Testing of identity, strength, quality and purity of pharmaceuticals such as drug substances, excipients and products requires them to be compared with a substance which is highly purified and characterized. 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”. The purpose of establishing reference standards is to achieve accuracy and reproducibility of the analytical results required by pharmacopeial testing and pharmaceutical control in general.

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.

 

What are various types of reference standards?

 

  • Primary Reference Standard: A standard which 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. These standards are normally prepared and issued by the regional or national pharmacopoeia commission or the regional or national quality control laboratory on behalf of the drug regulatory authority such as the National Institute for Standards and Testing (NIST), Indian Pharmacopoeia Commission (IPC) etc. These standards meet the requirements as laid under various pharmacopoeias such as the United States Pharmacopeia (USP), European Pharmacopoeia (EP), Japanese Pharmacopoeia (JP), or Indian Pharmacopeia (IP).

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.

  • Secondary Reference Standard (may also be referred as working standard): A standard is a substance whose characteristics are assigned and/or calibrated by comparison with a primary chemical reference standard. The extent of characterization and testing of a secondary chemical reference standard may be less than for a primary chemical reference standard. The working standards are often provided by raw material manufacturers, contract manufacturers or may be developed in-house by the user himself.

 

What are various uses of Reference Standards?

 

The intended use of a reference standard is an important criterion to determine the extent of characterization. A chemical reference substance proposed for an identification test does not require meticulous purification, since the presence of a small percentage of impurities in the substance often has no noticeable effect on the test.

On the other hand, chemical reference substances that are to be used in assays should possess a high degree of purity. As a guiding principle, a purity of 99.5% or higher is desirable, calculated on the basis of the material in its anhydrous form or free of volatile substances. However, where the selectivity of the analytical procedure for which the chemical reference substance is required is low, such a degree of purity may not be necessary. The various uses of reference standards are:

    1. For Identification: Reference standards are used to identify the material present in a drug substance/ drug product. For use in identification tests (IR spectrophotometry and/or chromatographic methods), a batch of good quality material selected from the normal production process is satisfactory if it is of acceptable purity.
    1. For Purity: Reference standards used to determine purity, or to identify impurities and quantify the same of a drug substance/ drug product. The characterization of a chemical reference standard for use in the determination of a specific impurity is more extensive, especially when used in a limit test. If the technique employed is thin-layer chromatography (TLC), an acceptable minimum purity is recommended (normally at least 90%), but purer material (at least 95%) may be required for liquid chromatography (LC) or gas chromatography (GC).
    1. For assay: Reference standards used to determine potency. If the chemical reference standard is to be used in an assay (colorimetry, LC, GC or UV spectrophotometry), the extent of testing is much greater. The relative reactivity or relative absorbance of the impurities present must be checked when a nonspecific assay method is employed, e.g. by colorimetry or UV spectrophotometry. When a selective assay method is employed, it is particularly important to determine the quantity of impurities. In such a case, it is best to examine the proposed reference standard by as many methods as practicable including, where possible, absolute methods. The total of the determinations of water content, organic solvents, mineral impurities and organic components should amount to 100%. For most chemical reference standard intended for assays, the content may be expressed “as is”. When establishing the chemical reference standard, it is, therefore, essential to determine the content of water and residual solvents for a non-specific assay, and also to determine the content of impurities for a selective assay.
    1. For instrument calibration: Reference standard may also be used as calibration material. In such cases, the extent of testing is similar to that for a reference standard used in assays.
    1. For Degradation products: Reference standards used to identify and possibly quantify the degradation products.

 

How to produce reference standard?

The production, validation, maintenance and distribution of chemical reference standard is a costly and time-consuming process.

Source material of satisfactory quality can be selected from a batch (lot) of the substance originating from the normal production process, if the purity is acceptable. However, it may require further purification to render the material acceptable for use as a chemical reference standard. When source material to be used as a chemical reference standard is obtained from a supplier, the following should be supplied with the material:

    1. certificate of analysis with complete information on test methods employed, values found and number of replicates used, where applicable, and relevant spectra and/or chromatograms;
    2. results of any accelerated stability studies;
    3. information on optimal storage conditions required to ensure stability (temperature and humidity considerations);
    4. results of any hygroscopicity study and/or statement of the hygroscopicity of the source material;
    5. identification of impurities detected and/or specific information on the relative response factor as determined in compendial methods concerning the principal component, and/or the percentage mass of the impurity;
    6. updated material safety data sheet outlining any health hazards associated with the material.

The extent of the analysis required depends on the purpose(s) for which the chemical reference standard is to be employed which has already been explained above.

 

How to evaluate reference standards?

