BASIC TESTS FOR PHARMACEUTICAL SUBSTANCESThe World Health Organization is a specialized agency of the United Nations with primary responsibility for international health matters and public health. Through this organization, which was created in 1948, the health professions of some 165 countries exchange their knowledge and experience with the aim of making possible the attainment by all citizens of the world by the year 2000 of a level of health that will permit them to lead a socially and economically productive life By means of direct technical cooperation with its Member States, and by stimulating such cooperation among them, WHO promotes the development of comprehensive health services, the prevention and control of diseases, the improvement of environmental conditions, the development of health manpower, the coordination and development of biomedical and health services research, and the planning and implementation of health programmes. These broad fields of endeavour encompass a wide variety of activities, such as developing systems of primary health care that reach the whole population of Member countries; promoting the health of mothers and children; combating malnutrition; controlling malaria and other communicable diseases, including tuberculosis and leprosy; having achieved the eradication of smallpox, promoting mass immunization against a number of other preventable diseases, improving mental health; providing safe water supplies; and training health personnel of all categories. Progress towards better health throughout the world also demands international cooperation in such matters as establishing international standards for biological substances, pesticides, and pharmaceuticals, formulating environmental health criteria, recommending international nonproprietary names for drugs, administering the International Health Regulations; revising the International Classification of Diseases, Injuries, and Causes of Death; and collecting and disseminating health statistical information. Further information on many aspects of WHO's work is presented in the Organization's publications.Basic tests for pharmaceutical substancesWORLD HEALTH ORGANIZATION PUBLICATIONS RELATING TO QUALITY ASSURANCE OF PHARMACEUTICAL PRODUCTS The International Pharmacopoeia A collection of recommended methods of analysis and specifications for pharmaceutical preparations. Third Edition. Volume 1, General methods of analysis 1979, 223 pages (E/F/S/R) Sw. fr, 24— Volume 2, Quality specifications 1981, 342 pages (E/F/S) Sw. fr. 36.— Volume 3, Quality specifications, in press, 1986 WHO Technical Report Series, No. 704 (Twenty-ninth report of the WHO Expert Committee on Specifications for Pharmaceutical Preparations) 1984 (E, F, S) Sw. fr. 6— Annex 1 of this report gives advice on the organization and starting of national laboratories for drug quality surveillance and control. Information on a certification scheme on the quality of pharma- ceutical products moving in international commerce and good practices in the manufacture and quality control of drugs is given in the unpublished WHO document, PHARM/82.4/Rev. 2, available on request from Pharmaceuticals, World Health Organization, 1211 Geneva 27, Switzerland.BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES @ World Health Organization GENEVA 1986ISBN 92 4 154204 7 © World Health Organization, 1986 Publications of the World Health Organization enjoy copyright protection in accord- ance with the provisions of Protocol 2 of the Universal Copyright Convention. For rights of reproduction or translation of WHO publications, in part ot in toto, application should be made to the Office of Publications, World Health Organization, Geneva, Switzerland. The World Health Organization welcomes such applications. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the Jegal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. ‘TYPESET IN INDIA PRINTED IN ENGLAND 85/5956 Macmillan/Spottiswoode 7000. Introduction CONTENTSThe tests described in this manual are intended only to verify the identity of pharmaceutical substances and to detect or exclude gross degradation. They should not be used to replace the monographs contained in the International Pharmacopoeia. As a first step, tests have been devised for the majority of drug substances contained in the WHO Model List of Essential Drugs as well as for a number of other widely used drug substances. Analogous tests for substances in dosage forms are in preparation.1. INTRODUCTION The simplified tests (basic tests) for drug substances described in this manual represent one element of quality assurance in the pharmaceutical supply system. They have been devised with the following objectives: (a) to provide a simple and readily applicable method for verifying the identity of a drug substance, using a limited range of widely available reagents, when the labelling and physical attributes of the substance do not provide adequate confirmation; (6) to provide a practicable means for confirming the identity of a drug, when a fully equipped laboratory is not available; (c) to indicate if gross degradation has occurred in certain sub- stances that are known to decompose readily under adverse conditions. Basic tests are not, in any circumstances, intended to replace the requirements of pharmacopoeial monographs. The latter give an assurance of quality whereas basic tests merely confirm identity. In several European countries, simple tests have already been endorsed by the national pharmaceutical associations for use at peripheral levels of distribution (wholesale premises, pharmacies) to verify the identity of pharmaceutical substances whenever the possibility of confusion arises and sometimes to exclude gross degradation or adulteration. Degradation during storage and transportation is of particular importance in tropical countries. Indeed, an expiry date determined for a temperate climate may be inappropriate in a tropical region even when adequate containers are used. For this reason, the stability charac- teristics of most of the substances referred to in this manual have been determined and tests to indicate gross degradation are presented for the least stable substances. Basic tests need not be carried out by fully qualified pharmacists or chemists, but they should be performed by persons who have some understanding of analytical chemistry such as that acquired in courses for pharmaceutical assistants. Several tests are described for most substances, Not all of these need to be applied to any one sample. One test concerned with melting2 BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES behaviour, together with two test-tube reactions, will suffice in most circumstances, If, however, there is any reason to suspect that the product is mislabelled or substandard, all tests described should be performed. By their nature, simplified tests cannot be completely reliable. An adverse result, even in one test, should be taken as a warning of potential unsuitability of a drug. In these circumstances, a final conclusion should not be drawn until a full analytical examination has been carried out in a properly equipped analytical laboratory. The reagents and equipment required for these tests have been kept to a minimum. Reagents that are unstable, corrosive, expensive or difficult to obtain have been excluded. The use of visual or similar characteristics for initial assessment is self-evident. Attributes such as colour and characteristic odour of the sample should always be noted. Some of the individual tests, such as those referring to a change in the physical aspect of the test substance, call for comparison against a standard. Each laboratory where basic tests are routinely performed should therefore retain a collection of authentic samples of frequently analysed substances. Such a collection may be established from small amounts of substances previously tested and found to be satisfactory. These provide a standard of comparison both for visual characteristics and test-tube reactions. In general, the following types of test have been preferred: —classical chemical techniques such as colour reactions, the formation of precipitates with specific reagents, the evolution of gas and its identification, and the behaviour of substances