11 USP REFERENCE STANDARDS
USP Reference Standards are highly characterized specimens of drug substances, excipients, reportable impurities, degradation products, compendial reagents, and performance calibrators.
They are explicitly required in many Pharmacopeial assays and tests and are provided solely for such use. Assessment of the suitability for use in other application(s) rests with the purchaser.
AUTHORITY FOR ESTABLISHMENT AND RELEASE
USP Reference Standards are established and released under the authority of the USPC Board of Trustees upon recommendation of the USP Reference Standards Expert Committee, which approves each lot as being suitable for use in its compendial applications. For some Reference Standards a preliminary review and approval is sought from other Expert Committees of the Council of Experts.
The distribution of controlled substances is subject to the regulations and licensing provisions of the Drug Enforcement Administration of the Department of Justice.
Industry Advisory Panels and other expert groups (such as Project Teams) may be assembled to advise USP on various aspects of the Reference Standards Program.
Future availability of the first USP Reference Standards was announced in 1926 (USP X) in order to facilitate the adoption of the biological assay standards of the Pharmacopoeia, and to provide a greater degree of uniformity in their application. The list of USP Reference Standards that in 1936 comprised 6 items has grown to almost 1650 in 2004, and the collection has tracked the progress in pharmaceutical sciences: The first vitamins (Cod Liver Oil) and the first enzyme (Pepsin) in 1936; the first sulfonamide (Sulfanilamide) and the first hormones (Insulin; Posterior Pituitary) in 1942; the first performance standards (Melting Point Standards) in 1947; the first penicillin (Penicillin G Sodium) in 1950; the first recombinant-DNA technology protein (Insulin Human) in 1985, etc.
The continuous increase in the number of USP Reference Standards (over 100 new standards are being developed yearly) reflects not only the increase in the number of monographs and General Chapters, but also the development and extensive use of modern analytical methodology (such as chromatography, spectrophotometry, biological and biochemical assays, etc.) which require measurements relative to a reference standard.
Standards designated as USP Reference Standards (USP RS) are, with a few exceptions, required for use in USPNF monographs or General Chapters. The exceptions include current lots of USP and NF Reference Standards for which uses are no longer specified in the current USP or NF but for which sufficient demand remains (upon depletion of the current lots, future lots will be designated as Authentic Substances), Reference Standards specified in monographs developed by USP that are not intended for publication in the USPNF, Reference Standards specified in the current edition of the Food Chemicals Codex (labeled with an additional designation FCC), and Fluoride Dentifrices (evaluated and distributed by agreement with the FDA and the Cosmetics, Toiletries, and Fragrances Association). A USP Reference Standard required in a monograph or General Chapter proposed in Pharmacopeial Forum may be released in advance of the official date of the proposed PF revision.
Reference Standards currently labeled as NF Reference Standards will eventually all be designated and labeled as USP Reference Standards pursuant to the consolidation of USP and NF within the USPC as of January 2, 1975. Meanwhile, where a USP Reference Standard is called for, the corresponding substance labeled as an NF Reference Standard may be used.
As a service, the USPC tests and distributes additional Authentic Substances (designated by AS) not currently required for use in a USP monograph or General Chapter. These also are provided under the supervision of the USP Reference Standards Expert Committee. They are highly characterized samples of chemicals, including substances of abuse, which are collaboratively tested and made available as a service primarily to analytical, clinical, pharmaceutical, and research laboratories. Such materials may be used for identification, method development, evaluation of method performance, or other applications as found suitable and validated by the user.
Authentic Visual References
Unlike chemical reference standards, Authentic Visual References (AVRs) are not used in chemical analyses. Instead, the AVRs are visual images used by analysts to compare certain test articles to ensure that they meet compendial requirements and are incorporated by reference into the monograph. Approval of AVRs for use in a monograph is the decision of the Expert Committee that approves the specific monograph.
DIVERSITY AND IMPLICATIONS
The USP Reference Standards collection is very diverse in terms of appearance, chemical structure, composition, and uses. This diversity has significant implications for the way the materials are tested, packaged, stored, and utilized.
