Qualification of Secondary Reference Standard

Standards are primarily used for the following types of analytical studies:

1. Assay
2. Identification tests
3. Limit tests for related substances
4. Analytical method validation
5. System suitability for analysis techniques and in particular spectroscopic and chromatographic analysis

It becomes necessary to understand the fine differences between different categories of standards used for chemical analysis. The standards are commonly grouped into two groups:

1. Primary standards or certified reference materials
2. Secondary standards or working standards

1. Primary Standard

A primary standard reference material is an ultra-high purity grade compound used in analysis involving assay, identification, or purity tests. It can be a single compound or a mixture having the analyte of interest in a specified and certified amount.

The impurities, if any, should be identified and controlled for use in assay studies. The material selected as a primary standard should be highly stable, free from the water of hydration and bear traceability to a national or international standards body.

In many cases it may not be possible to procure reference material from such sources because of the following reasons :

• The new molecule is encountered in R&D activity. In such case, a reference may not be available from standard bodies. A laboratory can use a reference compound whose purity is established for its routine use.
• In-house primary reference material can be selected from a particular batch in manufacturing industries. After assigning its batch number its characteristic properties are documented for reference purposes and comparison with future production lots.

2. Secondary or working standard

Primary standards come with a certificate of analysis and bear traceability to a globally recognized standards body. The cost is often too high for even milligram-range quantities. Secondary standards or working standards are also high-purity grade materials that are quantified in relation to primary standards and put to routine use in laboratories. Such working standards are assigned a limited validity depending on the stability of the material and before expiry fresh working standards should be prepared for future use. It is important to realize that if an expired working standard is used in analysis then no credibility can be placed on the reported results.

The definitions given below apply to the terms used. They may have different meanings in other contexts.

ALSO READ: SOP for Reference Standard Control

Chemical Reference Substance

The term chemical reference substance refers to an authenticated, uniform material that is intended for use in specified chemical and physical tests, in which its properties are compared with those of the product under examination, and which possesses a degree of purity adequate for its intended use.

Primary Chemical Reference Substance

A designated primary chemical reference substance is one that is widely acknowledged to have the appropriate qualities within a specified context, and whose assigned content when used as an assay standard is accepted without requiring comparison with another chemical substance.

Secondary Chemical Reference Substance

A secondary chemical reference substance is a substance whose characteristics are assigned and/or calibrated by comparison with a primary chemical reference substance. The extent of characterization and testing of a secondary chemical reference substance may be less than for a primary chemical reference substance.

International Chemical Reference Substance

International Chemical Reference Substances (ICRS) are primary chemical reference substances established on the advice of the WHO Expert Committee on Specifications for Pharmaceutical Preparations. They are supplied primarily for use in physical and chemical tests and assays described in the specifications for quality control of drugs published in The International Pharmacopoeia or proposed in draft monographs. The ICRS may be used to calibrate secondary standards.

Pharmacopeial Reference Standards

Pharmacopoeial standards and substances are established and distributed by pharmacopeial authorities following the general principles of this Guide. It should be noted, however, that a different approach is used by the pharmacopeial authorities to give the user the information provided by the certificate of analysis and expiration dates.

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?

A 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 Standards, Reference Standards, or Pharmacopeial Standards are well-characterized and certified materials 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. 

1. Select the drug substance lot recently manufactured with an adequate validity period i.e. essential retest period or with a suitable expiry date.
2. Availability of drug substance with valid COA and must comfortably comply with the assigned specification.
3. Analyze 3 different batches of drug substances with the verified or validated test procedure.
4. All three batches must comply with per assigned in-house or pharmacopeial specifications.
5. Select the single batch of drug substance that must be available in sufficient quantity.
6. The selected batch sample must comply with all test specifications comfortably and the assay value close to 100 %.
7. 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.
8. The 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:

1. Identification: By HPLC, TLC, IR, UV-VIS Spectrophotometer, and Chemical methods,
2. Loss of drying / Water content,
3. Related substances: By TLC, GC, HPLC,
4. Residual solvent analysis by HSGC,
5. Assay: By HPLC, UV-VIS Spectrophotometer, and 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 drying the standard before use with clear temperature conditions or depending on the Pharmacopoeial 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:

1. Name of the compound,
2. Physical Description,
3. LOD/water content result,
4. Assay or Purity on an as-is basis, (Must have clear instruction on vial about using purity on an as-is basis or Dry before use with clear temperature condition)
5. Vial number,
6. Date of preparation,
7. Validity or Retest date,
8. 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.

Is it acceptable to qualify secondary reference standard with the titration method?

Any chemical analysis can be considered valid only if the method of analysis is validated before adoption and results are reported against internationally recognized standard reference materials. The results of such analysis can be relied upon by consumers as well as other laboratories across the world.

For substances that are acidic or basic a titration with alkali or acid is simple, but other reactions which are known to be stoichiometric may be used. Phase solubility analysis and differential scanning calorimetry may also be employed in certain cases.

Handling and storage of standards

Standards play a crucial role in analysis so require to be preserved under specified conditions so that their authenticity is preserved over the prescribed storage periods.

The following recommendations can prove useful:

1. Store in amber-colored glass vials or bottles that are properly sealed or capped and stored in controlled humidity conditions.
2. Reseal the vial or cap the bottle securely after use
3. Temperature-sensitive standards should be stored in cooling chambers or dedicated refrigerators between 2-8℃.
4. Ensure that the standard is within its validity before use
5. Do not return a new standard to the original container to prevent contamination of the original stock.

The significant contribution of standards in any chemical analysis is emphasized in the article. It is indeed difficult to imagine a laboratory functioning without making use of standards and reference materials.

ALSO READ: SOP for Preparation of Data Sheets for Working Standard