on heating. In some instances these reactions lack specificity since drugs with similar functional groups may not be distinguishable by simple chemical reactions; —the appearance of a concentrated solution of a substance in selected solvents. This can be useful for detecting degradation products and the presence of some other impurities. The techniques used to determine melting characteristics are described in particular detail. They provide a basis not only for confirming the identity of a substance but also for detecting possible contamination, whether arising from poor manufacture, adulteration, cross-contamination during storage, or degradation. The capillary method has been selected for use in basic tests. Other techniques, such as the microscopic hot bench or melting block, are applicable, but the results of different methods are not directly comparable. It should be noted that the terms used to describe the melting characteristics are different from those adopted in the International Pharmacopoeia, since pharmacopoeial specifications are based on more rigorous methods.1. INTRODUCTION Basic tests for finished pharmaceutical forms are planned to follow this publication. Comments on the tests described are invited and should be addressed to: Pharmaceuticals, World Health Organization, 1211 Geneva 27, Switzerland.2. DETERMINATION OF MELTING CHARACTERISTICS 2.1 Determination of melting point 2.1.1 Definition The melting point is determined in a capillary tube. The expression “melts about...” means that the temperature at which the substance is completely melted, as indicated by the disappearance of the solid, will be in the range +4°C from the stated value, unless otherwise indicated. 2.1.2 Details of the procedure The following technique is adequate for the determination of melting point: Grind about 50 mg of the substance to be tested in a small mortar. Place the ground substance in a vacuum desiccator over silica gel or phosphorus pentoxide at room temperature and dry for about 24 hours (unless another drying procedure is given in the test sheet). Place the substance in a dry capillary tube of I-mm internal diameter forming a column about 3mm high. Heat the melting-point apparatus to a temperature 5-10°C below the expected temperature of melting and adjust the heating so that the temperature in the chamber rises about 1°C per minute. Introduce the capillary with the substance into the heated chamber, and note the temperature when the sintered substance becomes completely transparent; this is considered to be the melting point, as defined in section 2.1.1. 2.1.3 Discussion The difference between the purely theoretical definition of the melting temperature and the results obtained in practice is now widely recognized. A precise physical definition exists only for the so-called triple point, i.e., the temperature at which all three phases (solid, liquid and gaseous) are in equilibrium. The measurement of the triple point is done in a highly complicated experiment. Many compendia do not use this temperature, but describe melting intervals as observed in practice,2. DETERMINATION OF MELTING CHARACTERISTICS 5 when the formation of droplets, the softening of the substance o: sintering are considered to be the beginning of the melting process, while the formation of a clear and transparent drop of liquid is taken to be the end of the melting process. In the case of pure substances that melt without decomposition, the beginning of melting can be observed with some certainty. For impure substances, the beginning of the melting process will vary, depending on the nature of the impurities. Therefore it has been proposed that in the basic tests the following definition of melting point be used. This definition is similar to that used in the /nternational pharmacopoeia,’ to describe melting temperature: The melting point denotes the temperature at which the substance has just completely melted; this is indicated by the disappearance of the solid phase and complete transparency of the melt. This approach has the disadvantage that, if impurities are present, their presence can only be deduced from the lowering of the melting- point value, as no observation is made of the melting interval. An increase in the latter usually indicates low purity of a substance. These considerations, however, have less importance for basic test identifi- cation, where this disadvantage is fully offset by increased reproducibility of the values of melting point determined according to the above procedure. 2.2 Melting behaviour 2.2.1. Definition The expression “melting behaviour” used in the basic tests denotes the melting point of substances that melt with decomposition. It is also used for melting points above 250°C to indicate that the reproducibility of the value may be low. 2.2.2 Discussion It is necessary to bear in mind that a difference exists between true melting points (or melting ranges) and the temperature of decomposition. Ideally, in the case of a true melting point, no chemical change occurs in the substance. However, when some substances are heated, de- composition takes place either before or during the process of melting, being indicated by a change in the colour of the substance or by the evolution of a gas. In such situations, the observed temperature of 4 The international pharmacopoeia. Third edition. Volume 1: General methods of analysis. Geneva, World Health Organization, 1979; Volume 2: Quality specifications. Geneva, World Health Organization, 19816 BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES melting is not a true melting point of the substance but the melting point of a mixture with decomposition products. It is obvious that the temperature of decomposition cannot be considered as a physical property of a substance as the amount of decomposition products, and consequently the temperature of decomposition, depend on the length of the period of heating and therefore have low reproducibility, even if a standardized procedure is used. 2.3 Determination of eutectic temperature 2.3.1 Definition Eutectic temperature is given as a single value only and designates the beginning of melting, ie., the temperature at which the solid collapses or forms drops on the wall of the capillary tube. The mixture to be used in the test is usually prepared by thorough mixing of approximately equal parts of the test substance and the accessory substance, unless the use of strictly equal amounts of both substances is specially indicated in the test procedure. 2.3.2 Details of the procedure The following technique is adequate for the determination of eutectic temperature: Grind equal parts (by weight) of the substance to be tested and the accessory substance, both of them previously dried for about 24 hours at room temperature in a vacuum desiccator over silica gel or phosphorus pentoxide. Fill a dry capillary tube, of 1-mm internal diameter, with the mixture, forming a column about 3mm high. Heat the melting-point apparatus to a temperature 5—10°C below the expected temperature of melting and adjust the heating so that the temperature in the chamber rises about 1°C per minute. Introduce the capillary with the mixture into the heated chamber, and note the temperature at which the solid collapses or forms droplets on the wall of the capillary tube. 2.3.3 Discussion The measurement of eutectic temperature has been introduced in the basic tests as an additional criterion of identity. An exact determination of the eutectic melting point requires a set of measurements carried out on mixtures prepared in different ratios, The eutectic melting point thus measured is thermodynamically exactly defined and may be used as a criterion of both identity and purity. Such a procedure is not, however, practical for the basic test project, as it requires a long time and adequate laboratory facilities. For the purpose of basic tests, the determination is carried out at a constant ratio of 1:1. This has the disadvantage that in some cases the melt will not become transparent, so2, DETERMINATION OF MELTING CHARACTERISTICS, 7 that the reproducibility of the measurement is low owing to individual errors. Nevertheless, the eutectic temperatures given in the basic tests are usually reproducible to within +5 °C. It should be noted that during eutectic temperature determination the beginning of the process of melting is observed, whereas during melting- point determination it is the end of the process that is surveyed. 