The USP Reference Standards may be crystalline or amorphous powders, volatile or viscous liquids, solutions or suspensions, gels or pastes, plastic sheets, etc. In chemical structure they vary from simple inorganic salts to proteins produced by recombinant DNA technology. Some are highly-purified single components, while others are complex mixtures (in most cases extracted from plant or animal sources).
USES OF USP REFERENCE STANDARDS
The official and authorized uses of USP Reference Standards are specified in the USP monographs and General Chapters and they include the following:
As discussed under Nomenclature, USP also establishes and distributes standards not specified for use in a USP monograph or General Chapter.
The most frequent applications of USP Reference Standards (USP RS) are in chromatographic and spectroscopic methodologies. However, they are also widely used in biological and biochemical applications, such as microbial assays for antibiotics, enzymatic reactions, cell-culture tests, whole-animal tests; in thermal analysis for polymers; and in titration, etc. Some of the most frequently used USP RS are those utilized in General Chapters tests such as Dissolution 711, Bacterial Endotoxins Test 85, Total Organic Carbon 643, and Particulate Matter in Injections 788.
STEPS IN ESTABLISHING A USP REFERENCE STANDARD
The establishment of a new USP Reference Standard is triggered by the proposal of a new monograph or of a revision of an existing monograph by the inclusion of a test requiring a new USP RS. The need for a new lot of an existing USP Reference Standard is identified when its inventory reaches a pre-established threshold. The new lot is designated as a replacement lot if a new bulk material is to be procured or as a continuation lot if the candidate material is another portion of the bulk used for the existing official lot.
USP scientists generate a set of documents including procurement specifications and a testing protocol. A bulk material is obtained, generally from a major manufacturer of the article. The material is tested and characterized in an inter-laboratory collaborative study organized according to the protocol designed at USP Headquarters. The results are evaluated by USP Staff, additional testing or investigations are performed when necessary, and a report is compiled and presented for review and approval to the USP Reference Standards Expert Committee. After approval the material is subdivided (if not packaged prior to the collaborative study), labeled, quality checks are performed, and the standard is made available for distribution. If a candidate material is found to be unsuitable by USP scientific staff or by the Reference Standards Committee, a new bulk is procured and tested.
COLLABORATIVE STUDY FOR THE EVALUATION OF A USP RS CANDIDATE MATERIAL
The goals of the evaluation study are to confirm the identity and assess the purity of the material, to determine its suitability for use in the official applications, to provide the user with all the necessary information and directions for use, and to acquire time-zero information for future continued-suitability-for-use studies.
USP scientists design a detailed testing protocol that includes the following elements: types of tests, number of tests, number of collaborators, number of replicates, and references to the procedures to be used.
The following factors are considered when designing the study protocol: the compendial status of the standard, its official uses, the history of the standard, its composition and complexity, the characteristics of the methodology, and the availability of material and of competent laboratories.
The testing protocol may comprise visual and microscopic evaluation; identification tests (more elaborate for first-time standards); determination of physicalchemical constants (e.g., melting range, specific rotation, refractive index, specific gravity, etc); chromatographic and electrophoretic purity tests; inorganic contaminants determination; volatile tests (water, solvents); functional group analysis (such as titrations, UV absorptivity, elemental analysis); thermal analysis; and assays against another well-characterized standard (a previous lot, an international standard, etc). Specialized testing is implemented where appropriate, such as for dissolution calibrators and the particle count set, for standards that define an attribute (negative and positive bioreaction, ion-exchange capacity, permeability diameter), and for biological standards that define a Unit of activity (heparin, endotoxin, enzymes, complex antibiotics). Vapor sorption analysis may also be performed to assist in determining packaging and storage conditions, and directions for use. For lyophilized single-use USP RS, acceptable vial content reproducibility and stability of the lyophilized form are demonstrated.
