WHO Expert Committee on Specifications for Pharmaceutical Preparations - WHO Technical Report Series, No. 885 - Thirty-fifth Report
(1999; 168 pages) [Spanish] View the PDF document
Table of Contents
View the documentWHO Expert Committee on Specifications for Pharmaceutical Preparations
View the document1. Introduction
Open this folder and view contents2. The international pharmacopoeia and related issues
Open this folder and view contents3. International Chemical Reference Substances and Infrared Reference Spectra
Open this folder and view contents4. Quality control - national laboratories
Open this folder and view contents5. Good manufacturing practices
Open this folder and view contents6. Quality systems and inspection
Open this folder and view contents7. Other quality assurance topics
Open this folder and view contents8. Nomenclature and terminology
Open this folder and view contents9. Legal aspects of pharmaceuticals
Open this folder and view contents10. Regulatory issues
Open this folder and view contents11. Training activities
View the document12. Pharmaceuticals contaminated with diethylene glycol
View the documentAcknowledgements
View the documentReferences
View the documentAnnex 1. List of available International Chemical Reference Substances1
View the documentAnnex 2. List of available International Infrared Reference Spectra1
Close this folderAnnex 3. General guidelines for the establishment, maintenance and distribution of chemical reference substances
View the documentIntroduction
Close this folderPart A. Primary chemical reference substances
View the document1. Assessment of need for the establishment of chemical reference substances
View the document2. Obtaining source material
Open this folder and view contents3. Evaluation of chemical reference substances
Close this folder4. Chemical and physical methods used in evaluating chemical reference substances
View the document4.1 Methods used to verify the identity of chemical reference substances
View the document4.2 Methods used to determine the purity of chemical reference substances
View the document5. Assignment of content
Open this folder and view contents6. Handling and distribution of chemical reference substances
View the documentPart B. Secondary chemical reference substances
View the documentReferences
Open this folder and view contentsAnnex 4. Good manufacturing practices: authorized person - role, functions and training
Open this folder and view contentsAnnex 5. Good manufacturing practices: supplementary guidelines for the manufacture of pharmaceutical excipients
Open this folder and view contentsAnnex 6. Guidelines for inspection of drug distribution channels
View the documentAnnex 7. Good pharmacy practice in community and hospital pharmacy settings
Open this folder and view contentsAnnex 8. National drug regulatory legislation: guiding principles for small drug regulatory authorities
Open this folder and view contentsAnnex 9. Provisional guidelines for developing training programmes: inspection and examination of counterfeit pharmaceuticals
View the documentWorld Health Organization Technical Report Series
View the documentSelected WHO Publications of Related Interest
View the documentBack Cover
4.2 Methods used to determine the purity of chemical reference substances

The analytical methods to be employed in examining a substance should be considered in relation to its intended use. These analytical methods may be divided into three broad categories: those that require comparison with an external chemical reference substance (e.g. chromatographic or spectrophotometric methods), those that depend solely on an intrinsic dynamic property (e.g. phase solubility analysis and differential scanning calorimetry) and other methods.

4.2.1 Separation techniques

The methods used for the determination of purity should be established and validated with system suitability requirements as appropriate.

Chromatographic methods. Methods of analysis based on chromatographic separation are especially useful for detecting and determining impurities in chemical reference substances. High performance liquid chromatography (HPLC) is the most widely used chromatographic method, but TLC and GC are also used. The individual components separated by chromatographic methods may sometimes be recovered for characterization.

The selectivity of HPLC and of GC usually exceeds that of TLC. Both the first two methods also have the advantage of being readily applicable on a quantitative basis, but they require more complex equipment. HPLC, employing a spectrophotometric method of detection, is of particular value in the examination of chemical reference substances intended for use in UV spectrophotometric assays. The UV wavelength of detection employed for determining the impurity content of the chemical reference substance should be chosen so that the detection responses of the substance and its known impurities are similar. When the response factors are significantly different at the optimal wavelength of detection, appropriate corrections must be made to estimate the content of impurities. LC with diode-array detection is very useful for recording the UV spectra of both the main peak and the impurities. LC with MS detection is used for identification of separated impurities as well as for the main component, and is particularly important for chemical reference substances where no other reference standards or IR reference spectra are available.

In a GC method used for an assay, as with LC, the detection responses of the known impurities are determined. Generally, GC monograph methods are of particular value in detecting and determining volatile impurities, including solvent residues, in chemical reference substances.

TLC uses apparatus that is simple and cheap; the technique is easy to carry out and is readily applicable even in the microgram range. It can separate closely related compounds, such as geometric isomers and the members of a homologous series. All the constituents of a substance submitted to chromatography appear somewhere on the chromatogram. However, some constituents may remain on the starting line, some may move with the solvent front, some may migrate at the same rate as the main component, and some may remain undetected. For this reason, the usefulness of the method may be greatly enhanced by means of two-dimensional chromatography and by using a number of different solvent systems and a variety of detection methods. In some cases the method may be used quantitatively with acceptable accuracy by using a densitometer.

Capillary electrophoresis. Capillary electrophoresis is an increasingly common method. It may be considered as complementary to LC for detecting impurities.

4.2.2 Methods based on intrinsic thermodynamic properties

Methods in this group measure total impurity levels in absolute terms.

Differential scanning calorimetry. This technique is used to check the presence of different polymorphic forms and to determine the total amount of solid impurities. Purity estimation is based on determination of the heat of fusion of the sample and of the change in its melting point caused by the presence of impurities. This analytical method can be performed rapidly and with high precision. However, it is not applicable if the substance decomposes on melting. This limits its value as a general procedure for purity estimation of chemical reference substances. It is also inapplicable if solid solutions are formed.

Phase solubility analysis. The method has occasionally been used, but its value is limited and the procedure is time consuming. It may be employed to detect contaminating substances, including isomeric species, and to estimate their concentration. Some factors that may make the method inapplicable are degradation of the substance during the course of analysis, formation of a solid solution, and polymorphism in the main component.

4.2.3 Other methods

Spectrophotometric methods. UV spectrophotometry is occasionally used to determine purity. Since it depends upon the presence of a characteristic chromophore, it can detect impurities that contribute excessively to the absorbance value and may indicate the presence of impurities that have a negligible or distinctive absorbance.

However, the utility of the method is limited by the small number of absorption maxima in the UV range, the large numbers of compounds containing similar characteristic chromophores, and the need for an external chemical reference substance.

IR spectrophotometry may be used to identify and determine the proportions of geometric isomers. NMR spectroscopy, a powerful spectroscopic identification tool, is also occasionally useful in the determination of purity.

Titrimetric methods. Titrimetric methods provide a valuable means of confirming the identity and purity of a proposed chemical reference substance and are useful in confirming purity values obtained by other methods.

Optical rotation methods. Many chemical reference substances are optically active and the relative proportion of optical isomers can sometimes be determined by an optical rotation method, but generally such methods lack sensitivity. However, the quantitative use of these techniques is well established and can yield results of high precision, depending on the solvent and the wavelength chosen for measurement. Chiral chromatography and NMR are becoming increasingly important.

Determination of water and organic volatiles. It is essential that an accurate assessment of the moisture content and the content of volatile contaminants be made. These total values may often be obtained by drying under defined conditions that are appropriate to the proposed substance. Sometimes this may not be possible or may yield misleading results. In such cases, thermogravimetric analysis may be used to determine the water and volatile content. Alternatively, the water content may be determined by Karl Fischer titration and the content of volatile solvents by GC. Without an accurate assessment of these values at the time that other determinations are being made, judgements of the acceptability of the proposed chemical reference substance will be invalid.

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