2.4 Mixed-melting point 2.4.1 Procedure The determination of a mixed-melting point is carried out in a glass capillary as described in section 2.1.2. Equal amounts of the substance to be tested and the authentic substance are mixed and placed in a capillary. A separate capillary is filled with the substance to be tested and a further capillary with the authentic substance. All three capillaries are simultaneously heated in the melting-point apparatus. The melting point of the mixture should not differ by more than +4°C from the melting points of the single substances. 2.4.2 Discussion Although mixed-melting-point determinations are not included in the basic tests, this procedure is a highly reliable criterion in deciding whether two substances are identical. The general introduction of mixed- melting-point determination as an identity test would require a wide accessibility of appropriate reference substances, which can sometimes only be arranged on a national basis. Each laboratory can, however, gradually create for itself a collection of authentic substances from incoming consignments of materials of good quality and can then use the mixed-melting point as a strong additional criterion of identity. Such a collection, once established, may further be used in identity tests using the thin-layer chromatography technique. 2.5 Melting-point apparatus 2.5.1 Type of apparatus A number of types of melting-point apparatus are produced. A review of those that are commercially available is given by Bichi & Hasler.* The apparatus employed in the determination should be equipped with a magnifying glass, have a controlled heating arrangement that * BUcHI, J. & HASLER, C. Pharmaceutica acta Helvetiae, 49: 47 (1974).8 BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES: permits a heating rate of 1-2 °C/min around the temperature of melting, and be equipped to be used with capillaries of 1-mm inner diameter. The heating arrangement can take the form of a stirred bath, such as the Thiele apparatus and its modifications,* or a heated block, e.g., the Lindstrom or Culatti modifications.’ 2.5.2 Calibration of thermometers For the various measurements of melting characteristics to be of any value, it is essential to use accurate thermometers. The thermometer used should preferably be certified by a duly recognized body. Alternatively, it could be calibrated against such a thermometer. Another method of checking the accuracy of the thermometer is by measuring the melting points of a set of WHO melting point reference substances using a 1-mm capillary; if the observed melting points of the reference substances lie within 2°C of the melting temperature indicated for that substance, the thermometer may be considered satisfactory. An important requisite, however, is that the geometrical arrangement of the thermometer and capillaries in the apparatus is practically identical in every determination. The length of the column of mercury in the thermometer exposed to room temperature can introduce significant error particularly at high temperature. It is therefore desirable to use thermometers with narrow ranges of temperature such as 0-110°C, 110-210°C or 200-300°C. If this is not possible, a correction factor should be introduced according to the formula given in the International pharmacopoeia, third edition (volume 1, p. 22). 2.6 Heating behaviour The expression “heating behaviour” used in the basic tests denotes the behaviour of the substance (such as colour changes or evolution of gas) when heated in an open test-tube in a flame or in an electrical heater. * SKAN, EL. & ARTHUR, J. C. R. In: Weissberger, A., ed. Technique of organic chemisiry, New York, Interscience, 1971, vol. I, p. 105. ® Kienitz, H. In: Houben-Weyl, Methoden der organischen Chemie, Stuttgart, Georg Thieme Verlag, 1953, Vol. II, p. 788.3. TESTS ACETAZOLAMIDE Identity tests Description. A white, or almost white, crystalline powder; odourless. Melting behaviour. About 255°C with decomposition. Colour and other reactions 1, Triturate 0.5 g with a mixture of 5 ml of water and 0.5 ml of sodium hydroxide (~ 80 g/l) TS, add 0.2 g of zinc R powder and about 0.5 ml of hydrochloric acid (~ 420 g/l) TS; hydrogen sulfide is evolved and may be detected by its odour (proceed with caution), or by the use of a strip of lead nitrate paper R, which turns black on exposure. 2. Dissolve 25 mg in 5 ml of water and add 0.10 ml of sodium hydroxide (~ 80 g/l) TS and 0.05 ml of copper(II) sulfate (160 g/l) TS; a bluish green colour or precipitate is formed. ACETYLSALICYLIC ACID Identity tests Description. Colourless crystals or a white, crystalline powder; odour- less or almost odourless. Eutectic temperature. With acetanilide R, about 85 °C. Heating behaviour. Heat a small quantity in a test-tube; it melts quickly. The melt has a strong odour of acetic acid. On further heating, the colour of the melt changes from yellow to brown and finally to black. Colour and other reactions Heat 0.05 g in 2.0 ml of water for several minutes, cool and add | or 2 drops of ferric chloride (25 g/l) TS; a violet-red colour is produced which does not change on the addition of ethanol (~ 750 g/l) TS.10 BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES, Degradation test A strong odour of acetic acid on opening the container of the test substance and non-compliance with the following test usually indicate gross degradation: Dissolve 0.10 g in sufficient ethanol (~ 750 g/l) TS to produce 50 ml. Transfer 5 ml to a comparison tube. To serve as a reference, dissolve separately 10 mg of salicylic acid R in sufficient ethanol (~ 750 g/l) TS to produce 100ml. Transfer 1.0ml of this solution to a second comparison tube and add 4 ml of ethanol (~ 750 g/l) TS. Add 15 ml of water to both tubes. Dilute separately 1.0 ml of ferric chloride (25 g/l) TS to 5 ml and transfer 0,05 ml of this reagent solution to both tubes. Mix and allow to stand for 1 minute; the violet colour produced in the solution of the test substance should not be more intense than that obtained with the reference solution. ACRIFLAVINIUM CHLORIDE Identity tests Description. An orange-red powder; odourless. Melting behaviour. About 260°C with decomposition. Colour and other reactions 1. Dissolve 10 mg in 100 ml of water; an intense yellow colour with a green fluorescence is produced. Add a few drops of hydrochloric acid (~ 70 g/l) TS; the fluorescence disappears. Then add a few drops of sodium nitrite (10 g/l) TS; an intense purple colour is produced. 2. Dissolve 0.04 g in 10 ml of water and use this solution for tests 2, 3 and 4. Dilute 2.0 ml with 6 ml of water and add a few drops of methyl orange/ethanol TS; a red solution is produced. 3. To 2.0 ml of the solution prepared in test 2 add a few drops of sodium salicylate (100 g/l) TS; an orange-yellow precipitate is formed (distinction from fluorescein). 4. To Sml of the solution prepared in test 2 add a few drops of formaldchyde TS and 5ml of sodium nitrite (100 g/l) TS; a brownish precipitate is produced. Allow the mixture to stand for 5 minutes and then filter; the filtrate acquires a cherry red colour (distinction from the methylated diaminoacridine compounds). 5. Dissolve 5 mg in 5 ml of water and filter. To the filtrate add 1.0 ml of nitric acid (~ 130 g/l) TS and a few drops of silver nitrate (40 g/l) TS; a white, curdy precipitate is produced. Separate the precipitate, wash it with water and add an excess of ammonia (~ 100 g/l) TS; the precipitate dissolves.3. TESTS ll AJMALINE Identity tests Description. A white, or almost white, crystalline powder; odourless. Colour and other reactions 1. To 10 mg add about 0.5 ml of nitric acid (~ 1000 g/l) TS; a deep red colour is produced, which remains on the addition of 10 ml of water. 2. To 10mg add 1 ml of sulfuric acid (~ 1760 g/l) TS and 1.0 ml of ammonium molybdate (95 g/l) TS; a red-violet colour is produced, which turns to blue-violet. 