Assays and Tests

Assays and Tests
The assays and tests described are the official methods upon which the standards of the Pharmacopoeia depend. The analyst is not precluded from employing alternative methods, including methods of micro-analysis, in any assay or test if it is known that the method used will give a result of equivalent accuracy. Local reference materials may be used for routine analysis, provided that these are calibrated against the official reference materials. In the event of doubt or dispute, the methods of analysis, the reference materials and the reference spectra of the Pharmacopoeia are alone authoritative.
Where the solvent used for a solution is not named, the solvent is Purified Water.
Unless otherwise prescribed, the assays and tests are carried out at a temperature between 15 °C and 25 °C.
A temperature in a test for Loss on drying, where no temperature range is given, implies a range of ±2 °C about the stated value.
Visual comparative tests, unless otherwise prescribed, are carried out using identical tubes of colourless, transparent, neutral glass with a flat base. The volumes of liquid prescribed are for use with tubes 16 mm in internal diameter; tubes with a larger internal diameter may be used but the volume of liquid examined must be increased so that the depth of liquid in the tubes is not less than that obtained when the prescribed volume of liquid and tubes 16 mm in internal diameter are used. Equal volumes of the liquids to be compared are examined down the vertical axis of the tubes against a white background or, if necessary, against a black background. The examination is carried out in diffuse light.
Where a direction is given that an analytical operation is to be carried out 'in subdued light', precautions should be taken to avoid exposure to direct sunlight or other strong light. Where a direction is given that an analytical operation is to be carried out 'protected from light', precautions should be taken to exclude actinic light by the use of low-actinic glassware, working in a dark room or similar procedures.
For preparations other than those of fixed strength, the quantity to be taken for an assay or test is usually expressed in terms of the active ingredient. This means that the quantity of the active ingredient expected to be present and the quantity of the preparation to be taken are calculated from the strength stated on the label.
In assays the approximate quantity to be taken for examination is indicated but the quantity actually used must not deviate by more than 10% from that stated. The quantity taken is accurately weighed or measured and the result of the assay is calculated from this exact quantity. Reagents are measured and the procedures are carried out with an accuracy commensurate with the degree of precision implied by the standard stated for the assay.
In tests the stated quantity to be taken for examination must be used unless any divergence can be taken into account in conducting the test and calculating the result. The quantity taken is accurately weighed or measured with the degree of precision implied by the standard or, where the standard is not stated numerically (for example, in tests for Clarity and colour of solution), with the degree of precision implied by the number of significant figures stated. Reagents are measured and the procedures are carried out with an accuracy commensurate with this degree of precision.
The limits stated in monographs are based on data obtained in normal analytical practice; they take account of normal analytical errors, of acceptable variations in manufacture and of deterioration to an extent considered acceptable. No further tolerances are to be applied to the limits prescribed to determine whether the article being examined complies with the requirements of the monograph.
In determining compliance with a numerical limit, the calculated result of a test or assay is first rounded to the number of significant figures stated, unless otherwise prescribed. The last figure is increased by 1 when the part rejected is equal to or exceeds one half-unit, whereas it is not modified when the part rejected is less than a half-unit.
In certain tests, the concentration of impurity is given in parentheses either as a percentage or in parts per million by weight (ppm). In chromatographic tests such concentrations are stated as a percentage irrespective of the limit. In other tests they are usually stated in ppm unless the limit exceeds 500 ppm. In those chromatographic tests in which a secondary spot or peak in a chromatogram obtained with a solution of the substance being examined is described as corresponding to a named impurity and is compared with a spot or peak in a chromatogram obtained with a reference solution of the same impurity, the percentage given in parentheses indicates the limit for that impurity. In those chromatographic tests in which a spot or peak in a chromatogram obtained with a solution of the substance being examined is described in terms other than as corresponding to a named impurity (commonly, for example, as any (other) secondary spot or peak) but is compared with a spot or peak in a chromatogram obtained with a reference solution of a named impurity, the percentage given in parentheses indicates an impurity limit expressed in terms of a nominal concentration of the named impurity. In chromatographic tests in which a comparison is made between spots or peaks in chromatograms obtained with solutions of different concentrations of the substance being examined, the percentage given in parentheses indicates an impurity limit expressed in terms of a nominal concentration of the medicinal substance itself. In some monographs, in particular those for certain formulated preparations, the impurity limit is expressed in terms of a nominal concentration of the active moiety rather than of the medicinal substance itself. Where necessary for clarification the terms in which the limit is expressed are stated within the monograph.
In all cases where an impurity limit is given in parentheses, the figures given are approximations for information only; conformity with the requirements is determined on the basis of compliance or otherwise with the stated test.
The use of a proprietary designation to identify a material used in an assay or test does not imply that another equally suitable material may not be used.
Biological Assays and Tests
Methods of assay described as Suggested methods are not obligatory, but when another method is used its precision must be not less than that required for the Suggested method.
For those antibiotics for which the monograph specifies a microbiological assay the potency requirement is expressed in the monograph in International Units (IU) per milligram. The material is not of pharmacopoeial quality if the upper fiducial limit of error is less than the stated potency. For such antibiotics the required precision of the assay is stated in the monograph in terms of the fiducial limits of error about the estimated potency.
For other substances and preparations for which the monograph specifies a biological assay, unless otherwise stated, the precision of the assay is such that the fiducial limits of error, expressed as a percentage of the estimated potency, are within a range not wider than that obtained by multiplying by a factor of 10 the square roots of the limits given in the monograph for the fiducial limits of error about the stated potency.
In all cases fiducial limits of error are based on a probability of 95% (P = 0.95).
Where the biological assay is being used to ascertain the purity of the material, the stated potency means the potency stated on the label in terms of International Units (IU) or other Units per gram, per milligram or per millilitre. When no such statement appears on the label, the stated potency means the fixed or minimum potency required in the monograph. This interpretation of stated potency applies in all cases except where the monograph specifically directs otherwise.
Where the biological assay is being used to determine the total activity in the container, the stated potency means the total number of International Units (IU) or other Units stated on the label or, if no such statement appears, the total activity calculated in accordance with the instructions in the monograph.
Wherever possible the primary standard used in an assay or test is the respective International Standard or Reference Preparation established by the World Health Organization for international use and the biological activity is expressed in International Units (IU).
In other cases, where Units are referred to in an assay or test, the Unit for a particular substance or preparation is, for the United Kingdom, the specific biological activity contained in such an amount of the respective primary standard as the appropriate international or national organisation indicates. The necessary information is provided with the primary standard.
Unless otherwise directed, animals used in an assay or a test are healthy animals, drawn from a uniform stock, that have not previously been treated with any material that will interfere with the assay or test. Unless otherwise stated, guinea-pigs weigh not less than 250 g or, when used in systemic toxicity tests, not less than 350 g. When used in skin tests they are white or light coloured. Unless otherwise stated, mice weigh not less than 17 g and not more than 22 g.
Certain of the biological assays and tests of the Pharmacopoeia are such that in the United Kingdom they may be carried out only in accordance with the Animals (Scientific Procedures) Act 1986. Instructions included in such assays and tests in the Pharmacopoeia, with respect to the handling of animals, are therefore confined to those concerned with the accuracy and reproducibility of the assay or test.