products may be found after long-term storage. In preformulation studies, interactions between excipients and the drug substance are studied under stress conditions to access compatibility.

The design of a complete stability testing program for a drug or nutraceutical product is based upon an understanding of the behavior, properties and stability of the drug substance or active ingredient and the experience gained from preformulation studies and early clinical formulations. Products are analyzed at specific intervals to evaluate a variety of parameters, such as the identity of the active ingredient, potency, measurement of degradation products, dissolution time, physical properties and appearance. Samples from production lots of approved products are retained for stability testing and for comparison testing in the case of product failure. Testing of retained samples alongside returned samples is key to ascertaining whether the product failure was manufacturing or storage related.

The objective of analytical testing during preclinical evaluation and Phase I clinical development is to evaluate the stability of the investigational formulations used in initial clinical trials, to obtain information needed to develop a final formulation, and to select the most appropriate container and closure (e.g., compatibility studies of potential interactive effects between a drug substance and other components). Information from the experiments listed in Table 3 under Discovery to Phase I is summarized in the Investigational New Drug application (IND) with the initiation of Phase I clinical trials. When the delivery mechanism of the drug is an integral part of creating the therapeutic effect and must be used in the Phase I trials, formulation data, container closure data and corresponding short-term accelerated stability data should be included in the IND prior to Phase I trials.

Analysis studies on formulations should be underway by the end of Phase II and the stability protocol for study of both the drug substance and drug product should be defined. This will help assure that analytical chemistry data generated during Phase III are appropriate for submission. Prior to Phase I, stability of the drug substance and the formulation to be used must be evaluated. Impurities from the manufacturing and degradants that form are quantitated and tracked to ensure safety prior to moving into the Phase I clinical trials and continuity of material used for laboratory safety testing and clinical trials.

Stability testing done during Phase III studies focuses on testing final formulations in the proposed packaging produced at the manufacturing site. It is recommended that the stability protocol is defined prior to the initiation of Phase III studies. In this regard, consideration should be given to establish appropriate linkage between the non-clinical and clinical batches of the drug substance and drug product and those of the primary stability batches in support of the proposed expiration dating period. Factors to be considered include the source, quality and purity of various components of the drug product, manufacturing process and production facility for the drug substance and the drug product, as well as use of same containers. Data obtained on tests done under controlled conditions replicating conditions recommended for long-term storage and slightly elevated temperatures are used to determine a product's shelf life and expiration dates. In some cases, comprehensive stability testing must also be conducted after approval (Table 4).

A Focus on Stability
The stability of a product may be defined as the extent to which a product retains, within specified limits, throughout its period of storage and use, the same properties and characteristics possessed at the time of its packaging. Stability testing provides evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity and light. These studies are designed to determine if a drug substance will remain within specifications during its shelf life if stored under recommended storage conditions.

Stability testing focuses on the chemical (i.e., integrity, potency, degradation) and physical properties (e.g., appearance, hardness, particle size, solubility) of active pharmaceutical ingredients (API) and products (Table 5). In addition, microbiological testing is done to ensure the substance and product maintain their resistance to microbial and bacterial growth. Assuring the physical/chemical properties and effectiveness properties of a pharmaceutical is critical for labeling and marketing purposes. A wide range of testing is used to evaluate and verify the identity, potency and availability of the API in the product (Table 6). Stability testing is done at all phases of the development, production and marketing process for quality control and monitoring purposes. A wide scope of analytical methodologies is used, including high-performance liquid, gas and thin-layer chromatography (HPLC, GC, TLC) as well as IR and LC/mass spectrometry.

Stability testing requires the use of specialized environmental chambers that can simulate long-term storage conditions. The stress conditions in the chambers include heat, humidity and light. These chambers enable researchers to evaluate product stability based on real-time, accelerated and long-term protocols and are available in both walk-in and reach-in styles. Chambers are engineered and qualified to ensure uniform exposure of the stress conditions to all material in the chamber. Early in the development of the drug product, purposeful degradation studies are done as a means to predict possible degradation pathways of an API. This information is used in the validation of stability indicating analytical methods and in pre-formulation studies. Degradation studies include stress conditions such as heat, oxidative, light, acidic conditions, basic conditions and heat/humidity.

Physical-Chemical Properties
The physical-chemical properties of the substance are analyzed to verify the identity/structure of the drug substance or product API. Many of these tests require specialized instrumentation and laboratory expertise. In addition, the organoleptic properties—including appearance, hardness and moisture—are evaluated. For quality control purposes, the potency, availability and microbial quality are monitored. All of these factors are key ingredients in stability evaluations.

Testing to assure that products meet specifications for the presence of degradation and impurities are usually intensive chromatographic separations with detection down to the 0.01% levels. Typically, impurities and degradation products that are 0.1% and above need to be evaluated for identity and chemical structure. The level of the impurities allowed depends on the toxicity of the impurity and the daily dose levels of the drug.

Identity information on the stability of a drug substance under defined storage conditions is an integral part of the systematic approach to stability evaluation. Stress testing helps to determine the intrinsic stability characteristics of a molecule by establishing degradation pathways to identify the likely degradation products and to validate the stability indicating power of the analytical procedures used.

Microbiological
Along with chemical and physical testing, a number of microbiological tests must be performed based on the dosage form. For sterile products, the microbiological tests performed include sterility, bioburden and bacterial endotoxins. These tests must be validated to show that the compendial tests are suitable. For example, to validate sterility, the test for bacteriostasis and fungistasis (BF) is performed at time of set down. The BF test ensures that any BF activity does not adversely affect the reliability of the sterility test. Bioburden requires validation to show that the test article will not adversely affect the growth of positive controls.

Non-sterile products have different testing requirements depending on if preservatives are used. Orally administered suspensions or liquids with a preservative are evaluated for microbial limits, total yeasts and molds and antimicrobial preservative effectiveness test. The variations of container closure systems will determine the frequency of testing during the stability study. For example, the sterility of a formulation in a sealed glass ampule need not be tested after sterility is established. For most container closure systems, microbiological testing is performed initially, at 12 months and annually thereafter. For accelerated conditions, testing is minimally performed at end of the storage time.

Stress Testing
The severe conditions encountered during distribution are covered by stress testing of definitive batches of the drug substance. Stress testing provides data on forced decomposition products and mechanisms. These studies establish the inherent stability characteristics of the molecule (e.g., degradation pathways) and lead to identification of degradation products and support the suitability of the proposed analytical procedures. The detailed nature of the studies will depend on the individual drug substance and type of drug product.

Testing is carried out on a single batch of a drug substance and includes the effects of temperatures in 10°C increments above the accelerated temperature test condition and humidity, where appropriate (e.g., 75 % or greater). In addition, one must evaluate oxidation and photolysis on the drug substance, plus its susceptibility to hydrolysis across a wide range of pH values when in solution or suspension.

Photostability (i.e., light) testing is an integral part of stress testing. Some degradation pathways can be complex and, under forced conditions, decomposition products may be observed that are unlikely to be formed under accelerated or long-term testing. This information is useful in developing and validating suitable analytical methods, but may not be necessary to examine specifically for all degradation products if it has been demonstrated that in practice these are not formed. Information obtained from photostability is key in choosing appropriate container/closure systems.

Dosage Form/Delivery System Requirements
The route of administration and delivery system used are key components to the successful development of new drugs and therapies. In addition, these choices have a significant impact on the scientific and regulatory aspects of a stability protocol. The diversity of testing needed for all dosage forms and delivery systems requires a broad range of expertise and methodologies.

In general, all dosage forms are evaluated for appearance, assay and degradation products. Additional tests (i.e., potency) are needed for specific dosage forms. For example, sterility is needed for sterile products but not for tablets or capsules. In addition, not every test will be performed at each time point.

The evaluation of inhalation powders includes aerodynamic particle size distribution of the emitted dose, microscopic evaluation, microbial limit, moisture content, foreign particulates, content uniformity of the emitted dose and number of medication doses per device that meets content uniformity of the emitted dose (metered dose products). The unique characteristics of metered-dose and dry-powder inhalers can affect the product's efficacy as well as the ability of the product to deliver reproducible doses. These factors must be considered during development with respect to formulation, stability, manufacturing, container and closure system and quality control (Table 7).

Stability data for products supplied in closed-end tubes should support the maximum anticipated use period after the tube seal is punctured, allowing product contact with the cap. Ointments, pastes, gels and creams in large containers, including tubes, should be assayed by sampling at the surface, top, middle and bottom of the container. In addition, tubes should be sampled near the crimp.

Evaluation of ophthalmic or optic products (e.g., creams, ointments, solutions and suspensions) includes sterility, particulate matter and extractables. Evaluation of nonmetered topical aerosols includes appearance, assay, degradation products, pressure, weight loss, net weight dispensed, delivery rate, microbial limits, spray pattern, water content and particle size distribution (for suspensions).

Studies of drugs for injection (i.e., parenterals) include monitoring for appearance, clarity, color, reconstitution time and residual moisture content. The stability of drug for injection products must also be evaluated after reconstitution, according to the recommended labeling. Small volume parenterals (SVPs) are a wide range of injection products (e.g., drugs for injection, drugs for injectable suspension and drugs for injectable emulsion). Large volume parenterals (LVPs) studies include evaluation of product stability following exposure to at least the maximum specified process lethality. Interaction of administration sets and dispensing devices are considered to ensure that absorption and adsorption during dwell time do not occur. In veterinary applications, some LVPs are designed for multiple use. These products are evaluated for stability after opening with part of the content removed. The "in-use" studies last from seven days to four weeks.

