Reference-Standard and Working Standards
The US Food and Drug Administration defines a reference-standard material as a “highly purified compound that is well characterized”
(1). The US Pharmacopeia (USP) defines reference-standard materials as “highly characterized specimens of drug substances, excipients, reportable impurities, degradation products, compendial reagents, and performance calibrators”
(2). Scientists performing analytical testing use reference standards to determine quantitative (e.g., assay and impurity) as well as qualitative (e.g., identification tests) data, performance standards, and calibrators (e.g., melting point standards). The quality and purity of reference standards, therefore, are critical for reaching scientifically valid results.
Reference standards can be segregated into two groups: chemical and nuclidic.
Chemical purity must be determined for both groups.
Nuclidic reference standards, however, also need to be evaluated for radionuclidic and radiochemical purity.
Reference standards are authentic substances approved by the reference standard approving authority as a preferred method for use. Comparison Standards are described as individual monographs and methods of analysis. The purpose of this is to define the procedure for preparation, standardization, and usage of a working standard.
The precautions that have to be taken like using a clean and dry amber-colored vial for storage of working standards. All reference standards or impurity standards have to be stored as per the storage condition mentioned in MSDS from the supplier. Remember before usage, the temperature of the vials must reach room temperature. In the case that potency of the working standard is found more than 100.0% during the standardization, as a policy cut off the potency of the working standard will be considered as 100.0%.
Maintenance of Reference Standard
According to pharmaceutical standards, reference substances or standards are authentic purified chemicals supplied by the official pharmacopeia commission. These are generally used for comparison to determine the purity of the test specimen. In case the reference standard isn’t available in the pharmacopeial catalog, the manufacturer shall be asked for their reference standard or working standard with a certificate of analysis. As reference standards are usually available only in small quantities, working standards can be prepared to act as a substitute, these are prepared from approved raw materials and validated against authentic reference standards or substances.
All standardized records of working standards shall be maintained in a QC laboratory. The Inventory and consumption of reference standards shall be maintained, and the validity of reference standards shall be verified on a quarterly basis, and before preparing for a working standard.
Now, in case the reference standard is found to be consumed or expired during verification, then the indent for procurement of a fresh lot shall be initiated by the Manager Q.C.
Storage and Expiry Working Standards
All working standards are to be stored under refrigeration, in case a specific storage condition is mentioned then the material should be stored according to that. All working standards shall be brought to ambient temperature before use and the details are to be recorded in the usage log book. The expiry period for the working standard is one year from the day of preparation, and for a vial is one month, the expiry period should not exceed the retest period of the selected API batch.
Types of reference-standard materials
Reference-standard materials can be broadly categorized as such:
- Assays—used to determine potency for active pharmaceutical ingredients (APIs) and salts.
- Degradation products—used to identify and possibly quantitate degradation products.
- Process impurities—used to identify and possibly quantitate process-related compounds.
- Resolution—used to determine assay performance or impurity method.
- Metabolites—used to identify and possibly quantitate substances generated through a metabolic process.
Sources of reference-standard materials
Reference standards can be compendial or non-compendial and are typically obtained from the following sources.
- Pharmacopeias such as the United States Pharmacopeia (USP), European Pharmacopoeia (EP), or Japanese Pharmacopoeia (JP).
- Nationally recognized standard institutions such as the National Institute for Standards and Testing (NIST).
- The user (custom manufactures or synthesizes the reference standard)
- Contract manufacturer
- Companies such as chemical suppliers.
Pharmaceutical Secondary Standards
Our secondary standards have multi-traceability to the USP, EP, and BP primary standards; in addition, they are manufactured according to ISO/IEC 17025 and ISO Guide 17034.
The FDA and EP all recognize the use of secondary standards or working standards that are established with reference to the corresponding primary standard.
FEATURES & BENEFITS
The important product features are:
- Traceability to United States Pharmacopeia (USP); also to European Pharmacopoeia (EP) and British Pharmacopoeia (BP), if available
- Analysis performed on instruments validated according to GMP using pharmacopeia monograph methods
- Certified value according to ISO Guide 17034 and ISO 17025 using mass balance approach
The main benefit is:
- Eliminate the time and effort involved with preparing and validating your own working standards.
The values on the certificate are always traceable to the current pharmacopeia lots.
Note: If a valid pharmacopeia lot changes, then the corresponding secondary standard will be recertified with traceability to the new lot and a new certificate will be made available online.
For this reason, the certificate always needs to be downloaded from the website prior to the use of the material!
The certificate contains the following data:
- Traceability assay results versus pharmacopeia primary standards
- Value by mass balance
- Handling and storage instructions
- Analytical data
An excerpt of the certificate is shown in the image below, highlighting some of the key information that is displayed.
REGULATORY RECOGNITION OF SECONDARY STANDARDS
(1) Human Drug CGMP Notes, Vol 9, Number 3, 2001 (Internal FDA Publication):
Q: Can a company use reference standards from sources other than the USP?
A: Yes. Using a source other than USP can be acceptable provided the reference standard incorporates the critical characteristic properties, is suitable for the intended purpose, and is supported by complete documentation…..The supplier’s certificate of analysis should report the results of testing to adequately characterize the material across a complete range of quality characteristics.
(2) FDA: Guidance for Industry; Analytical Procedures and Methods Validation, August 2000:
“A reference standard (i.e., primary standard) may be obtained from the USP/NF or other official sources (e.g., CBER, 21 CFR 610.0). A working standard (i.e., in-house or secondary standard) is a standard that is qualified against and used instead of the reference standard.”