3. Dissolve 10 mg in 0.10 ml of hydrochloric acid (~ 70 g/l) TS and add 5 ml of water and 0.1 ml of ferric chloride (25 g/l) TS; a red colour is produced. ALLOPURINOL Identity tests Description. A white, or almost white, crystalline; powder; odourless or almost odourless. Colour and other reactions 1. Dissolve 0.05 g in 5 ml of sodium hydroxide (~ 80 g/l) TS, and 1.0 ml of alkaline potassio-mercuric iodide TS, heat to boiling and allow to stand; a yellow, flocculent precipitate is produced. 2. Dissolve 0.20 g in a mixture of 2.0 ml of sodium hydroxide (~ 80 g/1) TS and 2.0 ml of water. Add 3.0 ml of citric acid (90 g/l) TS and shake vigorously; a white precipitate is produced. ALUMINIUM DIACETATE Identity tests Description. A white, fine powder; slight odour of acetic acid. Colour and other reactions 1. Dissolve 0.5 g by heating in 5 ml of sodium hydroxide (~ 80 g/l) TS. To the clear solution add 0.5g of ammonium chloride R; a white, gelatinous precipitate is produced. 2. Heat 0.10g with 5ml of sulfuric acid (~ 100 g/l) TS; an odour of acetic acid is evolved. 3. Shake 1.0g with 20ml of freshly boiled and cooled water forn BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES 1 minute; the filtrate is slightly acid when tested with pH-indicator paper R. ALUMINIUM HYDROXIDE Identity tests Description. A white, fine, amorphous powder; odourless. Colour and other reactions 1, Dissolve 0.10 g by heating in 5 ml of sodium hydroxide (~ 80 g/l) TS. To the clear solution add 0.5g of ammonium chloride R; a white, gelatinous precipitate is produced. 2. Shake 1.0g with 20ml of freshly boiled and cooled water for 1 minute and filter; the filtrate is neutral when tested with pH-indicator paper R. AMILORIDE HYDROCHLORIDE Identity tests Description. A pale yellow to greenish yellow powder; odourless or almost odourless. Melting behaviour, About 292°C with decomposition (turns brown- k). Eutectic temperature. With dicyandiamide R, about 178°C; with phenolphthalein R, about 241 °C. Colour and other reactions 1. Dissolve 10 mg in 2.0 ml of water, add 1.0 ml of hydrochloric acid (~ 70 gf) TS and 4—5 drops of sodium nitrite (10 g/l) TS, shake for 2 minutes and add a solution of 10 mg of 2-naphthol R dissolved in 2.0 ml of sodium hydroxide (~ 80 g/l) TS; a reddish brown precipitate is produced. Add a few drops of hydrochloric acid (~ 70 g/l) TS; a greenish yellow precipitate separates. 2. Dissolve 20 mg in 50 ml of freshly boiled and cooled water and add 0.5 ml of nitric acid (~ 130 g/l) TS and a few drops of silver nitrate (40 g/l) TS; a white precipitate is produced. Separate the precipitate, wash it with water and add an excess of ammonia (~ 100 g/l) TS; the precipitate dissolves. 3. Dissolve 0.20 g in 25 ml of water and add 4 ml of sodium hydroxide (~ 80 g/l) TS; a light yellow precipitate is produced. Filter, wash the3. TESTS 13 precipitate twice with 10 ml portions of water and once with 10ml of acetone R and dry at 105°C for 2 hours; melting behaviour, about 245°C with decomposition (turns brown at 200°C). (Keep the remain- ing free base for test 4.) 4. In a test-tube fuse 0.05 g of the free base obtained in test 3 with 0.05 g of potassium nitrate R and 0.10g of potassium hydroxide R. Allow to cool, dissolve the residue in 15 ml of water and filter. To 5 ml of the filtrate add 2.0 ml of nitric acid (~ 130 g/l) TS and a few drops of silver nitrate (40 g/l) TS; a white precipitate is formed. AMINOCAPROIC ACID Identity tests Description. Colourless crystals or a white powder; odourless. Melting behaviour. About 205°C with decomposition. Colour and other reactions 1. Dissolve 2.0 g in water and dilute to 20.0 ml and use this solution for tests 1 and 2. To 2.0 ml add 0.1 m! of ferric chloride (25 g/l) TS; an orange-brown colour is produced. 2. To 2.0 ml of the solution prepared in test 1 add 0.1 ml of copper(II) sulfate (160 g/l) TS; a deep blue colour is produced. Add 1.0 ml of sodium hydroxide (~ 80 g/l) TS; the solution remains clear at first with a deep blue colour, but on standing turns turbid. AMINOPHENAZONE Identity tests Description. Colourless, glittering small crystals or a white powder; odourless. It is affected by light, the colour changing to yellow. Melting point. About 108°C. Colour and other reactions 1. Dissolve 0.05 g in Sml of water and add a few drops of ferric chloride (25 g/l) TS; a blue-violet colour is produced, which on the addition of a few drops of sulfuric acid (~ 100 g/l) TS, changes to red- violet. 2. Dissolve 0.05 g in 5 ml of water and add 10 drops of silver nitrate (40 gf) TS; a blue-violet solution is produced, that develops after 1 minute into a greyish black precipitate of metallic silver. 3. Dissolve 0.05g in Sml of water and add 1.0 ml of sulfuric acid14 BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES. (~ 100g/l) TS and 5 drops of freshly prepared sodium nitrite (10/1) TS; a blue colour is produced which fades quickly. Warm the solution; no green colour is observed. AMINOPHYLLINE Identity tests Description. White or slightly yellowish granules or a powder; odour, slightly ammoniacal. Eutectic temperature. With dicyandiamide R, about 184°C; with phenolphthalein R, about 220°C. Colour and other reactions 1, Dissolve 1.0g in 10 ml of water and, while shaking, add 2.0 ml of hydrochloric acid (~ 70 g/l) TS drop by drop. Collect the precipitate on a filter, wash it with water and dry at 105°C for 1 hour; melting point, about 272°C. (Keep the remaining precipitate for test 2.) 2. To 10mg of the precipitate obtained in test 1, contained in a porcelain dish, add 1 ml of hydrochloric acid (~ 250 g/l) TS and about 0.5 ml of hydrogen peroxide (~ 330 g/l) TS, and evaporate to dryness on a water-bath. Add 1 drop of ammonia (~ 100 g/l) TS; the residue acquires a purple colour which is destroyed by the addition of a few drops of sodium hydroxide (~ 80 g/l) TS. Degradation test If the test substance does not pass the following test, this usually indicates that gross degradation has occurred: Dissolve 0.20 g in 5ml of water; a clear and colourless solution is produced. AMITRIPTYLINE HYDROCHLORIDE Identity tests Description. Colourless crystals or a white, or almost white, powder; odourless or almost odourless. Melting point. About 197°C. Colour and other reactions 1. To Smg add 2ml of sulfuric acid (~ 1760 g/l) TS; an orange-red3. TESTS 15 colour is produced. Add 0.1 ml of formaldehyde TS; the colour turns to brown. 2. Dissolve 0.10g in Sml of water and add 1.0ml of nitric acid (~ 130 g/l) TS and 2.0 ml of silver nitrate (40 g/l) TS; a white, curdy precipitate is produced. Separate the precipitate, wash it with water and add an excess of ammonia (~ 100 g/l) TS; the precipitate dissolves. Degradation test If the test substance does not pass the following test, this usually indicates that gross degradation has occurred: Dissolve 2.0 g in 5 ml of water; a clear and almost colourless solution is produced. AMMONIUM CHLORIDE Identity tests Description. Colourless crystals or a white powder; odourless. Colour and other reactions 1. To 0.10 g add 2.0 ml of sodium hydroxide (~ 80 g/l) TS; an odour of ammonia is perceptible. Insert moistened pH-indicator paper R into the vapour; its coloration is changed to the alkaline range. 2. Dissolve 0.10g in Sml of water and add 1.0 ml of nitric acid (~ 130 g/l) TS and 2.0 ml of silver nitrate (40 g/l) TS; a white, curdy precipitate is produced. Separate the precipitate, wash it with water and add an excess of ammonia (~ 100 g/l) TS; the precipitate dissolves. Degradation test Discoloration and a change in the physical aspect of the test substance usually indicate gross degradation. AMOBARBITAL Identity tests Description. A white powder, odourless. Melting point. About 156°C.16 BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES Colour and other reactions 1. Dissolve 20 mg in 2.0 ml of methanol R and add 1 drop of cobalt(II) chloride (30 g/l) TS and 1 drop of ammonia (~ 100 g/l) TS; a violet colour is produced. 2. Dissolve 10 mg in 10 ml of water by warming. After cooling add the solution to a mixture composed of 0.5 ml of potassium bromate (15 g/!) TS, 0.05 of potassium bromide R and 1.0 ml of hydrochloric acid (~ 70 g/l) TS; a stable brown colour is produced. AMODIAQUINE HYDROCHLORIDE Identity tests Description. A yellow, crystalline powder; odourless. Melting behaviour. About 158°C with slight evolution of gas. Eutectic temperature. With benzanilide R, about 139°C. Colour and other reactions 1. Add 0.10 g to 2 ml of nitric acid (~ 1000 g/l) TS; a red solution is produced. 