15
determination is not accompanied by destruction to a degree that renders them extremely dependent on the actual mode of operation. The possible existence of polymorphism must also be taken into account; differences in the melting point must be indicated even when given under CHARACTERS. In exceptional cases, when the distinction of a specific crystalline form is necessary, determination of the melting point can aid in excluding the unwanted form(s). However, it should be kept in mind that an apparent melting point may be observed: a solid- solid polymorphic transition may take place during testing and the melting point of the resultant form is measured.
For first identification, neither the melting point alone nor the addition of a chemical reaction is sufficient to verify identity of a substance. However, the addition of another identification test such as TLC will often suffice. For second identifications, please refer to part II.4.2.
The melting point determined by the capillary method is defined in the Ph. Eur. (see method 2.2.14. Melting point – capillary method) as the last particle melting point (i.e. clear point or liquefaction point). It must not be confused with the melting interval even though both are given as a range.
II.4.6. Specific optical rotation
When an enantiomer is described in a monograph, a test for optical rotation is given in the IDENTIFICATION section or a cross-reference is made to the test for enantiomeric purity in the TESTS section. When both the racemate (or the racemic mixture) and the enantiomer are available, then, in the monograph of the racemate, an angle of rotation will be given in the TESTS section and will be referred to in the IDENTIFICATION section. When only the racemate is available the angle of rotation will be given in the TESTS section, provided the specific optical rotation of the chiral form is known and is of sufficient magnitude to provide a meaningful test for racemic character.
II.4.7. Thin-layer chromatography
This identification method requires the use of reference substances. Selectivity may be improved by combining TLC with chemical reactions in situ, i.e. by employing appropriate spray reagents. In the latter case, the same or a similar reaction is not to be repeated on a test-tube scale.
It is very important to ensure the separation of a critical pair in a related substances test but such a separation plays a minor role in an identification test. The separation of a critical pair in the individual identification tests is no longer required but the separation of a critical pair in the TESTS section is maintained. However, during development and validation, separation of the substance from similar substances must be demonstrated.
A chromatographic separation test for TLC plates is described in general chapter 4.1.1. Reagents to verify the performance of the plate type concerned. The test is intended to be a quality control procedure, carried out from time to time by the TLC plate user. It is clear that such a general procedure is not appropriate for every thin-layer separation problem and that the description of a separation criterion might still be necessary to ensure the identification of the substance. In these exceptional cases, a separation criterion is described in the IDENTIFICATION section.
16
A TLC system applied to purity testing in a monograph is preferred for identification when suitable. For the latter purpose the concentration of the solution to be examined and the corresponding reference solution is generally reduced so that 5 to 20 μg of each is deposited on the plate or sheet. It may also be necessary to change from a general to a more discriminating detection system.
Further technical requirements on these chromatographic methods are to be found in part II.5.8 on related substances.
II.4.8. Gas chromatography and liquid chromatography
The basic principles mentioned under thin-layer identification apply mutatis mutandis. Gas and liquid chromatography are rarely used for identification and where they are applied, it is by reference to a test or assay that applies the method elsewhere in the monograph. These methods are used only if there is no suitable alternative and are not used as the only identification test.
Further technical requirements on gas and liquid chromatography are to be found in part II.5.8 on related substances.
II.4.9. Chemical reactions
Several commonly applied identification reactions of a chemical nature are included amongst the general methods of the Ph. Eur. and these are to be used, whenever appropriate. Where several reactions for an ion or group are given in general chapter 2.3.1. Identification reactions of ions and functional groups, it is normally necessary to prescribe only one in the monograph. Note the need to specify the amount of material, or solution of it, to be taken for the identification test in question. The same holds true for tests that have to be described in full in the monograph. Identification reactions using toxic reagents are being slowly phased out; special care should be taken when choosing a chemical reaction to be added to a monograph.
Identification criteria that call for the recognition of an odour or a taste are to be avoided.
Each chemical reaction is to be chosen to demonstrate the presence of a different part of the molecule to be identified.
To differentiate substances within a group (family) which differ either by the extent of condensation or by the length of the hydrocarbon chain (e.g. fatty acids), it is necessary to cross-reference to the appropriate purity test(s) where values are determined (e.g. iodine value, saponification value, etc.).
II.5. TESTS II.5.1. General
The TESTS section is principally directed at limiting impurities in chemical substances. General chapter 5.10. Control of impurities in substances for pharmaceutical use gives details of the policy to be applied.
17
While the monograph must ensure adequate purity in the interests of public health, it is not the aim of the Ph. Eur. to impose excessive requirements that restrict unnecessarily the ability of manufacturers to produce compliant products.
In the interests of transparency, information is included wherever possible on: the impurities controlled by a test; the approximate equivalent (percentage, ppm, etc.) of the prescribed limit in terms of the defined impurities or class of impurities. The information on the limit imposed may be a nominal content inferred from the conditions of the test or may be based on data from recovery experiments.
Acceptance criteria and limits are set on the basis of analytical data at hand i.e. batch results provided by manufacturers and data produced during monograph elaboration by the testing laboratories. In order to define limits for tests (e.g. loss on drying, residual water, etc), the empirical “3-sigma” rule may be used. In a normal distribution of values the confidence interval defined by the mean of the values ± 3 times the standard deviation accounts for 99.7 % of the data population. A minimum of 10 test results must be available to calculate the mean. However this rule is not systematically applied, especially for the related substances test where impurity limits should reflect more closely their real content in substances used in approved medicinal products.