The functionality and integrity of parenterals in prefilled syringe delivery systems needs to be evaluated throughout the expiration dating period with regard to factors, such as the applied extrusion force, syringeability, pressure rating and leakage. Continued assurance of sterility for products is by a variety of means, including evaluation of the container and closure integrity.

Specific parameters to be examined for reconstituted drug products include appearance, clarity, color, pH, assay (i.e., potency), preservative, degradation products/aggregates, sterility, pyrogenicity and particulate matter. Studies for drug injectable suspension and drug for injectable suspension also include particle size distribution, redispersibility and rheological properties. The studies for drug injectable emulsion products also include phase separation, viscosity and mean size and distribution of dispersed phase globules.

When a drug product or dilutent is intended for use as an additive to another product, the potential for incompatibility exists. In these cases, the drug product labeled to be added to another (e.g., parenterals, inhalation solutions) should be evaluated for stability and compatibility in the mixture both in upright and inverted/on-the-side orientations. The tests should provide for tests to be conducted at appropriate time points over the intended use period at the recommended storage and use conditions.

Package Extraction and Migration
A widely overlooked factor in pharmaceutical analysis testing is the determination of potential impurities resulting from migration from packaging components. This includes testing for nitrosamine residue testing as well as both quantitative and qualitative techniques for nitrosamines and olefin polymers used in packages and closures. The 1998 draft stability guidance recommends performing extractable studies on the container/closure (C/C) system using sensitive and quantitative methods even if the C/C system meets compendial suitability tests. Concern over extractables/leachables from the C/C system depends on the route of administration and the likelihood of a packaging component-dosage form interaction. For example, routes of adminstration such as inhalation aerosals and injectables are of highest concern, whereas orally administered solid dosage forms are of lower priority.

An Integral Component
Although stability is an integral component of a CMC program, a comprehensive testing regimen includes a broad scope of analytical evaluations. The importance of assuring the physical and chemical properties throughout the development and commercialization of a compound is key to effectively managing resources and costs. The inclusion of a well-designed chemistry, manufacturing and controls process in the development pathway can help alleviate devastating pitfalls and facilitate a cost-effective process.

References

1. U.S. Department of Health and Human Services, "Guidance for Industry: Q1A Stability Testing of New Drug Substances and Products." Food and Drug Administration, August 2001.

2. International Conference on Harmonisation of Technical Requirements for Registration of Pharamceuticals for Human Use (ICH), "Stability Testing of New Drug Substances and Products (ICH Q1A)." ICH, September 1993.

3. Gallanger, Maxine M.; A Comparative Analysis of International Regulations and Guidances presented at PDA Scientific Forum: The Extractables Puzzle, November 2001

Excipient Quality in Pharmaceutical Development

Understanding their functions benefits process control

By Lokesh Bhattacharyya, Ph.D.
US Pharmacopeia

Excipients interact with the actives in the final formulated dosage form and/or provide a matrix that affects the critical quality attributes of the actives, including stability and bioavailability.

Is compliance with compendial monographs sufficient for complete characterization of an excipient, particularly for understanding its processability?

Excipients are the materials (components)—other than the active ingredient(s)—intentionally incorporated into pharmaceutical dosage forms to play specific functional roles. Almost all pharmaceutical dosage forms include excipients. Indeed, in most dosage forms the amounts of one or more excipients are greater than the amounts of the active pharmaceutical ingredients (APIs) present in them. As with APIs, excipients are derived from natural sources, synthesized chemically, or prepared semisynthetically starting from a natural-sourced materials, and range from simple, usually well-characterized, organic or inorganic molecules to highly complex materials that are difficult to fully characterize.

Excipients play a wide variety of functional roles in pharmaceutical dosage forms, including:

• modulating solubility and bioavailability of APIs,

• increasing the stability of active ingredients in dosage forms,

• helping active ingredients maintain preferred polymorphic forms or conformations,

• maintaining the pH and/or osmolarity of liquid formulations,

• acting as antioxidants, emulsifying agents, aerosol propellants, tablet binders, and tablet disintegrants,

• preventing aggregation or dissociation (e.g., of protein and polysaccharide actives),

• modulating immunogenic responses of active ingredients (e.g., adjuvants), and more.

USP 29–NF 24 includes more than 40 functional categories of excipients in pharmaceuticals, and many more may be added over time to meet the needs of new drug delivery systems and biotechnology-derived products.1 The large number of functional categories represents the variety of applications of excipients in both pharmaceutical and biotechnological products.

More than 800 excipients are used currently in marketed pharmaceutical products in the U.S. This number is expected to grow rapidly as new drug delivery technologies are developed to address challenges of drug development such as poor solubility, permeability and bioavailability, along with the growth of the biotechnology industry, including gene and cell therapy products that have different drug delivery requirements compared to traditional small-molecule pharmaceuticals. Thus, there is a significant need to enhance awareness about new excipient development.

Excipient Selection and Use

An excipient is selected and used because it contributes one or more functional attributes to the product characteristics. The excipient interacts with the active in the final formulated dosage form and/or provides a matrix that affects the critical quality attributes of the actives, including stability and bioavailability. It follows logically that the quality of an excipient and its function play critical roles in the effectiveness, safety, potency, purity, and quality of a product. Thus, it is necessary to understand the function of an excipient in order to fully characterize, understand, and control the process as well as the product quality, particularly in the new era of Quality by Design.2 The lack of understanding of the function of an excipient may lead to a situation in which process control—and hence product quality—may be compromised, particularly when the impact of normal variation of the excipient quality on process control has not been established. The need for complete characterization of an excipient and understanding its functional role in the formulated product is far greater when the excipient is used in a more complex product, such as a monoclonal antibody, a vaccine, or a gene therapy/cell therapy product, or in a new/novel drug delivery system such as an inhalation product.

Excipients also influence the safety and effectiveness of drugs depending on the route of administration, so qualitative and quantitative understanding of the excipient’s composition is critically important to the understanding of a dosage form’s bioavailability and bio-equivalence. For orally administered dosage forms, excipients can affect safety and effectiveness outcomes by promoting or delaying gastrointestinal release. The same appears to be true also for certain injections, for which excipients can modify release patterns in much the same way they do for orally administered modified-release dosage forms. For locally acting products—topical applications, products for oral inhalation, nasal administrations, otic products, and ophthalmic dosage forms—excipients are also widely acknowledged to modify the effectiveness outcomes by influencing the pharmacodynamic properties of the actives. Adjuvants, which are excipients required for protein and conjugate vaccines, play a critical role in the immunologic characteristics of vaccines.

Because excipients can affect the safety and effectiveness of dosage forms, manufacturers should understand the functional contributions of the excipients, that is, their “processability.” Unless manufacturers have a good understanding of the processability of the excipients used in their products, it is difficult to see how the manufacturers can reliably demonstrate pharmaceutical equivalence among product(s) synthesized or perhaps formulated differently at different manufacturing sites, using excipients that possibly are sourced from different suppliers or vendors. Such excipients are likely to have been manufactured by different processes, with starting materials whose qualities may be different from and/or sourced differently than those referenced in the original New Drug Application (NDA). Thus, it is likely that the quality of the excipients used by different product manufacturers or at different manufacturing sites of the same product manufacturer may be different, particularly if the manufacturer engages in multisourcing. In the latter case, interchangeability of excipients cannot necessarily be taken for granted. These factors, together with the potential variation in equipment, processing operations, and personnel who may have different backgrounds, training, and levels of expertise, may present a complex multivariate situation that may render very difficult adequate control of the product quality. The variation could range from minor to significant depending upon the function of the excipient used in the product, excipient interaction with the actives(s), and the characteristics of the product, including its route of administration and other factors.

Legal Factors and Release Testing

The US Code of Federal Regulations (CFR) indicates the requirements for excipient (component) release testing in 21 CFR 211.84(6)(d)(2) as follows:

Each component shall be tested for conformity with all appropriate written specifications for purity, strength, and quality. In lieu of such testing by the manufacturer, a report of analysis may be accepted from the supplier of a component, provided that at least one specific identity test is conducted on such component by the manufacturer, and provided that the manufacturer establishes the reliability of the supplier’s analyses through appropriate validation of the supplier’s test results at appropriate intervals.

The second sentence of the CFR requirement above for release testing of excipients is clearly deficient in addressing excipient quality and processibility issues. The concept and the recognition of the need to understand the processability of an excipient and its relation to the quality of a product are relatively recent and require further discussion. In theory, an excipient manufacturer could perform suitable tests to demonstrate appropriate quality and processability of its excipients, provided the company knew the appropriate tests. However, it is not clear how excipient manufacturers would know what the appropriate tests should be, because the tests depend on the nature of the excipient, the characteristics of the active, nature of the product (e.g., solid, liquid, aerosol; therapeutic, prophylactic), the route of administration of the dosage form, and other factors.

In addition, it is conceivable that the same excipient may have different processabilities and functional contributions in different types of dosage forms. For example, the knowledge of the particle size distribution of lactose, a frequently used excipient, is critical for tablets but is unnecessary for an injectable product. Consequently, the tests that are appropriate for one type of product may not be appropriate for another type. For more common types of products (e.g., solid oral, parenteral), one solution to these quandaries is an excipient qualification agreement concluded between excipient vendors and manufacturers according to which vendors agree to make no process changes or other alterations that would affect the specifications of the finished excipient or its impurity profile, and/or to notify the manufacturer of such changes—or some variation of such agreements.