2. Dissolve 0.3 g in 50 ml of water and filter. Make the filtrate alkaline with about 1 ml of ammonia (~ 100 g/l) TS and allow to stand for 30 minutes. Filter, wash the filter with water until free from chlorides and dry the residue at 105°C for 2 hours; the residue melts at about 205 °C with decomposition and discoloration. 3. Dissolve 0.05g in 5ml of water and add 0.5 ml of nitric acid (~ 130 g/l) TS and 0.5 ml of silver nitrate (40 g/l) TS; a white, curdy precipitate is produced. Separate the precipitate, wash it with water and add an excess of ammonia (~ 100 g/l) TS; the precipitate dissolves. AMPHOTERICIN B Identity tests Description. A yellow to orange powder; odourless or almost odourless. Melting behaviour. It decomposes above 170°C but does not melt below 320°C. Colour and other reactions 1, Dissolve about 1 mg in 2.0 ml of dimethyl sulfoxide R and introduce 5ml of phosphoric acid (~ 1440 g/l) TS to form a lower layer; a blue ring is immediately formed at the interface of the two liquids. Mix the3. TESTS 7 solution; a strong blue colour is produced. Add 10 ml of water and mix; the colour of the solution changes to pale yellow. 2. Dissolve 5mg in Sml of dimethyl sulfoxide R and add 0.5 ml of bromine TS; the colour of the bromine is discharged. Degradation test If the test substance does not pass the following test, this usually indicates that gross degradation has occurred: Dissolve 30 mg in 10 mi of methanol R; a clear and not darker than light yellow coloured solution is produced. AMPICILLIN Identity tests Description. A white, or almost white, crystalline powder; odourless or almost odourless. Melting behaviour. About 204°C with decomposition, discoloration and evolution of gas. Eutectic temperature. With dicyandiamide R, about 172°C. Colour and other reactions 1. Dissolve Smg in 3ml of water, add 0.10g of hydroxylamine hydrochloride R and 1.0m of sodium hydroxide (~ 80g/1) TS and allow to stand for 5 minutes. Then add 1.3 ml of hydrochloric acid (~ 70 gil) TS and 10 drops of ferric chloride (25 g/l) TS; a violet-red to violet-brown colour is produced. 2. Dissolve 10mg in 1.0ml of water and add 2.0ml of a mixture composed of 2.0 ml of potassio-cupric tartrate TS and 6 ml of water; a red-violet colour is produced. 3. Dissolve 5 mg in 1.0 ml of water and add 1-2 drops of ferric chloride (25 gf) TS; a clear, yellowish solution is produced. 4. (@) Add about 1 mg to a solution of 10 mg of paraformaldehyde R in 1 ml of sulfuric acid (~ 1760 g/l) TS; a colourless solution is produced. (6) Heat the solution from test 4(a) in a water-bath for 2 minutes and cool; a yellow colour is produced. Degradation test If the test substance does not pass the following test, this usually indicates that gross degradation has occurred:18 BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES Dissolve 0.10 g in 40 ml of water; a clear and colourless solution is produced. AMPICILLIN SODIUM Identity tests Description. A white, or almost white, powder; odourless; hygroscopic. Melting behaviour. About 220°C with decomposition (turns black) and evolution of gas. Eutectic temperature. With dicyandiamide R, about 163°C. Colour and other reactions 1, Dissolve Smg in 3ml of water, add 0.10g of hydroxylamine hydrochloride R and 1.0m! of sodium hydroxide (~ 80 g/l) TS and allow to stand for 5 minutes. Then add 1.3 ml of hydrochloric acid (~ 70 g/l) TS and 10 drops of ferric chloride (25 g/l) TS; a violet-red to violet-brown colour is produced. 2. Dissolve 10mg in 1.0 ml of water and add 2.0ml of a mixture composed of 2.0 ml of potassio-cupric tartrate TS and 6 ml of water; a red-violet colour is immediately produced, which changes to green after a few seconds. 3. Dissolve 5 mg in 1.0 ml of water and add 1-2 drops of ferric chloride (25 g/l) TS; a clear, yellowish solution is produced. 4. (a) Add about 1 mg to a solution of 10 mg of paraformaldehyde R in 1 mi of sulfuric acid (~ 1760 g/l) TS; a colourless solution is produced. (6) Heat the solution from test 4(a) in a water-bath for 2 minutes and cool; a yellow colour is produced. 5. Dissolve 20 mg in 2.0 ml of water, acidify with 2-4 drops of glacial acetic acid R, filter and add 1.0 ml of magnesium uranyl acetate TS to the filtrate. Scratch the inside of the tube with a glass rod to induce crystallization; a yellow, crystalline precipitate is produced. Degradation test Discoloration and a change in the physical aspect of the test substance (in particular, appearance of a hard, yellow mass) and non- compliance with the following test usually indicate gross degradation: Dissolve 0.05g in 1.0m! of water, shake and add 0.10ml of hydrochloric acid (~ 70 g/l) TS; no precipitate is observed.3. TESTS: 19 AMPICILLIN TRIHYDRATE Identity tests Description. A white, or almost white, crystalline powder; odourless or almost odourless. Melting behaviour. About 204°C with decomposition, discoloration and evolution of gas. Eutectic temperature. With dicyandiamide R, about 172°C. Colour and other reactions 1. Dissolve Smg in 3ml of water, add 0.10g of hydroxylamine hydrochloride R, 1.0 ml of sodium hydroxide (~ 80 g/l) TS and allow to stand for 5 minutes. Then add 1.3 ml of hydrochloric acid (~ 70 g/l) TS and 10 drops of ferric chloride (25 g/l) TS; a violet-red colour is produced. 2. Dissolve 10mg in 1.0ml of water and add 2.0m! of a mixture composed of 2.0 ml of potassio-cupric tartrate TS and 6 ml of water; a red-violet colour is produced. 3. Dissolve 5 mg in 1.0 ml of water and add 1-2 drops of ferric chloride (25 g/l) TS; a clear, yellowish solution is produced. 4. (a) Add about 1 mg to a solution of 10 mg of paraformaldehyde R in 1 ml of sulfuric acid (~ 1760 g/l) TS; a colourless solution is produced. (6) Heat the solution from test 4(a) in a water-bath for 2 minutes and cool; a yellow colour is produced. 5. Dry about 0.5g at 80-100°C for 2 hours; the loss of weight is betwen 12 and 14% (theoretical value = 13.4%) (distinction from ampicillin). Degradation test If the test substance does not pass the following test, this usually indicates that gross degradation has occurred: Dissolve 0.10 g in 40 ml of water; a clear and colourless solution is produced. ANTIMONY POTASSIUM TARTRATE Identity tests Description. Colourless, transparent crystals or a white, granular powder; odourless or almost odourless; the crystals effloresce upon exposure to air.20 BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES Colour and other reactions 1. Dissolve 0.05 g in 1.0 ml of hydrochloric acid (~ 70 g/l) TS and add 1,0 ml of hydrogen sulfide TS; an orange-red precipitate is produced which is soluble in ammonium sulfide TS and in sodium hydroxide (~ 80 gf) TS. 2, Dissolve 20 mg in 2.0 ml of water and add 2-3 drops of glacial acetic acid R and 1.0 ml of sodium cobaltinitrite (100 g/l) TS; an orange-yellow precipitate is produced. 3. Moisten 10 mg with 1 drop of hydrochloric acid (~ 70 g/l) TS and introduce the mixture into a nonluminous flame, using a magnesia stick, or a nichrome or platinum wire sealed to a glass rod; the flame acquires a violet colour. 4. Dissolve 20mg in 0.2 ml of water and add 2ml of sulfuric acid (~ 1760 g/l) TS and several crystals of resorcinol R. Heat slowly in a flame; after a few minutes a cherry-red colour appears. ANTI INY SODIUM TARTRATE Identity tests Description. Colourless, transparent scales or an almost white powder; odourless; hygroscopic. Colour and other reactions 1. Dissolve 0.05 g in 1.0 ml of hydrochloric acid (~ 70 g/l) TS and add 1.0 ml of hydrogen sulfide TS; an orange-red precipitate is produced which is soluble in ammonium sulfide TS and in sodium hydroxide (~ 80 g/l) TS. 2. Dissolve 20 mg in 2.0 ml of water and add 2 or 3 drops of glacial acetic acid R and 1.0 ml of magnesium uranyl acetate TS. Scratch the inside of the tube with a glass rod to induce crystallization; a yellow, crystalline precipitate is produced. 3. Moisten 10mg with 1 drop of hydrochloric acid (~ 70 g/l) TS and introduce the mixture into a nonluminous flame, using a magnesia stick, or a nichrome or platinum wire sealed to a glass rod; the flame acquires a yellow colour. 