The number of collaborators is generally not less than three (two outside of USP); but it can increase significantly, especially when the methodology is complex or does not have a high level of precision or when potential users express an interest in participating in the evaluation of the candidate. (Participation in all evaluation studies is open to all competent, interested parties.) Where appropriate, statistical control is exercised in the design of the evaluation study and in the analysis of the results. The USP Reference Standards Laboratory and the FDA laboratories participate in almost all evaluation studies. Other collaborators include Health Canada, the USP Research and Development Laboratory, and industrial and academic laboratories from the United States and from abroad.
BIOLOGICAL REFERENCE STANDARDS
The World Health Organization, an agency of the United Nations, manages a program providing International Standards for biological materials.
USP collaborates closely with the WHO in the harmonization of analytical methodology, in the definition of the units of potency, and in some cases to share in the preparation of a reference standard. In many cases the USP Units and the International Units of potency are identical.
SUITABILITY FOR USE AND PURITY ASSIGNMENT
The data collected in the collaborative evaluation study are analyzed to determine whether the material is suitable for its monograph-designated use. Characterization data and results must be considered as a whole when evaluating suitability for intended use, assignment strategy, and assigned value. For Reference Standards used in quantitative applications, this includes the determination of a calculation value to be used in the compendial utilization of the standard.
The method of choice in computing the assigned value is a mass balance analysis using independently determined components such as moisture, solvent residues, inorganic residues, chromatographic impurities, and ion content. The assay results against a previous lot or against another validated standard and the results of the functional group analysis are for confirmatory purposes only. Exceptions to the mass balance approach include many biological Reference Standards, especially those which define the Unit of activity.
The number of significant figures in the labeled calculation value is a function of the use of the standard and the number of significant figures in the acceptance range or limit. Generally, Reference Standards used in assays are labeled with three significant figures and standards used in limit tests with two significant figures. Reference Standards having multiple applications in different methodologies may require separate assay-specific assignments.
The assigned value is labeled without any associated uncertainty. However, for calibration standards, the labeled value is a range, determined by a statistical analysis of the results.
Previous approaches used a purity threshold above which the content was no longer labeled, and the analyst was directed to use a default value of 100.0%. This approach is no longer in use, but older lots of standards have not been re-labeled, and users should continue to apply the default 100.0% value for compendial quantitative applications.
For antibiotics, the designation µg/mg is sometimes used as a unit of biological activity, and values greater than 1000 µg/mg may be assigned to some of these standards. This can happen when the first standard is assigned a value higher than its actual purity and subsequent standards of higher purity are defined relative to the previous lot. A relatively overstated assigned value can also result when less selective separation techniques are replaced with more selective modern methodologies. As a result, the original content might have been assumed to be higher than the actual level.
No value is assigned to standards having only qualitative applications.
A report compiling the results of the evaluation study and including the proposed label text is submitted for review and approval to the USP Reference Standards Expert Committee.
The label text is designed to provide the user with all the information needed for the correct storage and usage of the Reference Standard in monograph application(s). The label includes directions for use, safety warnings, required information for controlled substances, and a calculation value for standards with quantitative applications. For calibrators, acceptance ranges are provided. Where necessary, USP Reference Standards are accompanied by additional documentation, such as Technical Sheets or Typical Chromatograms. USP generally does not provide Certificates of Analysis because all the information that the user needs for the official or authorized applications of the standard is provided in the label text and, where necessary, in the additional documentation provided.
Directions for use are lot-specific, and they take precedence over any other indication in the compendium.
Material Safety Data Sheets are generated for every standard that USP distributes. They are available on the USP website.
USP REFERENCE STANDARDS EXPERT COMMITTEE
The USP Reference Standards Expert Committee comprises professionals from industry, government agencies and academia from the United States and abroad. It is organized in groups and may be assisted in the review of the evaluation studies by an Industry Advisory Panel. The approval of the evaluation report has to be unanimous.
The USP Reference Standard production process operates under a registered ISO 9001:2000 Quality System and appropriate cGMP principles.