There is general agreement that if there is a pharmacopeial monograph (USP–NF, European Pharmacopoeia, and Japanese Pharmacopoeia) for an excipient, the tests described in the monograph should be performed. This is consistent with the CFR requirements mentioned above because the compendial monographs have written specifications for identity, purity, strength and quality. Therefore, it is critical to examine the roles and limitations of USP–NF (and other pharmacopeial) monographs in excipient testing. USP–NF standards are authoritative, science-based, and are established by a transparent and credible process with established integrity.3

The transparency and credibility of the monographs come from the open review and comment process that takes place when proposed monographs are published in Pharmacopeial Forum, USP’s bimonthly journal of standards development and compendial revision. Anyone interested can provide scientific and regulatory comments regarding the new monographs or revisions to existing monographs published in Pharmacopeial Forum. The quality standards and any public comments are evaluated by the members of the Expert Committees of the USP Council of Experts, who are unpaid volunteers and are recognized experts in their respective fields. Expert Committee members participate in the USP process as individual scientists and not as representatives of their employers or any trade association, thereby providing unbiased, authoritative, and science-based quality standards. Expert Committee members may agree with public comments regarding monographs published in Pharmacopeial Forum and may decide to revise and republish the monographs for further public review and comment. If the monograph is approved, it is published in and becomes official in USP–NF or in the next semiannual Supplement of USP–NF. Therefore, the quality values of monographs for excipients and other materials expressed in USP–NF are indisputable.

However, the question remains if compliance with compendial monographs is sufficient for complete characterization of an excipient, particularly for understanding its processability. This point is critical not only to their functionalities but also to the impurities that may be present in an excipient, which could under certain circumstances affect the quality, safety, and effectiveness of the dosage forms. An example may help clarify the point. A manufacturer changed the vendor of an excipient, and the product showed adverse reactions. However, the materials from both vendors complied with the USP–NF monograph of the excipient. Subsequent investigation showed that the impurity profiles of the excipients from two vendors were different.

Due considerations also need to be given to the concomitant components, i.e., the impurities that are necessary and desired components required to ensure the proper performance of an excipient in a drug formulation. The role and regulation of the concomitant components must be distinguished from other impurities, which are not intended to be present in the finished product, but are present because it is not possible, and often not necessary, in practice to remove them completely through the manufacturing process steps.

The previous example clearly illustrates the need for characterization and qualification of excipients, including evaluation of the processability. A USP–NF monograph, however authoritative and science-based, does not (and cannot) provide any information about the processability of an excipient. Processability depends on the nature of the active and its interaction with the excipient, the manufacturing process, and the route of administration of the dosage form. Neither USP–NF nor any other pharmacopeia has any knowledge of the manufacturing process used by a manufacturer. Furthermore, different manufacturers may use the same excipient to manufacture different dosage forms, so the processability of the excipient may be different.

Thus, the excipient user (product manufacturer) should develop a comprehensive and scientifically sound excipient characterization/qualification program to ensure purity, quality, strength, consistency, suitability, safety, traceability, and processability of the excipient. Although USP–NF monographs ensure purity, quality, strength, consistency, and freedom from bacteria, fungi, mycoplasma, and certain other adventitious agents, they do not ensure the processability and safety of the excipients, and their contributions to the effectiveness of drug products.

At present new excipients are allowed for use either as a part of an NDA process or via adoption of Generally Recognized as Safe (GRAS) status. Excipient standards are set by regional organizations such as USP, EP, and JP. The subtle differences in the requirements of the individual standards have resulted in added challenges for excipient manufacturers. Because the excipient supply chain is global and somewhat less regulated than that for finished pharmaceuticals, the increasing awareness of bioterrorism caused by product tampering has also enhanced the need for guidance for excipient manufacturing and supply chain control.

All these concerns have resulted in a strong need for additional information and guidelines (or guidances) about the development, characterization, and qualification of new excipients and new applications of current excipients. The importance of regulatory guidance for excipients is a concept that has emerged only in recent years. Hence very few standards (or guidances) are available that treat the subject in the manner outlined here. The pharmacopeias (USP, EP, and JP) and International Pharmaceutical Excipient Council (IPEC) have spearheaded some efforts to develop and harmonize the standards, as well as provide guidelines on good manufacturing and distribution practices for excipients.4 However, additional efforts are necessary to develop comprehensive and authoritative standards (or guidances) to promote innovation in the area of excipients, to improve understanding of the importance of excipients, and to forge new avenues for global regulatory review and approval. The pharmaceutical industry is globalizing. With the development of new concepts and new approaches to drugs and drug-delivery technologies, such standards (and guidances) are critical to the development of new excipients, sustaining excipient quality standards, and safe and optimum use of excipients in diverse types of drugs.

Acknowledgment

The author would like to thank Stefan Schuber, Ph.D., director of scientific reports at USP, for his editorial contributions to this paper.

References

1. United States Pharmacopeial Convention. USP 29–NF 24. Rockville, MD: United States Pharmacopeial Convention, Inc.; 2006.

2. Hussain, AS. Engineering a proactive decision system for pharmaceutical quality: integrating science of design, process analytical technology, and quality system. Available at: www.fda.gov/cder/OPS/hussain_1_2005.pdf. Accessed March 29, 2006.

3. Bhattacharyya, L. et al. The value of USP public standards for therapeutic products. Pharm. Res. 2004;21:1725–1731.

Infrared reference spectra: Amoxicillin trihydrate (RS006)

Instrument: Fourier Transform
Phase: Potassium bromide disc

Infrared reference spectra: Amodiaquine hydrochloride (RS005)

Instrument: Fourier Transform
Phase: Potassium bromide disc

Infrared reference spectra: Amitriptyline hydrochloride (RS004)

Instrument: Fourier Transform
Phase: Potassium bromide disc

Infrared reference spectra: Amidotrizoic acid (RS003)

Instrument: Fourier Transform
Phase: Potassium bromide disc

Infrared reference spectra: Allopurinol (RS002)

Instrument: Fourier Transform
Phase: Potassium bromide disc

Infrared reference spectra: Acetazolamide (RS001)

Instrument: Fourier Transform
Phase: Potassium bromide disc

Monographs: Pharmaceutical substances: Diethyltoluamidum - Diethyltoluamide

C₁₂H₁₇NO

Relative molecular mass. 191.3

Chemical name. N,N-Diethyl-m-toluamide; N,N-diethyl-3-methylbenzamide; CAS Reg. No. 134-62-3.

Description. Colourless or faintly yellow liquid.

Solubility. Practically immiscible in water and glycerol R; miscible with ethanol (~750 g/l) TS and ether R.

Category. Insect repellent.

Storage. Diethyltoluamide should be kept in a tightly closed container.

Additional information. CAUTION: Diethyltoluamide is an irritant to eyes and mucous membranes.

Requirements

Diethyltoluamide contains not less than 97.0% and not more than 103.0% of C₁₂H₁₇NO, calculated with reference to the anhydrous substance.

Identity tests

• Either test A alone or tests B, C, and D may be applied.

A. Carry out the examination as described under 1.7 Spectrophotometry in the infrared region. The infrared absorption spectrum is concordant with the spectrum obtained from diethyltoluamide RS or with the reference spectrum of diethyltoluamide.

B. Refractive index,

C. To about 2 ml, add 25 ml of hydrochloric acid (~250 g/l) TS and heat under a reflux condenser for 1 hour. Neutralize the solution with sodium hydroxide (~200 g/l) TS, cool, and extract with three quantities, each of 30 ml, of ether R. (Keep the aqueous layer for test D.) Carefully evaporate the ether layer to dryness on a water-bath, and dissolve the residue in 5ml of sodium nitrite (100 g/l) TS. Allow to stand at 5 °C for 10 minutes, add 10 ml of water, and extract with 20 ml of ether R. Evaporate the ether layer and add to the residue 1.0 g of phenol R. Cool and add about 1 ml of sulfuric acid (~1760 g/l) TS; an intense green solution is produced. Pour the mixture into water; the colour turns to red. Add sodium hydroxide (~80 g/l) TS; the colour changes to green.

D. Acidify the aqueous layer obtained in test C with hydrochloric acid (~70 g/l) TS, extract with two quantities, each of 20 ml of ether R, and carefully evaporate the ether layer. Dry the residue at 60 °C; the melting temperature of the residue is about 108 °C.

Mass density. ρ₂₀ = 0.996-1.002.

Sulfated ash. Not more than 1.0 mg/g.

Water. Determine as described under 2.8 Determination of water by the Karl Fischer method, Method A, using about 0.5 g of the substance; the water content is not more than 5.0 mg/g.

Acidity. Dissolve 10.0 g in 50 ml of neutralized ethanol TS, titrate with sodium hydroxide (0.01 mol/l) VS using phenolphthalein/ethanol TS as indicator; not more than 4.0 ml of sodium hydroxide (0.01 mol/l) VS is required to obtain the midpoint of the indicator (pink).

Assay. Carry out Method A as described under 2.10 Determination of nitrogen, using about 0.3 g, accurately weighed, and 7 ml of nitrogen-free sulfuric acid (~1760 g/l) TS, and proceed with the distillation. Titrate with sulfuric acid (0.05 mol/l) VS using methyl red/ethanol TS as indicator. Repeat the procedure without the Diethyltoluamide being examined and make any necessary corrections.

Each ml of sulfuric acid (0.05 mol/l) VS is equivalent to 19.13mg of C₁₂H₁₇NO.

