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This FAQ provides guidance on how to apply the test requirements and on data interpretation in the related substances test presented in an individual monograph.

The figures given in the examples below do not apply to biological and biotechnological products, oligonucleotides, products of fermentation and semi-synthetic semisynthetic products derived therefrom, to crude products of animal or plant origin or herbal products or excipients (see Substances for pharmaceutical use (2034)); however the principle remains valid.

As stated in chapter 2.2.46. Chromatographic separation techniques, disregard any peak due to the solvents and reagents or arising from the mobile phase or the sample matrix.

The signal-to-noise (S/N) ratio of the specified impurities is calculated on the solution used for reporting threshold / disregard limit.

QUANTITATIVE METHOD

• Reporting threshold: inject a solution of the substance to be examined at a concentration corresponding to the reporting threshold (e.g. 0.05% of the concentration of the test solution) and note the area of the principal peak.

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b) When an impurity requires a correction factor, report the impurity peak if its corrected area is greater than this peak area.

NB: instead  instead of a solution of the substance to be examined at a concentration corresponding to the reporting threshold, it is possible to use the reference solution used for the quantitation of the unspecified impurities (e.g. 0.10% of the concentration of the test solution) and to extrapolate the area of the principal peak to the reporting threshold (e.g. half the peak area obtained with the reference solution at 0.1%).

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• Disregard limit: inject a solution of the substance to be examined at a concentration corresponding to the disregard limit (e.g. 0.05% of the concentration of the test solution) and note the area of the principal peak. Disregard the peaks in the chromatogram obtained with the test solution with an area lower than or equal to this peak area. 

NB: instead  instead of a solution of the substance to be examined at a concentration corresponding to the disregard limit, it is possible to use the reference solution used for the quantitation of the unspecified impurities (e.g. 0.10% of the concentration of the test solution) and to extrapolate the area of the principal peak to the disregard limit (e.g. half the peak area obtained with the reference solution at 0.10%).

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2. Area comparison with a reference solution = dilution of test solution (for impurities having a response factor of 0.8 to 1.2)   + Area comparison with a reference solution = solution of an impurity CRS (for impurities having a response factor outside the range 0.8 to 1.2)

• Disregard limit: inject a solution of the substance to be examined at a concentration corresponding to the disregard limit (e.g. 0.05% of the concentration of the test solution) and note the area of the principal peak.

a) Disregard the peaks in the chromatogram obtained with the test solution having an area lower than or equal to this peak area. 

NB: instead  instead of a solution of the substance to be examined at a concentration corresponding to the disregard limit, it is possible to use the reference solution used for the quantitation of the unspecified impurities (e.g. 0.10% of the concentration of the test solution) and to extrapolate the area of the principal peak to the disregard limit (e.g. half the peak area obtained with the reference solution at 0.1%).

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      if an area comparison is stated, see A. )1.

B) CORRECTION FACTORS STATED FOR ONE OR MORE IMPURITIES

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b) When an impurity requires a correction factor, report the impurity peak if its corrected area is greater than this peak area.

NB: instead  instead of a solution of the substance to be examined at a concentration corresponding to the disregard limit, it is possible to use the reference solution used for the quantitation of the unspecified impurities (e.g. 0.10% of the concentration of the test solution) and to extrapolate the area of the principal peak to the disregard limit (e.g. half the peak area obtained with the reference solution at 0.10%)

• Specified and unspecified impurities: compare the (corrected) areas of the reported individual peaks with (x times) the area of the peak obtained with the relevant reference solution, as stated in the monograph.
• Total: sum up the areas of the reported individual peaks and compare the figure with (y times) the area of the peak obtained with the relevant reference solution, as stated in the monograph.

NORMALISATION PROCEDURE

• Disregard limit / reporting threshold: inject the solution prescribed in the monograph for the disregard limit/reporting threshold. Disregard the peaks in the chromatogram obtained with the test solution having an area lower than or equal to this peak area.
• Specified and unspecified impurities: divide the peak area due to each impurity, in the chromatogram obtained with the test solution, by the sum of the areas of all the reported peaks, including the peak due to the substance to be examined; multiply this result by 100.
• Total: sum up the areas of the reported peaks and divide by the sum of the areas of all the reported peaks, including the peak due to the substance to be examined; multiply this result by 100.

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