4. Dissolve 20mg in 0.2m! of water and add 2ml of sulfuric acid (~ 1760 g/l) TS and several crystals of resorcinol R. Heat slowly in a flame; after a few minutes a cherry-red colour appears. Degradation test Discoloration and a change in the physical aspect of the test substance usually indicate gross degradation; in particular, no odour should be discernible.3, TESTS 21 ASCORBIC ACID Identity tests Description. Colourless crystals or a white, or almost white, crystalline powder; odourless or almost odourless. Melting point. About 190°C with decomposition. Heating behaviour. Heat a small quantity in a test-tube; it melts and turns brown. The melt has an odour resembling caramel, When ignited it swells and burns. Colour and other reactions 1, Dissolve 0.04g in 4ml of water, and add 0.10g of sodium hydrogen carbonate R and about 20 mg of ferrous sulfate R, shake and allow to stand; a deep violet colour is produced which disappears on the addition of Sml of sulfuric acid (~ 100 g/l) TS. 2. Dissolve 20mg in 1.0 ml of water and add 1.0 ml of potassio-cupric tartrate TS; an orange-yellow precipitate is produced which on heating changes to brownish red. 3. Dissolve 0.10 g in 2.0 ml of water and add a few drops of nitric acid (~ 130 g/l) TS and a few drops of silver nitrate (40 g/l) TS; a dark grey precipitate is produced. Degradation test If the test substance does not pass the following test, this usually indicates that gross degradation has occurred: Dissolve 0.10 g in 10 ml of water and add 10.6 ml of iodine TS; a colourless solution is produced without any yellowish tinge. ATROPINE SULFATE Identity tests Description. Colourless crystals or a white, crystalline powder; odourless. Note. This substance is very toxic and should be handled with care. Melting behaviour. Determined immediately after drying at 120°C for 4 hours; about 190°C with decomposition. Colour and other reactions 1. To 10 mg add 3 drops of nitric acid (~ 1000 g/l) TS and evaporate to dryness on a water-bath. To the cooled residue add 2.0 ml of acetone R22 BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES. and 3-4 drops of potassium hydroxide/ethanol TS; a deep violet colour is produced. 2. Dissolve 10mg in 2.0ml of water and add a few drops of hydrochloric acid (~ 70 g/l) TS and a few drops of barium chloride (50 g/l) TS; a white precipitate is produced. AZATHIOPRINE Identity tests Description. A pale yellow powder; odourless. Note. This substance is very toxic and should be handled with care. Melting behaviour. About 236°C with decomposition (turns brown at 220 °C). Eutectic temperature. With dicyandiamide R, about 196°C (turns brown). Colour and other reactions 1. Heat 20 mg with 100 ml of water and filter. To 5 ml of the filtrate add 1.0 ml of hydrochloric acid ( ~ 70g/1) TS and 10mg of zinc R powder and allow to stand for 5 minutes; the solution becomes yellow. Filter, cool in ice and add 3-4 drops of sodium nitrite (10 g/l) TS and 5-6 drops of hydrochloric acid (~ 70 g/l) TS. Mix, and to this solution add 0.2-0.3g of urea R. Shake until the bubbles cease and add 0.5ml of 2-naphthol TS; a pink coloured precipitate is produced, which turns yellowish brown on shaking. 2. Fuse 0.05g with 0.05g of potassium nitrate R and 0.10g of potassium hydroxide R in a test-tube. Cool, dissolve the residue in 20 ml of water and filter. To 5ml of the filtrate add 1.5 ml of hydrochloric acid (~ 70 g/l) TS and 5-6 drops of barium chloride (50 g/l) TS; a white turbidity appears. BACITRACIN Identity tests Description. A white or pale brownish yellow powder; odourless or with a faint, characteristic odour; hygroscopic. Colour and other reactions Shake 5 mg with 1.0m of water, add 1.0 ml of triketohydrindene hydrate/ethanol TS and 0.5 ml of pyridine R and heat at 100°C for 5 minutes; a deep purple colour is produced.3. TESTS 23 Degradation test Discoloration of the test substance and non-compliance with the following test usually indicate gross degradation: Dissolve 0.4g in 10ml of water; a clear and almost colourless solution is produced. BARIUM SULFATE Identity tests Description. A white, heavy, fine powder; free from grittiness; odourless. Heating behaviour. Moisten a small quantity with hydrochloric acid (~70g/) TS and introduce it into a nonluminous flame using a magnesia stick, or a nichrome or platinum wire sealed to a glass rod; a pale green colour appears in the flame. Colour and other reactions 1. Boil 0.20 g for 5 minutes with 5 ml of a mixture of 2.5 g of sodium carbonate R dissolved in 5 ml of water, then add 10 ml of water and filter. To 5 ml of the filtrate add 5 ml of hydrochloric acid (~ 70 g/l) TS and 1.0ml of barium chloride (50 g/l) TS; a white precipitate is produced (keep the precipitate for test 2). 2. Wash the precipitate from test 1 with 3 successive small quantities of water. To the residue add 5 ml of hydrochloric acid (~ 70 g/l) TS, filter and to the filtrate add 0.3 ml of sulfuric acid (~ 100 g/l) TS; a white precipitate is produced. BECLOMETASONE DIPROPIONATE Identity tests Description. A white to creamy white powder; odourless. Melting behaviour. About 212°C with decomposition. Colour and other reactions 1. Dissolve about 2mg in 2ml of sulfuric acid (~ 1760 g/l) TS and allow to stand for 5 minutes; a purplish brown solution is produced. Very cautiously dilute the solution with 10ml of water; the colour changes to a very light bluish grey.24 BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES 2. Dissolve 5 mg in 5 drops of ethanol (~ 750 g/l) TS, add 10 drops of sulfuric acid (~ 1760 g/l) TS and mix; immediately a purple colour is produced. BENZATHINE BENZYLPENICILLIN Identity tests Description. A white powder; odourless or almost odourless. Melting behaviour. 129-133°C with decomposition. Colour and other reactions 1. To 5 mg add 3.0 ml of water, 0.10 g of hydroxylamine hydrochloride R and 1.0 ml of sodium hydroxide (~ 80 g/l) TS, mix and allow to stand for 5 minutes. Then add 1.3 ml of hydrochloric acid (~ 70 g/l) TS and 10 drops of ferric chloride (25 g/l) TS; a violet-brown colour is produced. 2. To about 2 mg add 1 drop of water followed by 2 ml of sulfuric acid (~ 1760 g/l) TS and mix; the solution is almost colourless. Heat in a water-bath for 1 minute; the solution remains almost colourless to slightly yellow. 3. To about 2mg add 1 drop of water and 2ml of sulfuric acid (~ 1760 gf) TS. After cooling, add 2 drops of formaldehyde TS; the solution is almost colourless. Heat in a water-bath for 1 minute; a reddish brown colour is produced. 4. Dissolve 10 mg in 1.0 ml of sodium hydroxide (~ 80 g/l) TS and add 3.0 ml of water and 1.0 ml of potassium permanganate (10 g/l) TS; a green colour is produced. Heat the solution; an odour of benzaldehyde is perceptible. Degradation test Discoloration and, in particular, a slight odour to the test substance indicate gross degradation. BENZOCAINE Identity tests Description. Colourless crystals or a white, crystalline powder; odourless.3. TESTS 25 Melting point. About 90°C. Heating behaviour. Place a few mg from a spatula into a nonluminous flame; it inflames producing a brownish black residue which on prolonged ignition disappears. Colour and other reactions 1. Dissolve 10 mg in 1.0 ml of hydrochloric acid (~ 70 g/l) TS and add 2-3 drops of freshly prepared sodium nitrite (10 g/l) TS and a solution of 10mg of 2-naphthol R in 2.0 ml sodium hydroxide (~ 80 g/l) TS; a red colour is produced. Add hydrochloric acid (~ 70 g/l) TS drop by drop; a red precipitate separates. 2. To 0.05 g add 1-2 drops of hydrochloric acid (~ 70 g/l) TS and then dissolve in 1.0 ml of water. Add 1-2 drops of potassio-mercuric iodide TS; no turbidity or precipitate is produced. 3. Dissolve 20mg in 2-3 ml of sodium hydroxide (~ 80 g/l) TS and boil; no odour is perceptible. BENZOIC ACID Identity tests Description. Colourless, light, feathery crystals or a white, crystalline powder; faint, characteristic odour. Melting point. About 123°C. Heating behaviour. Place a few mg from a spatula into a nonluminous flame; it melts and sublimes, then burns with a soot-producing flame. Colour and other reactions 1. Dissolve 0.10 g in 5 ml of water by warming. Cool the solution while shaking and filter. Insert moistened pH-indicator paper R into the filtrate; its coloration is changed to the acid range. (Keep the filtrate for test 2.) 