(3) European Pharmacopoeia, Chapter 5.12:
“Secondary standard: A standard established by comparison with a primary standard. A secondary standard may be used for routine quality control purposes for any of the uses described above for primary standards provided that it is established with reference to the primary standard.”
Qualification of Pharmaceutical Working Standards
The certified standard is very much important in pharmaceutical analysis. It ensures that the performed analysis is accurate and assures the safety of the drug product. The analysis is always carried out by using the working standard and results are reported against the working standard.
What is Working Standard?
Working standard is defined as, “A drug substance of established quality and purity as shown by comparison to the reference standards.” This working standard is used for routine analysis.
The working standard is qualified against the pharmacopeial reference standard or certified reference standard. Qualification of a working standard is done by using a verified or validated analytical method and its results are compared with the reference standard.
Why Use Working Standard?
Primary Standard, Reference Standard, or Pharmacopeial Standards is well characterized and certified material by standard regulatory agencies or appropriate laboratories. These reference standards are available with minimum pack quantity at a high cost. Due to the higher cost of the primary reference standard, it is not possible to use this standard for routine analysis. Due to this reason, the working standard is prepared for routine analysis. bearing reference to the original certified reference material.
Selection of Drug Substance:
The working standard must be high purity grade materials. Following are the selection criteria of the drug substance batch for the working standard preparation.
- Select the drug substance lot recently manufactured with an adequate validity period i.e. essential retest period or with a suitable expiry date.
- Availability of drug substance with valid COA and must comfortably comply with the assigned specification.
- Analyze 3 different batches of drug substance with the verified or validated test procedure.
- All three batches must comply as per assigned in-house or pharmacopeial specifications.
- Select the single batch of drug substance that must be available in sufficient quantity.
- The selected batch sample must comply with all test specifications comfortably and the assay value close to 100 %.
- Select the batch number for the working standard preparation and collect a sufficient amount of sample quantity for analysis as well as for use as a working standard. Different vials with a minimum quantity will be sufficient for 1 month. This quantity and number of vials again depend on the requirement of standard per analysis, nature of the drug substance, and stability of the drug substance.
- Batch selected for working standard preparation must be analyzed in triplicate.
Qualification of Working Standard:
- The use of primary reference standards for the working standard qualification must have characterization data with valid COA or a valid lot of pharmacopeial reference standards.
- Analysis of working standards should be carried out by using calibrated instruments.
- The selected drug substance shall be tested as per standard test specifications and standard test procedure of IP/ BP/EP/JP/USP or in-house criteria against the pharmacopeial reference standard or primary reference standards.
- Mainly following tests shall be performed for the preparation of the working standard:
- Identification: By HPLC, TLC, IR, UV-VIS Spectrophotometer, and Chemical methods,
- Loss of drying / Water content,
- Related substances: By TLC, GC, HPLC,
- Residual solvent analysis by HSGC,
- Assay: By HPLC, UV-VIS Spectrophotometer, Chemical methods. The assay performed in triplicate should not deviate ± 2 % and it should be within specification.
- There should not be any deviation, errors, or out-of-specification results at the time of analysis.
- If the assay is found more than 100 % then consider it as 100 %.
Assign the purity on an as-is basis and also need to check if there is any salt and base correction factor required. Also, it must have clear instructions on the vial about using purity on an as-is basis or dry the standard before use with clear temperature conditions or depending on the pharmacopeial monograph or as per primary standard test procedure.
After analysis assigns the validity depends upon the nature and stability of the drug substance. The validity of the working standard shall be assigned for 1 year or it must be within the retest date or expiry date of the drug substance assigned by the manufacturer.
After completion of the analysis and report generated, prepare the COA, and submit it for the QA review along with a hard copy or electronic data.
Storage of Working Standard:
After approval of the report label the working standards bottles properly with the following details:
- Name of the compound,
- Physical Description,
- LOD/water content result,
- Assay or Purity on as-is the basis, (Must have clear instruction on vial about Use purity on an as-is basis or Dry before use with clear temperature condition)
- Vial number,
- Date of preparation,
- Validity or Retest date,
- Storage Condition.
Store the working standard as per the storage condition of the drug substance. The validity of working standards shall be one year or depend on the nature and stability of the compound. Working standards shall be stored in amber-colored glass bottles with airtight closures. Each vial shall be used for a maximum of 2 months or depending on the nature of the standard.
One vial with an adequate quantity of the working standard shall be kept as a “Control sample”.
The working standards shall be revalidated at well-defined intervals to ensure their purity, integrity, and authenticity. Maintain the usage record of the working standard.
To ascertain the degree to which an analytical method is deemed suitable for its intended use, the validation parameters set forth in ICH Q2(R1) Validation of Analytical Procedures (6) stipulate the following criteria:
- Specificity—evaluation of interference from extraneous components.
- Linearity—linear range of the method.
- Range—the interval between the lower and upper concentration amounts of analyte in the sample.
- Accuracy—a measure of the closeness of agreement between the value obtained and the theoretical.
- Precision—a measure of the closeness of agreement (degree of scatter) of the data values over a number of measurements (i.e., injection repeatability, analysis repeatability (multiple measurements, same analyst) and intermediate precision (multiple measurements, different days, different analysts), reproducibility (precision between different labs).
- Detection limit—the lowest level the analyte can be detected.
- Quantitation limit—the lowest level the analyte can be quantitated.
- Robustness—effects of small changes in method parameters.
- System suitability testing—evaluation of the suitability of the equipment.
Expired working or reference standards should not be used. Instead, they should be discarded immediately in a proper way.