Monographs: Pharmaceutical substances: Diethylcarbamazini dihydrogenocitras - Diethylcarbamazine dihydrogen citrate

Molecular formula. C₁₀H₂₁N₃O,C₆H₈O₇ or C₁₆H₂₉N₃O₈

Relative molecular mass. 391.4

Graphic formula.

Chemical name. N,N-Diethyl-4-methyl-1-piperazinecarboxamide citrate (1:1); N,N-diethyl-4-methyl-1-piperazinecarboxamide 2-hydroxy-1,2,3-propanetricarboxylate (1:1); CAS Reg. No. 1642-54-2.

Description. A white, crystalline powder; odourless or almost odourless.

Solubility. Very soluble in water; soluble in 35 parts of ethanol (~750 g/l) TS; practically insoluble in ether R.

Category. Filaricide.

Storage. Diethylcarbamazine dihydrogen citrate should be kept in a tightly closed container, protected from light.

Additional information. Diethylcarbamazine dihydrogen citrate is hygroscopic; it has an acid and bitter taste. Even in the absence of light, Diethylcarbamazine dihydrogen citrate is gradually degraded on exposure to a humid atmosphere, the decomposition being faster at higher temperatures.

Requirements

Definition. Diethylcarbamazine dihydrogen citrate contains not less than 98.0% and not more than 101.0% of C₁₀H₂₁N₃O,C₆H₈O₇, calculated with reference to the anhydrous substance.

Identity tests

• Either tests A and D or tests B and C may be applied.

A. Dissolve 0.05 g in 25 ml of water. Add 1 ml of sodium hydroxide (~80 g/l) TS and 4 ml of carbon disulfide R, and shake for 2 minutes. Separate the aqueous layer. Centrifuge the lower layer if necessary, and filter through a dry filter, collecting the filtrate in a small flask provided with a glass stopper. Carry out the examination of the filtered solution using carbon disulfide R as the blank as described under 1.7 Spectrophotometry in the infrared region. The infrared absorption spectrum is concordant with the spectrum obtained from diethylcarbamazine dihydrogen citrate RS treated similarly or with the reference spectrum of diethylcarbamazine base.

B. Dissolve 0.5 g in 10 ml of water, add 10 ml of sodium hydroxide (1 mol/l)VS, and extract with 4 successive quantities, each of 5 ml of chloroform R. Retain the aqueous layer for test C. Wash the combined chloroform extracts with water, filter through a plug of cotton wool, and evaporate the chloroform. Add 1 ml of ethyl iodide R to the residue, and heat gently under a reflux condenser for 5 minutes. Cool, separate the viscous yellow oil, and dissolve it in ethanol (~750 g/l) TS. Add, with continuous stirring, sufficient ether R to precipitate the quaternary ammonium salt, and filter. Dissolve the precipitate in ethanol (~750 g/l) TS, reprecipitate with ether R, and dry at 105°C; melting temperature, about 152°C (1-diethylcarbamoyl-4-methylpiperazine ethiodide).

C. The aqueous layer from test B yields reaction B described under 2.1 General identification tests as characteristic of citrates.

D. Melting temperature, after drying at 80°C, about 137°C.

Heavy metals. Use 1.0 g for the preparation of the test solution as described under 2.2.3 Limit test for heavy metals, Procedure 1; determine the heavy metals content according to Method A; not more than 20 μg/g.

Sulfated ash. Not more than 1.0 mg/g.

Water. Determine as described under 2.8 Determination of water by the Karl Fischer method, Method A, using about 1 g of the substance; the water content is not more than 10 mg/g.

pH value. pH of a 30 mg/ml solution, 3.5-4.5.

N -Methylpiperazine. Carry out the test as described under 1.14.1 Thin-layer chromatography, using silica gel R1 as the coating substance and a mixture of 6 volumes of ethanol (~750 g/l) TS, 3 volumes of glacial acetic acid R and 1 volume of water as the mobile phase. Apply separately to the plate 5 μl of each of 2 solutions in methanol R containing (A) 50 mg of the test substance per ml and (B) 0.050 mg of N-methylpiperazine R per ml. After removing the plate from the chromatographic chamber, allow it to dry in air, spray with a mixture of 3 volumes of platinic chloride (60 g/l) TS, 97 volumes of water and 100 volumes of potassium iodide (60 g/l) TS, and examine the chromatogram in daylight. The spot obtained with solution B is more intense than any spot, corresponding in position and appearance, obtained with solution A.

Assay. Dissolve about 0.35 g, accurately weighed, in 30 ml of glacial acetic acid R1, and titrate with perchloric acid (0.1 mol/l) VS as described under 2.6 Non-aqueous titration, Method A. Each ml of perchloric acid (0.1 mol/l) VS is equivalent to 39.14 mg of C₁₀H₂₁N₃O,C₆H₈O₇.

Monographs: Pharmaceutical substances: Didanosinum - Didanosine

C₁₀H₁₂N₄O₃

Relative molecular mass. 236.2

Chemical name. 9-[(2R,5S)-5-(hydroxymethyl)tetrahydrofuran-2-yl]-1,9-dihydro-6H-purin-6-one; 9-(2,3-dideoxy-β-D-glycero-pentofuranosyl)-1,9-dihydro-6H-purin-6-one; 2',3'-dideoxyinosine (DDI); CAS Reg. No. 69655-05-6.

Description. A white to almost white powder.

Solubility. Sparingly soluble in water; slightly soluble in methanol R and ethanol (95 per cent) R

Category. Antiretroviral (Nucleoside Reverse Transcriptase Inhibitor).

Storage. Didanosine should be kept in a tightly closed container.

Requirements

Didanosine contains not less than 98.5% and not more than 101.0% of C₁₀H₁₂N₄O_3, calculated with reference to the dried substance.

Identity test

• Either tests A and B, or test C may be applied.

A. Carry out test A.1. or, where UV detection is not available, test A.2.

A.1. Carry out the test as described under 1.14.1 Thin-layer chromatography, using silica gel R6 as the coating substance and a mixture of 67 volumes of dichloromethane R, 20 volumes of acetonitrile R, 10 volumes of methanol R and 3 volumes of ammonia (~260 g/l) TS as the mobile phase. Apply separately to the plate 5 μl of each of 2 solutions in methanol containing (A) 5 mg of the test substance per ml and (B) 5 mg of didanosine RS per ml. After removing the plate from the chromatographic chamber, allow it to dry exhaustively in air or in a current of cool air. Examine the chromatogram in ultraviolet light (254 nm).

The principal spot obtained with solution A corresponds in position, appearance, and intensity with that obtained with solution B.

A.2. Carry out the test as described under 1.14.1 Thin-layer chromatography, using silica gel R5 as the coating substance and a mixture of 67 volumes of dichloromethane R, 20 volumes of acetonitrile R, 10 volumes of methanol R and 3 volumes of ammonia (~260 g/l) TS as the mobile phase. Apply separately to the plate 5 μl of each of 2 solutions in methanol containing (A) 5 mg of the test substance per ml and (B) 5 mg of didanosine RS per ml. After removing the plate from the chromatographic chamber, allow it to dry exhaustively in air or in a current of cool air. Spray with vanillin/sulfuric acid TS1. Heat the plate for a few minutes at 120°C. Examine the chromatogram in daylight.

The principal spot obtained with solution A corresponds in position, appearance, and intensity with that obtained with solution B.

B. The absorption spectrum of a 10 μg/ml solution in methanol R, when observed between 210 nm and 300 nm, exhibits one maximum at about 250 nm; the specific absorbance () is between 435 to 485.

C. Carry out the examination as described under 1.7 Spectrophotometry in the infrared region. The infrared absorption spectrum is concordant with the spectrum obtained from didanosine RS or with the reference spectrum of didanosine.

If the spectra are not concordant, use didanosine RS. Dissolve the sample in a small amount of methanol R, evaporate to dryness and carry out the IR spectrum with the residue as mentioned above. Treat didanosine RS in the same way. The infrared absorption spectrum is concordant with the spectrum obtained from didanosine RS.

Specific optical rotation. Use a 10 mg/ml solution and calculate with reference to the dried substance; .

Heavy metals. Use 1.0 g for the preparation of the test solution as described under 2.2.3 Limit test for heavy metals, Procedure 3; determine the heavy metals content according to Method A; not more than 20 μg/g.

Sulfated ash. Not more than 1.0 mg/g.

Loss on drying. Dry for 4 hours at 105°C; it loses not more than 5.0 mg/g.

Related substances

Prepare fresh solutions and perform the tests without delay.

Carry out the test as described under 1.14.4 High-performance liquid chromatography, using a stainless steel column (25cm x 4.6mm), packed with octadecylsilyl base-deactivated silica gel for chromatography R (5μm).

Maintain the column temperature at 20 - 25°C.

The mobile phases for gradient elution consist of a mixture of aqueous phase (Mobile phase A) and methanol (Mobile phase B), using the following conditions:

Mobile phase A: A 0.05 M solution of ammonium acetate R adjusted to pH 8.0 using ammonia (~100 g/l) TS.

Mobile phase B: Methanol R.

Time (min)	Mobile phase A (% v/v)	Mobile phase B (% v/v)
0	92	8
18	92	8
25	70	30
45	70	30
50	92	8
60	92	8

Prepare the following solutions in a mixture of 92 volumes of mobile phase A and 8 volumes of mobile phase B (dissolution solvent).

