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