2. To the filtrate obtained in test 1 add 4—5 drops of ferric chloride (25 g/l) TS; a reddish brown precipitate is produced (no violet colour). Add a few drops of hydrochloric acid (~ 70 g/l) TS; the precipitate dissolves. BENZYL BENZOATE Identity tests Description. A clear, colourless, oily liquid. It freezes to a colourless mass when cooled below 18°C. Odour, faintly aromatic.26 BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES Heating behaviour. Place a drop from a spatula into a nonluminous flame; it smokes, bursts into flames and burns with a rather sooty flame. Colour and other reactions Boil 2.0g with 50 ml of potassium hydroxide/ethanol TS for 10 minutes, evaporate the ethanol on a water-bath, cool, add 20 ml of water, extract with two successive 15 ml portions of chloroform R and proceed as follows: 1. Evaporate the chloroform layer on a water-bath; heat | drop of the oily liquid with 5ml of sodium carbonate (50 g/l) TS and 1.0 ml of potassium permanganate (10 g/l) TS; an odour of benzaldehyde is discernible. 2. Neutralize the aqueous layer with hydrochloric acid (~ 70 g/l) TS and add 1.0 ml of ferric chloride (25 g/l) TS; a reddish brown precipitate is formed. Shake the precipitous mixture with 10 ml of hydrochloric acid (~ 70 g/l) TS; a voluminous, white precipitate is formed. BENZYLPENICILLIN POTASSIUM Identity tests Description. A white, or almost white, crystalline powder; odourless or with a faint, characteristic odour; hygroscopic. Melting behaviour. About 225°C with complete decomposition (turns black). Eutectic temperature. With dicyandiamide R, 170°C; with phenol- phthalein R, about 198°C. Colour and other reactions 1. Dissolve Smg in 3ml of water, add 0.10g of hydroxylamine hydrochloride R and 1.0ml of sodium hydroxide (~ 80 g/l) TS and allow to stand for 5 minutes. Then add 1.3 ml of hydrochloric acid (~ 70 g/l) TS and 10 drops of ferric chloride (25 g/l) TS; a violet-red colour is produced. 2. Dissolve 5mg in a few drops of ethanol (~ 750 g/l) TS and add 1.0 ml of water and 1-2 drops of ferric chloride (25 g/l) TS; a yellowish precipitate is produced. 3. (a) Add about 1 mg to a solution of 10 mg of paraformaldehyde R in 1 ml of sulfuric acid (~ 1760 g/l) TS; a colourless solution is produced. (6) Heat the solution from test 3(a) on a water-bath for 2 minutes and cool; the colour of the solution changes to slightly pinkish. 4. Dissolve 20 mg in 2.0 ml of water and add 2-3 drops of glacial acetic acid R and 1.0 ml of sodium cobaltinitrite (100 g/l) TS; an orange-yellow precipitate is produced.3. TESTS 27 Degradation test Discoloration, a change in the physical aspect of the test substance and non-compliance with the following test usually indicate gross degradation: Dissolve 0.20g in 1.0ml of water; the solution is clear and colourless. BENZYLPENICILLIN SODIUM Identity tests Description. A white, or almost white, crystalline powder; odourless or with a faint characteristic odour. Melting behaviour. About 227°C with strong decomposition (turns black). Eutectic temperature. With dicyandiamide R, starting at 172°C; with phenolphthalein R, starting at about 198°C. Colour and other reactions 1. Dissolve Smg in 3ml of water, add 0.10g of hydroxylamine hydrochloride R and 1.0ml of sodium hydroxide (~ 80 g/l) TS and allow to stand for 5 minutes. Then add 1.3 ml of hydrochloric acid (~ 70 gf) TS and 10 drops of ferric chloride (25 g/l) TS; a violet-red colour is produced. 2. Dissolve 5mg in a few drops of ethanol (~ 750 g/l) TS and add 1.0 ml of water and 2 drops of ferric chloride (25 g/l) TS; a yellowish precipitate is produced. 3. (a) Add about 1 mg to a solution of 10 mg of paraformaldehyde R in 1 ml of sulfuric acid (~ 1760 g/l) TS; a light yellow colour is produced. (6) Heat the solution from test 3(a) in a water-bath for 2 minutes and cool; the colour of the solution changes to brown-red. 4. Dissolve 0.05 g in 5 ml of water, acidify with about 0.5 ml of glacial acetic acid R and filter. To 2 ml of the filtrate add 1.0 ml of magnesium uranyl acetate TS. Scratch the inside of the tube with a glass rod to induce crystallization; a yellow, crystalline precipitate is produced. Degi lation test Discoloration and a change in the physical aspect of the test substance and non-compliance with the following test usually indicate gross degradation: Dissolve 0.20g in 1.0m] of water; the solution is clear and colourless.28 BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES BEPHENIUM HYDROXYNAPHTHOATE Identity tests Description. A yellow to greenish yellow, crystalline powder; odourless or almost odourless. Melting behaviour. About 170°C with decomposition. Colour and other reactions Dissolve 0.20 g in 10 m! of warm dehydrated ethanol R, add 5 ml of trinitrophenol (7 g/l) TS and allow to stand for a few minutes; wash the formed precipitate with ethanol (~ 750 g/l) TS and water and dry at 105°C; melting temperature, about 134°C with decomposition. Degradation test Discoloration of the test substance usually indicates gross degradation. BETAMETHASONE Identity tests Description. A white or creamy white powder; odourless. Melting behaviour. After drying over silica gel, desiccant, R for 24 hours; 231-234°C with decomposition. Eutectic temperature. With phenolphthalein R, about 210°C. Colour and other reactions 1. Add 5 mg to a mixture of 2 drops of formaldehyde TS with 1 ml of sulfuric acid (~ 1760 g/l) TS and shake well; an orange colour is produced. Heat on a water-bath for 1 minute; the colour changes to brown. 2. Dissolve Smg in 0.5ml of methanol R and add 1.0 ml of hot potassio-cupric tartrate TS; a reddish yellow precipitate is formed. 3. Mix 3 drops of potassium dichromate (100 g/l) TS with about 0.5 ml of sulfuric acid (~ 1760 g/l) TS and heat on a water-bath for 5 minutes; the solution wets the sides of the tube. Add 10 mg of the test substance, shake well and heat again for 5 minutes in a water-bath; the colour turns dark brown and the solution no longer wets the sides of the tube.3. TESTS: 29 BETAMETHASONE VALERATE Identity tests Description. A white or creamy white powder; odourless. Melting point. About 190°C. Eutectic temperature. With benzanilide R, about 140°C. Colour and other reactions 1. Add 5 mg to a mixture of 2 drops of formaldehyde TS with 1 ml of sulfuric acid (~ 1760 g/l) TS and shake well; an orange colour is produced. Heat on a water-bath for | minute; the colour changes to brown. 2. Dissolve Smg in 0.5ml of methanol R and add 1.0ml of hot potassio-cupric tartrate TS; a reddish yellow precipitate is formed. 3. Mix 3 drops of potassium dichromate (100 g/l) TS with about 0.5 ml of sulfuric acid (~ 1760 g/l) TS and heat on a water-bath for 5 minutes; the solution wets the sides of the tube. Add 10 mg of the test substance, shake well and heat again for 5 minutes in a water-bath; the colour turns dark brown and the solution no longer wets the sides of the tube. 4. Heat 0.05g with 2.0 ml of potassium hydroxide/ethanol TS on a water-bath for 5 minutes. Cool, add 2.0 ml of sulfuric acid (~ 100 g/l) TS and boil gently for 1 minute; a pleasant odour of ethyl valerate is perceptible. BIPERIDEN Identity tests Description. A white, or almost white, crystalline powder; odourless. Melting point. About 114°C. Colour and other reactions 1. Dissolve 20 mg in 5ml of phosphoric acid ( ~ 1440 g/l) TS and allow to stand; a green colour is produced. 2. To 0.20 g add 80 ml of water, 0.5 ml of hydrochloric acid (~ 70 g/l) TS and warm until dissolved. Cool and to Sml of this solution add bromine TS drop by drop; a yellow precipitate is formed which dissolves on shaking. Upon the addition of more bromine TS, a permanent Precipitate is produced.30 BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES BIPERIDEN HYDROCHLORIDE Identity tests Description. A white, crystalline powder; odourless. Melting behaviour. About 275°C with slight decomposition. Colour and other reactions 1. Dissolve 5 mg in 1 ml of phosphoric acid (~ 1440 g/l) TS with gentle heat, and allow to stand; a green colour turning to grey is produced. 2. Dissolve 5 mg in 2.0 ml of warm water, cool to room temperature and add bromine TS drop by drop; a yellow precipitate is formed which dissolves on shaking. Upon the addition of more bromine TS, a permanent precipitate is produced. 