For solution (1) dissolve 5.0 mg of hypoxanthine R in the dissolution solvent and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml to 20.0 ml with the same solvent. For solution (2) dissolve 5 mg of didanosine for system suitability RS (containing impurities A to F) in the dissolution solvent and dilute to 10 ml with the same solvent. For solution (3) dissolve 25 mg of the test substance in the dissolution solvent and dilute to 50.0 ml with the same solvent. For solution (4) dilute 5.0 ml of solution (3) to 50.0 ml with the dissolution solvent. Then dilute 5.0 ml of this solution to 50.0 ml with the same solvent.

Operate with a flow rate of 1.0 ml per minute. As a detector use an ultraviolet spectrophotometer set at a wavelength of about 254 nm.

Use the chromatogram supplied with didanosine for system suitability RS and the chromatogram obtained with solution (2) to identify the peaks due to impurities A to F.

Inject 20μl of solution (2). The test is not valid unless the resolution factor between the peaks due to impurity (C) (2'-deoxyinosine) and impurity D (3'-deoxyinosine) is greater than 2.5, if necessary reduce the amount of methanol in the mobile phase and adjust the proportion of aqueous phase pH 8.0 accordingly.

Inject separately 20μl of solution (4) in replicate injections in the chromatographic system. The relative standard deviation for peak areas of didanosine in replicate injections of solution (4) is not more than 5.0%.

Inject separately 20μl each of solutions (1) and (3) and 20ml of dissolution solvent in the chromatographic system. Examine the mobile phase chromatogram for any extraneous peaks and disregard the corresponding peaks observed in the chromatogram obtained with solution (3).

In the chromatogram obtained with solution (2), the following peaks are eluted at the following relative retention with reference to didanosine (retention time about 13-15 min): impurity A about 0.3; impurity B about 0.4; impurity C about 0.44; impurity D about 0.48; impurity E about 0.5; impurity F about 0.8; impurity I about 1.4; impurity G about 1.6; impurity H about 2.0.

In the chromatogram obtained with solution (3) the area of any peak corresponding to impurity A (hypoxanthine) is not greater than the area of the principal peak obtained with solution (1) (0.5%). The area of any individual peak corresponding to impurities B, C, D, E, F or G is not greater than 0.2 times the area of the principal peak obtained with solution (4) (0.2%). The area of any other impurity peak is not greater than 0.1 times the area of the principal peak obtained with solution (4) (0.1%). The sum of the areas of all peaks, other than the principal peak, is not greater than the area of the principal peak obtained with solution (4) (1.0%). Disregard any peak with an area less than 0.05 times the area of the principal peak obtained with solution (4) (0.05%).

Assay

Dissolve about 0.200 g, accurately weighed, in 50 ml of glacial acetic acid R1 and titrate with perchloric acid (0.1 mol/l) VS as described under 2.6 Non-aqueous titration; Method A determining the end point potentiometrically.

Each ml of perchloric acid (0.1 mol/l) VS is equivalent to 23.62 mg of C₁₀H₁₂N₄O₃.

Impurities

The following list of known and potential impurities that have been shown to be controlled by the tests in this monograph is given for information.

A. 1,7-dihydro-6H-purin-6-one (hypoxanthine)

B. R1 = R2 = OH, R3 = H

9-β-D-ribofuranosyl-1,9-dihydro-6H-purin-6-one (inosine)

C. R1 = R3 = H, R2 = OH

9-(2-deoxy-β-D-erythro-pentofuranosyl)-1,9-dihydro-6H-purin-6-one (2'-deoxyinosine)

D. R1 = OH, R2 = R3 = H

9-(3-deoxy-β-D-erythro-pentofuranosyl)-1,9-dihydro-6H-purin-6-one (3'-deoxyinosine)

E. R1 + R2 = O, R3 = H

9-(2,3-anhydro-β-D-ribofuranosyl)-1,9-dihydro-6H-purin-6-one (2',3'-anhydroinosine)

F. R = H

9-(2,3-dideoxy-β-D-glycero-pent-2-enofuranosyl]-1,9-dihydro-6H-purin-6-one; (2',3'-didehydro-2',3'-dideoxyinosine)

G. R = OH

9-(2,3-dideoxy-β-D-glycero-pentofuranosyl)-9H-purin-6-amine (2',3'-dideoxyadenosine)

H. R = H

9-(2,3,5-trideoxy-β-D-glycero-pentofuranosyl)-9H-purin-6-amine (2',3',5'-trideoxyadenosine)

I. 9-(2,3-dideoxy-β-D-glycero-pent-2-enofuranosyl)-9H-purin-6-amine (2',3'-dideoxy-2',3'-didehydroadenosine)

J. structure as shown for impurities B to E where R1= R2 = H, R3 = CO-CH₃

9-(5-O-acetyl-2,3-dideoxy-β-D-glycero-pentofuranosyl)-1,9-dihydro-6H-purin-6-one (didanosine acetate)

K. structure as shown for impurity F where R = CO-CH₃

9-(5-O-acetyl-2,3-dideoxy-β-D-glycero-pent-2-enofuranosyl)-1,9-dihydro-6H-purin-6-one (2',3'-didehydrodidanosine acetate)

L.9-[2,3-O-[(1RS)-1-methoxyethylene]-β-D-ribofuranosyl]-1,9-dihydro-6H-purin-6-one (2',3'-O-(1-methoxyethylidene)inosine;("dioxalane")

M. mixture of 9-(3,5-di-O-acetyl-2-bromo-2-deoxy-β-D-arabinofuranosyl)-1,9-dihydro-6H-purin-6-one and 9-(2,5-di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-1,9-dihydro-6H-purin-6-one ("bromoesters")

Monographs: Pharmaceutical substances: Dicoumarolum - Dicoumarol

Molecular formula. C₁₉H₁₂O₆

Relative molecular mass. 336.3

Graphic formula.

Chemical name. 3,3'-Methylenebis[4-hydroxycoumarin]; 3,3'-methylenebis[4-hydroxy-2H-1-benzopyran-2-one]; CAS Reg. No. 66-76-2.

Description. A white or creamy white, crystalline powder; odour, characteristic, faint.

Solubility. Practically insoluble in water, ethanol (~750 g/l) TS and ether R.

Category. Anticoagulant.

Storage. Dicoumarol should be kept in a well-closed container, protected from light.

Requirements

Definition. Dicoumarol contains not less than 98.5% and not more than 101.0% of C₁₉H₁₂O₆, calculated with reference to the dried substance.

Identity tests

• Either test A alone or tests B and C may be applied.

A. Carry out the examination as described under 1.7 Spectrophotometry in the infrared region. The infrared absorption spectrum is concordant with the spectrum obtained from dicoumarol RS or with the reference spectrum of dicoumarol.

B. Fuse 0.2 g with 0.2 g of potassium hydroxide R, cool, stir with 5 ml of water, filter and acidify the filtrate with hydrochloric acid (~250 g/l) TS; a white, crystalline precipitate is obtained (salicylic acid). Retain the filtrate for test C.

C. To 1 ml of the filtrate from test B add 5 ml of water and a mixture of 1 drop of ferric chloride (25 g/l) TS and 2 drops of hydrochloric acid (~70 g/l) TS; a violet colour is produced.

Sulfated ash. Not more than 2.5 mg/g.

Loss on drying. Dry to constant weight at 105°C; it loses not more than 5.0 mg/g.

Acidity. Shake 0.5 g with 10 ml of carbon-dioxide-free water R for 1 minute and filter; titrate the filtrate with sodium hydroxide (0.1 mol/l) VS, methyl red/ethanol TS being used as indicator; not more than 0.1 ml is required to obtain the midpoint of the indicator (orange).

Assay. Dissolve about 0.35 g, accurately weighed, in 40 ml of 1-butylamine R, add 5 drops of azo violet TS and titrate with lithium methoxide (0.1 mol/l) VS to a deep-blue end-point, as described under 2.6 Non-aqueous titration, Method B. Each ml of lithium methoxide (0.1 mol/l) VS is equivalent to 16.82 mg of C₁₉H₁₂O₆.

Monographs: Pharmaceutical substances: Dicloxacillinum natricum - Dicloxacillin sodium

Molecular formula. C₁₉H₁₆Cl₂N₃NaO₅S,H₂O

Relative molecular mass. 510.3

Graphic formula.

Chemical name. Monosodium (2S,5R,6R)-6-[3-(2,6-dichlorophenyl)-5-methyl-4-isoxazolecarboxamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate monohydrate; monosodium [2S-(2α,5α,6β)]-6-[[[3-(2,6-dichlorophenyl)-5-methyl-4-isoxazolyl]carbonyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate monohydrate; monosodium [3-(2,6-dichlorophenyl)-5-methyl-4-isoxazolyl]penicillin monohydrate; CAS Reg. No. 13412-64-1 (monohydrate).

Description. A white or almost white, crystalline powder.

Solubility. Freely soluble in water and methanol R; soluble in ethanol (~750 g/l) TS.

Category. Antibacterial drug.

Storage. Dicloxacillin sodium should be kept in a tightly closed container, protected from light.

Additional information. Even in the absence of light, Dicloxacillin sodium is gradually degraded on exposure to a humid atmosphere, the decomposition being faster at higher temperatures.

Requirements

Definition. Dicloxacillin sodium contains not less than 88.0% of total penicillins calculated as dicloxacillin free acid (C₁₉H₁₇Cl₂N₃O₅S) and with reference to the anhydrous substance.

Identity tests

A. Carry out the examination as described under 1.7 Spectrophotometry in the infrared region. The infrared absorption spectrum is concordant with the spectrum obtained from dicloxacillin sodium RS or with the reference spectrum of dicloxacillin sodium.