Example 1: Determination of specification for water content (2.5.12) ? Batch data provided by manufacturer: 10 batches ? Min. value: 3.2 %, max. value: 5.4 % ? Mean + 3 sigma = 6.1 %
Conclusion: The limit for water is set at 6.1% according to “3-sigma” rule.
Example 2: Determination of specification for impurity X limit
? Batch data for level of impurity X provided by manufacturer: 57 batches ? 52 batches around or less 0.05 % , 4 batches about 0.08 %, 1 batch 0.09 %, ? Mean + 3 sigma = 0.11 %
Conclusion: The rule of “mean + 3 sigma” is not applied. The limit for impurity X is set at 0.10 %, based on batch data.
Certain tests may apply to special grades (parenteral, dialysis solutions, etc.) or a test may have a special limit for a particular use: the particular application of a test/limit is indicated within the test.
II.5.2. Title of tests
Wherever possible, the title includes the impurity or class of impurities limited by the test (e.g. Oxalic acid, Potassium, Copper, Chlorides, etc.). Non-specific tests carry a more general title appropriately chosen from the standard terminology of the Ph. Eur. (e.g. Appearance of solution, pH, Acidity or alkalinity, etc.) or a similar designation. Titles that merely refer to the methodology employed in the test (e.g. Absorbance, Specific optical rotation) are to be avoided wherever possible.
18
II.5.3. Solution S
A solution of the substance to be examined, designated “Solution S”, is prepared whenever this can be used to perform more than one test (and/or identification).
If necessary, several solutions S, (designated S1, S2...) may be prepared in various ways, each being used for at least 2 tests.
For insoluble substances, solution S may be prepared by an extraction process.
The solvent used depends on the purpose of the tests and the solubility of the substance to be examined and that of its potential impurities. It may be:
?
water (usually):
o carbon dioxide-free water R in cases where the presence of carbon dioxide can
appreciably influence the outcome of a test, e.g. for pH or Acidity or alkalinity (see part II.5.5);
o distilled water R if solution S is used in the tests for barium, calcium and sulfates; o carbon dioxide-free water R prepared from distilled water when both previous cases
apply;
a dilute acid or an alkaline solution;
more rarely, other solvents (alcohols, tetrahydrofuran...) that give solutions with a narrower field of application than aqueous solutions.
? ?
The solvent must make it possible to carry out the specified tests, either directly, or after suitable dilutions explicitly specified in each test. Generally the concentration is around 20 to 50 g/L but may be lower (e.g. 10 g/L) or higher (100 g/L and, exceptionally, more). The quantity of solution S prepared must be sufficient to carry out each of the tests for which it has been prepared. If solution S is to be filtered, account must be taken of the loss on filtering and when the insoluble portion thus separated is to be used for another test, this is clearly indicated. While several tests may be carried out on the same portion of solution S, this is only done for substances where there are good reasons to economise (expensive products or products whose use is subject to restrictions) and this is then clearly indicated in the monograph.
Depending on the particular tests, the concentration of solution S is defined with varying accuracy:
? ? ?
for “Appearance of solution”, “pH” and some identifications, an accuracy of 5 to 10 % is sufficient;
for most limit tests an accuracy of about 2 % is appropriate;
for some cases such as the determination of specific rotation, specific absorbance, various chemical values and, more generally, tests where the result is obtained by calculation, a greater accuracy is needed.
The accuracy with which the concentration of solution S is defined is that required by the most exacting test for which it is intended. The description of the preparation of solution S thus specifies:
?
the quantity of substance to be examined with the required accuracy (see General
19
?
Notices);
the volume, to 1 decimal place (10.0 mL, 25.0 mL...) when the concentration must be known to within less than 1 %, without a decimal (10 mL, 25 mL...) when a lower accuracy is adequate.
II.5.4. Appearance of solution
This test makes it possible to ascertain the general purity of a substance by the detection of impurities insoluble in the solvent selected, or of coloured impurities.
The “Appearance of solution” test is practically always prescribed for substances intended for preparations for parenteral use. Apart from this, it is to be applied only if it yields useful information about specific impurities.
It can comprise one or both of the following tests:
? clarity and degree of opalescence of liquids (2.2.1.); ? degree of coloration of liquids (2.2.2.).
The 2 tests are practically always carried out on identical solutions, usually solution S, but they may be performed on different solutions.
The solvent employed is usually water but other solvents may be preferred depending on the solubility of the substance to be examined.
When an organic solvent is used to prepare solution S, it may be necessary to ensure that the solvent also complies with the test, especially where there is a very stringent requirement. The more concentrated the solution the stricter the test. For very pure substances or those used in high doses, the concentration chosen is 50 to 100 g/L, whereas for less pure substances or substances administered in small doses the concentration is 10 to 20 g/L. II.5.4.1. Clarity and degree of opalescence (2.2.1.)
This test is mainly performed on colourless substances or those that give only slightly coloured solutions in order to permit valid comparison with reference suspensions. Newer instruments with ratio selection are capable to measure coloured substances
The quantity of solution required depends on the diameter of the comparison tubes used; it varies from 7 mL to 20 mL for tubes with a diameter of 15 mm to 25 mm prescribed in the general methods. It is therefore necessary to take the latter volume into account.
Most often the solution examined must be “clear” (as defined in the Ph. Eur.). However, in certain cases for substances that are not intended to be used in solution, a more marked opalescence may sometimes be permitted.
II.5.4.2. Degree of coloration (2.2.2.)
The test applies to essentially colourless substances that contain, or may degrade to form, coloured impurities that can be controlled by limiting the colour of solution of the substance. Two methods are described in general method 2.2.2. Degree of coloration of liquids :