USP Reference Standards are packaged in individual units designed to maintain the integrity of the contained Reference Standard material. The packaging and storage conditions for USP Reference Standards provide protection for all materials even though the material may not need such exceptional protection due to its inherent stability. The most common packaging configurations are vials for solid materials and ampuls for liquids. The packaging environment is determined by the sensitivity of the material to light, oxidation, or atmospheric humidity, and by its toxicity. Where appropriate, containers are filled in a glove box under inert gas and in conditions of controlled low residual humidity. (The need to store such standards under inert gas protection is indicated on the label.) They may also be sealed in a foil bag as an added protective barrier. Ampuls are filled and sealed on an automated device and are typically purged with an inert gas. The most common ampul sizes are 2 mL and 5 mL. Vials may be filled by manual, semi-automated, or fully automated operations. Vials may be of different sizes depending upon the amount of material. The amount of material per individual container depends on the compendial application of the standard and is generally sufficient for several replicates. Larger amounts are provided when additional experiments are required (such as a titrimetric determination of the water content at the time of use). In general, Reference Standard containers are slightly over-filled so that the user can retrieve the labeled, nominal amount of material. Vials are closed with Teflon-lined stoppers and secured with aluminum crimps and a USP-logo tamper-evident seal. Lots using prior vial closure configurations may still be in distribution.
Various considerations may determine the need to provide the standard in single-use containers, mainly for materials with significant handling issues or for those that are available only in small amounts. Such single-use containers are generally filled by lyophilization, and their content is labeled in mass or activity Units per container. If so labeled, the content of the container is to be reconstituted in its entirety without any additional weighing. Instructions for reconstitution are given either on the label or in the monographs where the standard is being used.
IMPURITY REFERENCE STANDARDS
The topic of impurities is addressed in several sections of USP, such as General Notices, General Chapters such as Ordinary Impurities 466, Impurities in Official Articles 1086, etc. In addition, most of the monographs for drug substances and many of those for formulations include specific tests for the identification or quantitation of impurities. Such tests generally require an official Reference Standard. The development of these impurity Reference Standards is one of the reasons for the continuous accelerated growth of the USP Reference Standards.
In many cases, the materials for impurity Reference Standards are expensive and difficult to procure. Only a limited amount of material may be availableprocurement might require custom synthesisand it may be of lesser quality than the Reference Standard for the official article, requiring purification. The limited amount of material available can affect the testing protocol and the packaging. Impurity Reference Standards might be available as purified single-component materials, solutions, or solid dispersions, or mixtures of more than one impurity. Other options include samples of the official article with a labeled content of impurity(ies), the in-situ generation of the impurity from the official article by a validated specified procedure, the use of relative chromatographic mobilities and relative response factors, or of theoretical values such as UV absorptivities at selective wavelengths.
In earlier editions of the compendium, the impurities were designated by their chemical names. For ease of indexing and searching, these have been gradually replaced with the designation X Related Compound Y RS, where X is the name of the official article and Y is a sequential alphabetical letter. Reference Standard impurity mixtures might be designated by their use, such as X System Suitability RS. The conventional names and the chemical names are cross-referenced in the final section of this chapter and in a special section of the Official USP Reference Standards Catalog.
CONTINUED SUITABILITY FOR USE PROGRAM
To ensure that the Reference Standards maintain the properties determined at the initial evaluation, USP maintains a Continued Suitability for Use Program. The retesting intervals and protocols are a function of the uses and properties of the standard and of the information available about its stability. Abbreviated protocols use the stability-indicating methodology employed in the initial characterization of the material to confirm the consistency of attributes such as appearance, chromatographic purity, or volatiles content.
Neither the Reference Standards nor the Authentic Substances are intended for use as drugs or as medical devices.
USP Reference Standards do not carry an expiration date as long as they are in distribution. A lot of USP RS may be used in its official applications as long as it is listed as Current Lot in the current (most recent) Official USP Reference Standards Catalog. Upon depletion, the lot is designated in the catalog as Previous Lot and a Valid Use Date is assigned. USP publishes the Official Catalog of Reference Standards (which also includes Authentic Substances) bimonthly as a separate brochure* . An updated version of the catalog can be found on the USP website at www.usp.org. It is the responsibility of the user to ascertain that a particular supply of USP Reference Standard has official status either as a Current Lot or as a Previous Lot within the valid use date.