B. To 10 mg of paraformaldehyde R dissolved in 1 ml of sulfuric acid (~1760 g/l) TS add about 1 mg of the test substance; a colourless solution is produced. Heat the solution in a water-bath for 2 minutes and cool; the solution remains colourless (distinction from cloxacillin sodium).

C. Ignite 20 mg and dissolve the residue in acetic acid (~60 g/l) TS. The solution yields reaction B described under 2.1 General identification tests as characteristic of sodium.

Specific optical rotation. Use a 10 mg/ml solution, and calculate with reference to the anhydrous substance; .

Water. Determine as described under 2.8 Determination of water by the Karl Fischer method, Method A, using about 0.25 g of the substance; the water content is not less than 30 mg/g and not more than 50 mg/g.

pH value. pH of a 10 mg/ml solution, 4.5-7.5.

Chlorine. Carry out the combustion as described under 2.4 Oxygen flask method, but using 25 mg of the test substance and 10 ml of sodium hydroxide (0.1 mol/l) VS as the absorbing liquid. When the process is complete, transfer the resulting solution to a titration vessel, heat on a water-bath for 30 minutes, cool to room temperature, add 20 ml of nitric acid (~130 g/l) TS, and titrate with silver nitrate (0.01 mol/l) VS, determining the end-point potentiometrically using a silver/silver chloride electrode system. Repeat the operation without the substance being tested. Each ml of silver nitrate (0.01 mol/l) VS is equivalent to 0.3546 mg of Cl. Calculate the total content of chlorine in mg/g and subtract from it the content of free chlorides as determined below; the content of chlorine is between 130 mg/g and 142 mg/g.

Free chlorides. Dissolve about 0.12 g, accurately weighed, in 10 ml of sodium hydroxide (0.1 mol/l) VS, add 20 ml of water, and heat on a water-bath for 30 minutes. Cool to room temperature, add 20 ml of nitric acid (~130 g/l) TS, and titrate with silver nitrate (0.01 mol/l) VS, determining the end-point potentiometrically using a silver/silver chloride electrode system. Repeat the operation without the substance being tested. Each ml of silver nitrate (0.01 mol/l) VS is equivalent to 0.3546 mg of Cl; the content of free chlorides is not more than 5 mg/g.

Assay. Dissolve about 50 mg, accurately weighed, in sufficient water to produce 1000 ml. Transfer two 2.0-ml aliquots of this solution into separate stoppered tubes. To one tube add 10.0 ml of imidazole/mercuric chloride TS, mix, stopper the tube and place in a water-bath at 60 °C for exactly 25 minutes. Cool the tube rapidly to 20 °C (solution A). To the second tube add 10.0 ml of water and mix (solution B).

Without delay measure the absorbance of a 1-cm layer at the maximum at about 343 nm against a solvent cell containing a mixture of 2.0 ml of water and 10.0 ml of imidazole/mercuric chloride TS for solution A and water for solution B.

From the difference between the absorbance of solution A and that of solution B, calculate the amount of C₁₉H₁₆Cl₂N₃NaO₅S in the substance being tested by comparison with dicloxacillin sodium RS, similarly and concurrently examined.

Monographs: Pharmaceutical substances: Diazoxidum - Diazoxide

Molecular formula. C₈H₇ClN₂O₂S

Relative molecular mass. 230.7

Graphic formula.

Chemical name. 7-Chloro-3-methyl-2H-1,2,4-benzothiadiazine 1,1-dioxide; CAS Reg. No. 364-98-7.

Description. A white, or almost white, crystalline powder; odourless.

Solubility. Practically insoluble in water and ether R; freely soluble in dimethylformamide R; slightly soluble in ethanol (~750 g/l) TS.

Category. Antihypertensive.

Storage. Diazoxide should be kept in a well-closed container.

Requirements

Definition. Diazoxide contains not less than 98.0% and not more than 101.0% of C₈H₇ClN₂O₂S, calculated with reference to the dried substance.

Identity tests

A. Carry out the examination as described under 1.7 Spectrophotometry in the infrared region. The infrared absorption spectrum is concordant with the spectrum obtained from diazoxide RS or with the reference spectrum of diazoxide.

B. See the test described below under "Related substances". The principal spot obtained with solution B corresponds in position, appearance, and intensity with that obtained with solution C.

Sulfated ash. Not more than 1.0 mg/g.

Loss on drying. Dry to constant weight at 105°C; it loses not more than 5.0 mg/g.

Related substances. Carry out the test as described under 1.14.1 Thin-layer chromatography, using silica gel R2 as the coating substance and a mixture of 17 volumes of ethyl acetate R, 4 volumes of methanol R, and 3 volumes of ammonia (~260 g/l) TS as the mobile phase. Apply separately to the plate 10 μl of each of 3 solutions in sodium hydroxide (0.1 mol/l) VS containing (A) 15 mg of the test substance per ml, (B) 0.15 mg of the test substance per ml and (C) 0.15 mg of diazoxide RS per ml. After removing the plate from the chromatographic chamber, allow it to dry in air until the odour of ammonia is no longer detectable, and examine the chromatogram in ultraviolet light (254 nm). Any spot obtained with solution A, other than the principal spot, is not more intense than that obtained with solution B.

Assay. Dissolve 0.45 g, accurately weighed, in 100 ml of a mixture of 2 volumes of dimethylformamide R and 1 volume of water, and titrate with sodium hydroxide (0.1 mol/l) VS, determining the end-point potentiometrically. Each ml of sodium hydroxide (0.1 mol/l) VS is equivalent to 23.07 mg of C₈H₇ClN₂O₂S.

Monographs: Pharmaceutical substances: Diazepamum - Diazepam

Molecular formula. C₁₆H₁₃ClN₂O

Relative molecular mass. 284.7

Graphic formula.

Chemical name. 7-Chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one; CAS Reg. No. 439-14-5.

Description. A white or almost white, crystalline powder; odourless or almost odourless.

Solubility. Very slightly soluble in water; soluble in ethanol (~750 g/l) TS.

Category. Tranquillizer.

Storage. Diazepam should be kept in a well-closed container, protected from light.

Requirements

Definition. Diazepam contains not less than 99.0% and not more than 101.0% of C₁₆H₁₃ClN₂O, calculated with reference to the dried substance.

Identity tests

• Either tests A and D or tests B, C and D may be applied.

• For tests B and C use low-actinic glassware and measure within 30 minutes.

A. Carry out the examination as described under 1.7 Spectrophotometry in the infrared region. The infrared absorption spectrum is concordant with the reference spectrum of diazepam.

B. The absorption spectrum of an 8.0 μg/ml solution in hydrochloric acid (0.1 mol/l) VS, when observed between 230 nm and 350 nm, exhibits maxima at about 241 nm and 286 nm; the absorbances of a 1-cm layer at the maximum wavelengths of 241 nm and 286 nm are about 0.80 and 0.38, respectively (preferably use 2-cm cells for the measurements and calculate the absorbances for 1-cm layers).

C. The absorption spectrum of a 0.030 mg/ml solution in hydrochloric acid (0.1 mol/l) VS, when observed between 325 nm and 400 nm, exhibits a maximum at about 362 nm; the absorbance of a 1-cm layer at this wavelength is about 0.44 (preferably use 2-cm cells for the measurement and calculate the absorbance of a 1-cm layer).

D. Carry out the combustion as described under 2.4 Oxygen flask method, using 20 mg of the test substance and 5 ml of sodium hydroxide (~80 g/l) TS as the absorbing liquid. When the process is complete, acidify with sulfuric acid (~100 g/l) TS and boil gently for 2 minutes; the solution yields reaction A, described under 2.1 General identification tests as characteristic of chlorides.

Melting range. 131-135°C.

Heavy metals. Use 1.0 g for the preparation of the test solution as described under 2.2.3 Limit test for heavy metals, Procedure 3; determine the heavy metals content according to Method A; not more than 20 μg/g.

Sulfated ash. Not more than 1.0 mg/g.

Loss on drying. Dry to constant weight at 50°C under reduced pressure (not exceeding 0.6 kPa or about 5 mm of mercury); it loses not more than 5.0 mg/g.

Related substances. Carry out the test in subdued light as described under 1.14.1 Thin-layer chromatography, using silical gel R2 as the coating substance and a mixture of 1 volume of dehydrated ethanol R and 24 volumes of ethyl acetate R as the mobile phase. Apply separately to the plate 10 μl of each of 2 freshly prepared solutions in chloroform R containing (A) 0.20 g of the test substance per ml and (B) 0.10 mg of 5-chloro-2-methylaminobenzophenone RS per ml. After removing the plate from the chromatographic chamber, allow it to dry in air, and examine the chromatogram in ultraviolet light (254 nm). Any spot obtained with solution A, other than the principal spot, is not more intense than that obtained with solution B.

Assay. Dissolve about 0.55 g, accurately weighed, in 30 ml of glacial acetic acid R1, and titrate with perchloric acid (0.1 mol/l) VS, determining the end-point potentiometrically as described under 2.6 Non-aqueous titration, Method A. Each ml of perchloric acid (0.1 mol/l) VS is equivalent to 28.47 mg of C₁₆H₁₃ClN₂O.

Additional requirements for Diazepam for parenteral use

Complies with the monograph for "Parenteral preparations".

Bacterial endotoxins. Carry out the test as described under 3.4 Test for bacterial endotoxins; contains not more than 11.6 IU of endotoxin RS per mg.

Monographs: Pharmaceutical substances: Dexamethasoni natrii phosphas - Dexamethasone sodium phosphate

Molecular formula. C₂₂H₂₈FNa₂O₈P

Relative molecular mass. 516.4

Graphic formula.