Reference standards need to be tested extensively to prove that they meet the acceptance criterion. The procedures used to evaluate reference standards are broadly classified as:

    1. Methods used to verify the identity of chemical reference standards: These may include but not limited to IR spectroscopy, NMR spectroscopy, MS, or X-ray diffraction crystallography etc. Special attention should be given in the cases where polymorphism exists.
    2. Methods used to determine the purity of chemical reference standards: The methods employed are categorized as follows:
      • Separation Techniques: This includes chromatographic methods such as HPLC, Gas chromatography and Thin Layer Chromatography. HPLC is widely used method. Capillary electrophoresis is also a common method which is considered as complimentary to Liquid Chromatography for detecting impurities.
      • Based on Intrinsic Thermodynamic properties: These methods measure total impurity levels in absolute terms. The methods include Differential Scanning Calorimetry (DSC) and phase solubility analysis. DSC is used to check for the presence of different polymorphic forms and to determine the total amount of solid impurities.
      • Other methods: These includes conventional methods such as UV spectrophotometry, titrimetric methods, optical rotation methods and by determination of water and organic volatiles.

This is to understand that with most methods, the percentage purity of a chemical reference substance cannot be expressed as an absolute value if the impurities have not been identified. The quoted purity is then an estimate based upon the data obtained by the various analytical methods.

 

How to identify the substances with same molecular constitutions as that of Reference standards?

 

In a situation where authentic specimen is available for comparison, it shall be ensured that the characteristic properties of the two specimens are identical. For this purpose, it is often sufficient to compare their IR absorption spectra.

However, in a situation where no authentic specimen of the proposed substance is available for comparison, and definitive data about its properties are lacking, it may be necessary to verify its identity by applying several of the analytical techniques such as elemental analyses, crystallographic studies, MS, NMR spectroscopy, functional group analyses, and IR or UV spectrophotometry, as well as other supplementary tests, as required, to establish that the proposed substance is fully characterized.

 

What are the precautions that should be taken while packing the reference standards?

Utmost care shall be taken while packing the reference standards. Any lapses may compromise with the strength, identity, purity and quality of the reference standard. Hence, the reference standards shall be packed in a GMP environment. Proper procedures shall be defined for performing packing of reference standards. The containers should protect their contents from moisture, light and oxygen and must be tested for permeability to moisture. The lack of permeability to moisture and the compatibility of the material of which the closure is made with the reference standard are important factors in determining the suitability of container closure systems. The use of multidose containers is not excluded, but is not recommended.


Can we pack the reference standards in glass ampoules?

Yes, however, risk of contaminating the substance with glass particles when the ampoules are opened cannot be ruled out.

 

What are the precautions that shall be taken while storing reference standards?

Mostly, we tend to store the reference standards in refrigerators. It is a generally understood phenomenon that the stability of the substances is enhanced at low temperatures but special precautions shall be taken for the substances which contain water.  Storage at or below 0 °C may impair the stability. Also, relative humidity in refrigerators is normally high, hence, improperly stored or loosely closed containers may absorb moisture, which may deteriorate the reference standard. Storage at about 5 °C, with precautions to prevent such absorption, has proved satisfactory for most reference standards. Vials should, however, not be opened until they have attained room temperature to prevent ingress of moisture by condensation.

 

What is the information to be supplied with reference standards?

The labels on chemical reference substances should give the following information:

    1. Name of the substance
    2. Name of the issuing body
    3. Approximate quantity of material in the container; and
    4. Batch or control number.

The following information should be given, as necessary, on the labels and/or in associated documents:

    1. Name and address of the issuing body;
    2. Recommended storage conditions;
    3. Intended use of the reference standard;
    4. Directions for use (e.g. storage and handling);
    5. Information about the assigned analytical value of the chemical reference substance;
    6. Disclaimer of responsibility in cases where chemical reference substances are misused, or stored under inappropriate conditions, or used for purposes other than those intended by the issuing body; and
    7. Health hazard information or warnings in conformity with national and regional regulations or international agreements.

 

Does reference standard have an expiry date?

Chemical reference substances do not carry an “expiry date” in the conventional sense. To avoid the unnecessary discarding of satisfactory substances, a mechanism for general control of the batch of a chemical reference substance may be used by the issuing body. If the issuing body applies stability considerations and a monitoring procedure to its collection based on its experience, this should be a guarantee to the user of the acceptability of the chemical reference substance for its intended use.

If, exceptionally, it is considered necessary to specify an expiry or re-test date, this should be stated on the label and/or in a document accompanying the chemical reference substances. Adequate shipping records should be kept to enable contact to be made with the purchaser of a batch for recall or other notification.

However, it should be noted that the storage and maintenance of unopened containers of the reference standard in accordance with the information provided are integral to its suitability for use.

 

Reference:

  1. Annex 3 of WHO TRS 943, General guidelines for the establishment, maintenance and distribution of chemical reference substances
  2. ICH, Q3A(R2) Impurities in New Drug Substances (Geneva, Switzerland), Oct. 25, 2006
  3. USP 30–NF 25 General Chapter <11>, “Reference Standards,” p. 1