Many Pharmacopeial tests and assays are based on comparison of a test specimen with a USP Reference Standard. In such cases, measurements are made on preparations of both the test specimen and the Reference Standard. Where it is directed that a Standard solution or a Standard preparation be prepared for a quantitative determination by stepwise dilution or otherwise, it is intended that the Reference Standard substance shall be accurately weighed (see Weights and Balances 41 and Volumetric Apparatus 31). Due account should also be taken of the relatively large errors associated with weighing small masses.
The label text provides the user with directions on the proper use of a Reference Standard. The directions include one of the following options. A Reference Standard may be used as follows:
Whenever the labeled directions for use require a preliminary drying or a correction for volatiles, it should be performed at the time of use. Further experimental details should be controlled by the user's Standard Operating Procedures and good laboratory practices.
To serve its intended purpose, each USP Reference Standard must be properly stored, handled, and used. Generally, Reference Standards should be stored in their original stoppered containers away from heat and protected from light. Avoid humid storage areas in particular. Where special storage conditions are necessary, directions are given on the label.
RELATIONSHIPS WITH OTHER STANDARDS-SETTING ORGANIZATIONS
USP maintains continuous contact with other organizations that establish Reference Materials for compendial and other purposes, such as the European and the Japanese Pharmacopoeias (through the Pharmacopeial Discussion Group), the World Health Organization, the National Institute for Science and Technology, the Reference Materials Committee of ISO (REMCO), etc.
The specific nature of pharmacopeial reference substances has been officially recognized by ISO-REMCO in the introduction of the ISO Guide 34General requirements for the competence of reference material producers (Second Edition 2000): Pharmacopoeial standards and substances are established and distributed by pharmacopoeial authorities following the general principle of this guide. It should be noted, however, that a different approach is used by the pharmacopoeial authorities to give the user the information provided by certificate of analysis and the expiration dates. Also, the uncertainty of their assigned values is not stated since it is negligible in relation to the defined limits of the method-specific assays of the pharmacopoeias for which they are used.
The USP Reference standards section of an individual USP or NF monograph or general chapter names each USP Reference Standard required for assay and test procedures and refers to this chapter for additional information and instructions. It is especially important to refer to the current Supplement to USP and to NF for official revisions listed in the following section.
USP REFERENCE STANDARDS SPECIFIED IN USP AND NF MONOGRAPHS AND GENERAL CHAPTERS
noteConsult the latest Supplement or Interim Revision Announcement pertaining to USP and to NF for revisions, additions, or deletions.
Revisions, additions, and deletions of individual USP Reference Standards are listed cumulatively in each Supplement to USPNF. As a consequence, therefore, it is necessary to consult only the current edition of USPNF and the latest Supplement for the complete list of USP Reference Standards currently specified in USPNF monographs and general chapters. The list provides up-to-date and complete names and applicable chemical information for the USP Reference Standards that are in distribution as of the official date of that Supplement.
Revisions of this chapter are implemented continuously via the Interim Revision Announcements that are published in Pharmacopeial Forum. Those interim revisions of USP Reference Standards are cumulatively included in the next USPNF Supplement.
The alphabetical list that follows constitutes an index of all revisions to this chapter. Thus, it is unnecessary to name repetitively the revised Reference Standards in the general index to the Supplement.
In the list that follows, chemical names are given for many substances (e.g., related compounds) that are not USP or NF monograph articles. Following the name of such a chemical substance RS, the empirical formula and molecular weight, separated by the symbol, may be given in parentheses if those data are available.
* For nonsubscribers, the most recent Official Catalog is available from: U.S. Pharmacopeial Convention, Inc., Reference Standards Order Department, 12601 Twinbrook Parkway, Rockville, MD 20852. Telephone 1-301-881-0666. FAX 1-301-816-8148. Toll-free telephone 1-800-227-USPC or access the Catalog on USP's website www.usp.org/dsd/refstd.