Chemical name. 9-Fluoro-11β,17,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione 21-(dihydrogen phosphate) disodium salt; 9-fluoro-11β,17-dihydroxy-16α-methyl-21-(phosphonooxy)pregna-1,4-diene-3,20-dione disodium salt; CAS Reg. No. 2392-39-4.

Description. A white or almost white, crystalline powder; odourless or with a slight odour of ethanol.

Solubility. Freely soluble in water; slightly soluble in ethanol (~750 g/l) TS; practically insoluble in ether R.

Category. Adrenal hormone.

Storage. Dexamethasone sodium phosphate should be kept in a tightly closed container, protected from light.

Additional information. Dexamethasone sodium phosphate is very hygroscopic. Even in the absence of light, it is gradually degraded on exposure to a humid atmosphere, the decomposition being faster at higher temperatures.

Requirements

Definition. Dexamethasone sodium phosphate contains not less than 96.0% and not more than 103.0% of C₂₂H₂₈FNa₂O₈P, calculated with reference to the anhydrous and ethanol-free substance.

Identity tests

A. Carry out the test as described under 1.14.1 Thin-layer chromatography, using silica gel R1 as the coating substance and a freshly prepared mixture of 3 volumes of 1-butanol R, 1 volume of acetic anhydride R, and 1 volume of water as the mobile phase. Apply separately to the plate 2 μl of each of 4 solutions in methanol R containing (A) 2.5 mg of the test substance per ml, (B) 2.5 mg of dexamethasone sodium phosphate RS per ml, (C) a mixture of equal volumes of solutions A and B, and (D) equal volumes of solution A and a solution of 2.5 mg of prednisolone sodium phosphate RS per ml of methanol R. After removing the plate from the chromatographic chamber, allow it to dry in air until the solvents have evaporated, spray it with a mixture of 10 ml of sulfuric acid (~1760 g/l) TS and 90 ml of ethanol (~750 g/l) TS, heat it at 120°C for 10 minutes, allow it to cool, and examine the chromatogram in ultraviolet light (365 nm). The principal spot obtained with solution A corresponds in position, appearance, and intensity with that obtained with solution B. The principal spot obtained with solution C appears as a single compact spot, whereas the chromatogram of solution D shows 2 closely running spots.

B. Place 0.5 ml of chromic acid TS in a small test-tube and heat in a water-bath for 5 minutes; the solution wets the sides of the tube but there is no greasiness. Add about 3 mg of the test substance and again heat in a water-bath for 5 minutes; the solution no longer wets the sides of the tube.

C. Heat carefully 0.04 g with 2 ml of sulfuric acid (~1760 g/l) TS until white fumes are evolved, add drop by drop nitric acid (~1000 g/l) TS until oxidation is complete, and cool. Add 2 ml of water, heat until white fumes are again evolved, cool, add 10 ml of water, and neutralize with ammonia (~100 g/l) TS, using pH-indicator paper R. Keep half of the solution for test D. The remaining solution yields reaction A described under 2.1 General identification tests as characteristic of orthophosphates.

D. The solution prepared in test C yields reaction B described under 2.1 General identification tests as characteristic of sodium.

Specific optical rotation. Use a 10 mg/ml solution and calculate with reference to the anhydrous and ethanol-free substance; .

Clarity of solution. A solution of 0.10 g in 10 ml of carbon-dioxide-free water R is clear.

Water. Determine as described under 2.8 Determination of water by the Karl Fischer method, method A, using about 0.3 g of the substance. The sum of the contents of water and ethanol (described below), both calculated in mg/g, is not more than 160 mg/g.

Ethanol. Carry out the test as described under 1.14.5 Gas chromatography, using 3 solutions in water containing (1) a mixture of 10 μl of 1-propanol R per ml serving as an internal standard and 10 μl of dehydrated ethanol R per ml, (2) 0.10 g of the test substance per ml, and (3) a mixture of 0.10 g of the test substance and 10 μl of the internal standard per ml. It may be necessary to adjust the content of dehydrated ethanol R in solution (1) to produce a peak of similar height to the corresponding peak in the chromatogram obtained with solution (2).

For the procedure use a column 1.5 m long and 4 mm in internal diameter packed with porous polymer beads (particle size 80-100 μm from a commercial source, is suitable). Maintain the column at 135 °C, use nitrogen R as the carrier gas and a flame ionization detector.

Calculate the content of ethanol in mg/g, assuming the weight per ml at 20 °C to be 0.790 g; not more than 80 mg/g.

pH value. pH of a 10 mg/ml solution in carbon-dioxide-free water R, 7.5-10.5.

Free dexamethasone and other related substances. Carry out the test as described under 1.14.1 Thin-layer chromatography, using silica gel R1 as the coating substance and methanol R as the mobile phase. Apply separately to the plate 2 μl of each of 2 solutions in methanol R containing (A) 10 mg of the test substance per ml, and (B) 0.20 mg of dexamethasone RS per ml. After removing the plate from the chromatographic chamber, allow it to dry in air for 5 minutes, spray it with a solution of 3 g of zinc chloride R in 10 ml of methanol R, heat it at about 125°C for 1 hour, and examine the chromatogram in ultraviolet light (365 nm). Any spot obtained with solution A, other than the principal spot, is not more intense than that obtained with solution B.

Assay. Dissolve about 0.2 g, accurately weighed, in sufficient water to produce 200 ml. Dilute 5 ml to 250 ml with water and measure the absorbance of this solution in a 1-cm layer at the maximum at about 241 nm. Calculate the content of C₂₂H₂₈FNa₂O₈P, using the absorptivity value of 29.7 .

Additional requirements for Dexamethasone sodium phosphate for parenteral use

Complies with the monograph for "Parenteral preparations".

Bacterial endotoxins. Carry out the test as described under 3.4 Test for bacterial endotoxins; contains not more than 31.3 IU of endotoxin RS per mg.

Monographs: Pharmaceutical substances: Dexamethasoni acetas - Dexamethasone acetate

Dexamethasone acetate, anhydrous

Dexamethasone acetate monohydrate

Molecular formula. C₂₄H₃₁FO₆ (anhydrous); C₂₄H₃₁FO₆,H₂O (monohydrate).

Relative molecular mass. 434.5 (anhydrous); 452.5 (monohydrate).

Graphic formula.

Chemical name. 9-Fluoro-11β,17,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione 21-acetate; 21-(acetyloxy)-9-fluoro-11β,17-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione; CAS Reg. No. 1177-87-3 (anhydrous).

9-Fluoro-11β,17,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione 21-acetate monohydrate; 21-(acetyloxy)-9-fluoro-11β,17-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione monohydrate; CAS Reg. No. 55812-90-3 (monohydrate).

Description. A white or almost white powder, odourless.

Solubility. Practically insoluble in water; soluble in 40 parts of ethanol (~750 g/l) TS; slightly soluble in ether R.

Category. Adrenoglucocorticoid.

Storage. Dexamethasone acetate should be kept in a tightly closed container, protected from light.

Labelling. The designation on the container of Dexamethasone acetate should state whether the substance is the monohydrate or is in the anhydrous form.

Requirements

Definition. Dexamethasone acetate contains not less than 96.0% and not more than 104.0% of C₂₄H₃₁FO₆, calculated with reference to the dried substance.

Identity tests

• Either tests A, B, C and E, or tests B, C, D and E may be applied.

A. Carry out the examination as described under 1.7 Spectrophotometry in the infrared region. For the anhydrous form the infrared absorption spectrum is concordant with the spectrum obtained from dexamethasone acetate RS or with the reference spectrum of dexamethasone acetate. For the monohydrate the infrared absorption spectrum is concordant with the spectrum obtained from dexamethasone acetate monohydrate RS or with the reference spectrum of dexamethasone acetate monohydrate.

B. Dissolve 22 mg in 20 ml of ethanol (~750 g/l) TS and dilute 2 ml to 20 ml with the same solvent. To 2 ml of this solution placed in a stoppered test-tube add 10 ml of phenylhydrazine/sulfuric acid TS, mix, heat in a water-bath at 60°C for 20 minutes and cool immediately. The absorbance of a 1-cm layer at the maximum at about 423 nm is not less than 0.42 (preferably use 2-cm cells for the measurement and calculate the absorbance of a 1-cm layer).

C. See the test described below under "Related steroids". The principal spots obtained with solutions A and C correspond in position with that obtained with solution B. In addition the appearance and intensity of the principal spot obtained with solution A corresponds with that obtained with solution B.

D. Carry out the combustion as described under 2.4 Oxygen flask method, using 7 mg of the test substance and a mixture of 0.5 ml of sodium hydroxide (0.01 mol/l) VS and 20 ml of water as the absorbing liquid. When the process is complete, add 0.1 ml to a mixture of 0.1 ml of freshly prepared sodium alizarinsulfonate (1 g/l) TS and 0.1 ml of zirconyl nitrate TS; the red colour of the solution changes to clear yellow.

E. Heat 0.05 g with 2 ml of potassium hydroxide/ethanol (0.5 mol/l) VS in a water-bath for 5 minutes. Cool, add 2 ml of sulfuric acid (~700 g/l) TS, and boil gently for 1 minute; ethyl acetate, perceptible by its odour (proceed with caution), is produced.

Specific optical rotation. Use a 10 mg/ml solution in dioxan R;

Sulfated ash. Weigh 0.1 g and use a platinum dish; not more than 5.0 mg/g.

Loss on drying. Dry to constant weight at 100°C under reduced pressure (not exceeding 0.6 kPa or about 5 mm of mercury). For the anhydrous form use about 0.5 g of the substance; it loses not more than 5.0 mg/g. For the monohydrate use about 0.15 g of the substance; it loses not less than 35 mg/g and not more than 45 mg/g.

Related steroids. Carry out the test as described under 1.14.1 Thin-layer chromatography, using silica gel R1 as the coating substance and a mixture of 77 volumes of dichloromethane R, 15 volumes of ether R, 8 volumes of methanol R, and 1.2 volumes of water as the mobile phase. Apply separately to the plate 1 μl of each of 2 solutions in a mixture of 9 volumes of chloroform R and 1 volume of methanol R containing (A) 15 mg of the test substance per ml and (B) 15 mg of dexamethasone acetate RS per ml; also apply to the plate 2 μl of a third solution (C) composed of a mixture of equal volumes of solutions A and B and 1 μl of a fourth solution (D) containing 0.15 mg of the test substance per ml in the same solvent mixture as used for solutions A and B. After removing the plate from the chromatographic chamber, allow it to dry in air until the solvents have evaporated and heat at 105°C for 10 minutes; allow to cool, spray with blue tetrazolium/sodium hydroxide TS, and examine the chromatogram in daylight. Any spot obtained with solution A, other than the principal spot, is not more intense than that obtained with solution D.

Assay

• The solutions must be protected from light throughout the assay.

Dissolve about 20 mg, accurately weighed, in sufficient aldehyde-free ethanol (~750 g/l) TS to produce 100 ml. Dilute 20 ml of this solution with sufficient aldehyde-free ethanol (~750 g/l) TS to produce 100 ml. Transfer 10.0 ml of the diluted solution to a 25-ml volumetric flask, add 2.0 ml of blue tetrazolium/ethanol TS and displace the air with oxygen-free nitrogen R. Immediately add 2.0 ml of tetramethylammonium hydroxide/ethanol TS and again displace the air with oxygen-free nitrogen R. Stopper the flask, mix the contents by gentle swirling and allow to stand for 1 hour in a water-bath at 30°C. Cool rapidly, add sufficient aldehyde-free ethanol (~750 g/l) TS to produce 25 ml, and mix. Measure the absorbance of a 1-cm layer at the maximum at about 525 nm against a solvent cell containing a solution prepared by treating 10 ml of aldehyde-free ethanol (~750 g/l) TS in a similar manner. Calculate the amount of C₂₄H₃₁FO₆ in the substance being tested by comparison with dexamethasone acetate RS, similarly and concurrently examined.

Monographs: Pharmaceutical substances: Dehydroemetini dihydrochloridum - Dehydroemetine dihydrochloride

Molecular formula. C₂₉H₃₈N₂O₄,2HCl

Relative molecular mass. 551.6

Graphic formula.

Chemical name. (±)-2,3-Didehydroemetine dihydrochloride; (±)-2,3-didehydro-6',7',10,11-tetramethoxyemetan dihydrochloride; (±)-(11bR*)-3-ethyl-1,6,7,11b-tetrahydro-9,10-dimethoxy-1-[[(1bS*)-1,2,3,4-tetrahydro-6,7-dimethoxy-1-isoquinolyl]methyl]-4H-benzo[a]quinolizine dihydrochloride; CAS Reg. No. 3317-75-7.

Description. A white to yellowish, crystalline powder; odourless.

Solubility. Sparingly soluble in water; soluble in methanol R.

Category. Antiamoebic drug.

Storage. Dehydroemetine dihydrochloride should be kept in a tightly closed container.

Requirements

Definition. Dehydroemetine dihydrochloride contains not less than 98.0% and not more than 101.0% of C₂₉H₃₈N₂O₄,2HCl, calculated with reference to the dried substance.

Identity tests

A. The absorption spectrum of a 0.040 mg/ml solution in hydrochloric acid (0.1 mol/l) VS, when observed between 240 nm and 350 nm, exhibits a maximum at about 282 nm. The absorbance of a 1-cm layer at this wavelength is about 0.49.

B. Sprinkle a small quantity of the powdered substance on the surface of 1 ml of sulfuric acid (~1760 g/l) TS containing 5 mg of molybdenum trioxide R; a bright green colour is produced.

C. A 0.1 g/ml solution yields reaction B described under 2.1 General identification tests as characteristic of chlorides.

Clarity and colour of solution. A solution of 0.30 g in 10 ml of water is clear and not more intensely coloured than standard colour solution Yw2 when compared as described under 1.11 Colour of liquids.

Sulfated ash. Not more than 1.0 mg/g.

Loss on drying. Dry to constant weight at 105°C; it loses not more than 70 mg/g.

pH value. pH of a 30 mg/ml solution, 3.5-5.0.

Related substances. Carry out the test as described under 1.14.1 Thin-layer chromatography, using silica gel R4 as the coating substance and a mixture of 9 volumes of ethyl acetate R and 1 volume of diethylamine R as the mobile phase. Apply separately to the plate 5 μl of each of 3 solutions in methanol R containing (A) 20 mg of the test substance per ml, (B) 0.10 mg of the test substance per ml, and (C) 0.10 mg of emetine hydrochloride RS per ml. After removing the plate from the chromatographic chamber, allow it to dry in air, spray it with mercuric acetate/acetic acid TS, heat it at 120 °C for 10 minutes, and examine the chromatogram in ultraviolet light (365 nm). Any spot obtained with solution A, other than the principal spot, is not more intense than that obtained with solution B or solution C, as appropriate.

Assay. Dissolve about 0.4 g, accurately weighed, in 75 ml of glacial acetic acid R1, add 10 ml of mercuric acetate/acetic acid TS, and titrate with perchloric acid (0.1 mol/l) VS as described under 2.6 Non-aqueous titration, Method A. Each ml of perchloric acid (0.1 mol/l) VS is equivalent to 27.58 mg of C₂₉H₃₈N₂O₄,2HCl.

Monographs: Pharmaceutical substances: Deferoxamini mesilas - Deferoxamine mesilate

Molecular formula. C₂₅H₄₈N₆O₈,CH₄O₃S

Relative molecular mass, 656.8

Graphic formula.

Chemical name. N-[5-[3-[(5-Aminopentyl)hydroxycarbamoyl]propionamido]pentyl]-3-[[5-(N-hydroxyacetamido)pentyl]carbamoyl]propionohydroxamic acid monomethanesulfonate (salt); N'-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-1,4-dioxobutyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-N-hydroxybutanediamide monomethanesulfonate (salt); CAS Reg. No. 138-14-7.

Other name. Desferrioxamine mesylate.

Description. A white to yellowish white powder; odourless or almost odourless.

Solubility. Soluble in 5 parts of water; soluble in ethanol (~750 g/l) TS; slightly soluble in methanol R; practically insoluble in ether R.

Category. Antidote to iron poisoning.

Storage. Deferoxamine mesilate should be kept in a well-closed container, protected from light, and stored at a temperature not exceeding 4 °C.

Requirements

Definition. Deferoxamine mesilate contains not less than 98.0% and not more than 102.0% of C₂₅H₄₈N₆O₈,CH₄O₃S, calculated with reference to the anhydrous substance.

Manufacture. The production method must be evaluated to determine the potential for formation of alkyl mesilates, which is particularly likely to occur if the reaction medium contains lower alcohols. Where necessary, the production method is validated to demonstrate that alkyl mesilates are not detectable in the final product.

Identity tests

A. Dissolve 5 mg in 5 ml of water, add 2 ml of trisodium orthophosphate (2 g/l) TS, mix, then add 1 ml of sodium 1,2-naphthoquinone-4-sulfonate (5 g/l) TS; a blackish brown colour is produced.

B. The titrated solution obtained in the assay is reddish brown in colour. To 5 ml of the titrated solution add 2 ml of benzyl alcohol R and shake; the colour is extracted. To a further 5 ml of the titrated solution add 2 ml of ether R and shake; the colour is not extracted.

Heavy metals. Use 1.0 g for the preparation of the test solution as described under 2.2.3 Limit test for heavy metals, Procedure 3; determine the heavy metals content according to Method A; not more than 20 μg/g.

Chlorides. Dissolve 0.7 g in a mixture of 2 ml of nitric acid (~130 g/l) TS, and proceed as described under 2.2.1 Limit test for chlorides; the chloride content is not more than 0.35 mg/g.

Sulfates. Dissolve 0.85 g in 40 ml of water, and proceed as described under 2.2.2 Limit test for sulfates; the sulfate content is not more than 0.6 mg/g.

Clarity and colour of solution. A solution of 1.0 g in 10 ml of water is clear; measure the absorbance of the solution in a 1-cm layer at 420 nm; not more than 0.10.

Sulfated ash. Not more than 1.0 mg/g.

Water. Determine as described under 2.8 Determination of water by the Karl Fischer method, Method A, using about 1 g of the substance; the water content is not more than 20 mg/g.

pH value. pH of a 0.10 g/ml solution in carbon-dioxide-free water R, 3.5-6.0.

Assay. Dissolve about 0.3 g, accurately weighed, in 15 ml of water and add 2 ml of sulfuric acid (0.05 mol/l) VS. Titrate slowly with ferric ammonium sulfate (0.1 mol/l) VS, determining the end-point potentiometrically using a platinum electrode and a calomel reference electrode. Each ml of ferric ammonium sulfate (0.1 mol/l) VS is equivalent to 65.68 mg of C₂₅H₄₈N₆O₈,CH₄O₃S. (Keep the titrated solution for identity test B.)