3. Dissolve 5mg in 2.0 ml of warm water, add 1 drop of nitric acid (~ 130 g/l) TS and 2 drops of silver nitrate (40 g/l) TS; a white precipitate is observed. Separate the precipitate, wash it with water and add an excess of ammonia (~ 100 g/l) TS; the precipitate dissolves. BUPIVACAINE HYDROCHLORIDE Identity tests Description. A white, crystalline powder; odourless. Melting behaviour. About 255°C with decomposition. Eutectic temperature. With phenolphthalein R, about 153°C. Colour and other reactions 1. Dissolve 5mg in 1.0 ml of pyridine R and add 0.5 ml of sodium hydroxide (~ 80 g/l) TS and 0.25 ml of benzenesulfonyl chloride R; a yellow-orange colour is produced. 2. Dissolve 5mg in a mixture of 1ml of sulfuric acid (~ 1760 g/l) TS and 0.1 ml of formaldehyde TS; at room temperature the solution is almost colourless. Immerse the test-tube in a water-bath for 1 minute; a brown to deep red colour is produced. 3. Dissolve 5mg in 0.1 ml of water and about 0.5 ml of sulfuric acid (~ 1760 g/l) TS. Heat to boiling for 1 minute, cool and add 0.5 ml of sodium nitrite (10 g/l) TS. Allow to stand for 1 minute, add 1.5 ml of sodium hydroxide (~ 80 g/l) TS and then Smg of 2-naphthol R; an intense orange colour is produced. 4. Dissolve 10mg in 4ml of water and add 0.5ml of nitric acid (~ 130 gf) TS and 0.5 ml of silver nitrate (40 g/l) TS; a white, curdy precipitate is produced. Separate the precipitate, wash it with water and add an excess of ammonia (~ 100 g/l) TS; the precipitate dissolves.3. TESTS 31 BUSULFAN Identity tests Description. A white, crystalline powder. Note. This substance is very toxic and should be handled with care. Melting point. About 116°C. Eutectic temperature. With phenacetin R, about 108 °C. Colour and other reactions 1. Fuse 0.10 g with a mixture of 0.10 g of potassium nitrate R and 0.20 g of potassium hydroxide R. Cool, dissolve the residue in 40 ml of water and filter. To Sml of the filtrate add 1.5 ml of hydrochloric acid (+70 g/l) TS and 0.5ml of barium chloride (50 g/l) TS; a white precipitate is produced. 2. Heat 0.10g with 10 ml of water and 2.5 ml of sodium hydroxide (~80 g/l) TS until a clear solution is obtained; a pungent, characteristic odour is perceptible. Cool the solution and divide into two portions for tests 3 and 4. 3. To one portion of the solution prepared in test 2, add 2-3 drops of potassium permanganate (10 g/l) TS; the purple colour changes to violet, then to blue and finally to emerald green. 4. Acidify the second portion of the solution prepared in test 2 with 2.0 ml of sulfuric acid (~100 g/l) TS, add 2-3 drops of potassium permanganate (10 g/l) TS and shake; the colour of the permanganate is slowly discharged. CAFFEINE Identity tests Description. Silky, colourless crystals or a white, crystalline powder; odourless. Eutectic temperature. With acetanilide R, about 108 °C. Colour and other reactions 1. To 10mg, contained in a porcelain dish, add 1 mi of hydrochloric acid (~ 250 g/l) TS and 1 drop of hydrogen peroxide (~ 330 g/l) TS and evaporate to dryness on a water-bath. Add 1 drop of ammonia (~ 100 gf) TS; the residue acquires a purple colour, which disappears upon the addition of 2-3 drops of sodium hydroxide (~ 80 g/l) TS. 2. Dissolve about 2 mg in 1.0 ml of water and add a few drops of silver nitrate (40 g/l) TS; no turbidity is produced.32 BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES 3. Shake 0.20g with 2.0ml of ammonia (~ 100 g/l) TS; the test substance remains undissolved. CALCIUM CARBONATE Identity tests Description A white, fine, crystalline powder; odourless, Colour and other reactions 1, Dissolve 20 mg in 0.3 ml of hydrochloric acid (~ 70 g/l) TS and 2 ml of water and filter. Add 1.0 ml of ammonium oxalate (25 g/l) TS to the filtrate; a white precipitate is produced which is insoluble in acetic acid (~ 300 g/l) TS and ammonia (~ 260 g/l) TS but soluble in hydrochloric acid (~ 70 g/l) TS. 2. Moisten 10mg with 1 drop of hydrochloric acid (~ 70 g/1) TS and introduce the mixture into a nonluminous flame using a magnesia stick, or a nichrome or platinum wire sealed to a glass rod; the flame acquires a brick-red colour. 3. To 0.1 g add 1 ml of acetic acid (~ 300 g/l) TS; a gas evolves which is colourless and odourless. Pass the evolved gas into calcium hydroxide TS; a white precipitate is produced immediately. CALCIUM FOLINATE Identity tests Description. A white or creamy white powder; odourless. Note. This substance is very toxic and should be handled with care. Melting behaviour. It decomposes above 280 °C (turns brown) but does not melt below 320°C. Eutectic temperature. With dicyandiamide R, about 194°C with decomposition. Colour and other reactions 1. Dissove 20 mg in 3.0 ml of water, add 0.5 ml of hydrochloric acid (~ 70 g/l) TS and 0.5 ml of sodium nitrite (100 g/l) TS. Shake for 2 minutes and add 1.5 ml of 2-naphthol TS; a yellow-brown coloured precipitate appears while the solution turns green. 2. Dissolve 20mg in 2.0 ml of water and add 1.0m] of ammonium oxalate (25 g/l) TS; a white precipitate is produced, which is insoluble in3. TESTS 33 acetic acid (~ 300 g/l) TS and ammonia (~ 260 g/l) TS but soluble in hydrochloric acid (~ 70 g/l) TS. 3. Dissolve 20mg in 5ml of water and add 1.0 ml of silver nitrate (40 g/l) TS; a white, curdy precipitate is produced. Add a few drops of nitric acid (~ 130 g/l) TS; the precipitate dissolves. CALCIUM GLUCONATE Identity tests Description. White, crystalline granules or a white, crystalline powder; odourless. Colour and other reactions 1, Add 5 mg to a solution of 5 mg of 2-naphthol R in 1 ml of sulfuric acid (~ 1760 g/l) TS. Heat for 1 minute on a water-bath; a dark blue- green colour is produced. 2. To 2.0 ml of silver nitrate (40 g/l) TS add ammonia (~ 100 g/1) TS, drop by drop, until the initially formed brown precipitate just dissolves. Add 5 mg of the test substance and heat to boiling for 1-2 minutes; a silver mirror is produced. 3. Dissolve 10 mg in 2.0 ml of water and add 5 drops of ammonium oxalate (25 g/l) TS; a white precipitate is formed. Add a few drops of hydrochloric acid (~ 70 g/l) TS; the precipitate dissolves. Add a few drops of acetic acid (~300g/l) TS; the precipitate is practically insoluble. Degradation test If the test substance does not pass the following test, this usually indicates that gross degradation has occurred: Mix 0.5g with 2ml of sulfuric acid (~ 1760 g/l) TS; a colourless suspension is obtained. CALCIUM PARA-AMINOSALICYLATE Identity tests Description. A white, slightly yellowish, or greyish, fine powder; odourless; hygroscopic. It is affected by air and light, changing to a dark powder.34 BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES Melting point of the sublimate. About 122°C (see Colour and other Teactions, test 1). Eutectic temperature of the sublimate. With phenacetin R, about 95°C (see Colour and other reactions, test 1). Colour and other reactions 1, Over a very low flame heat 0.20 g in a 50-ml porcelain crucible placed on an asbestos wire-net. As soon as white vapours evolve, cover the crucible with a watch-glass and continue heating carefully until a sufficient amount of the sublimate of m-aminophenol has settled on the watch-glass. 2. Dissolve about 1 mg in 1.0 ml of water and add 3-4 drops of ferric chloride (25g/l) TS; a strong violet-red colour is produced. 3. Dissolve 5 mg in 5 drops of nitric acid (~ 130 g/l) TS and add 1.0 m1 of water and 1.5 ml of sodium nitrite (10 g/l) TS; a yellow solution is produced. Add Sml of sodium hydroxide (~ 80 g/l) TS; the colour changes to orange-red. 4, Dissolve 20mg in 2.0 ml of water and add 1.0 ml of ammonium oxalate (25 g/l) TS; a white precipitate is produced which is insoluble in acetic acid (~ 300 g/l) TS and ammonia (~ 260 g/1) TS but soluble in hydrochloric acid (~ 70 g/l) TS. Degradation test Discoloration, a change in the physical aspect of the test substance and non-compliance with the following test usually indicate gross degradation: Dissolve 10 mg in 100 ml of water; the solution is clear and almost colourless. CARBAMAZEPINE Identity tests Description. A white, or yellowish white, crystalline powder; odourless or almost odourless. Melting point. About 191°C. Colour and other reactions 1. To Smg add 2ml of sulfuric acid (~ 1760 g/l) TS and 0.1 ml of formaldehyde TS; a yellow colour is produced which turns orange on standing. 2. Heat 0.10 g with 2ml of nitric acid (~ 1000 g/l) TS in a water-bath for 3 minutes; an orange-red colour is produced.3, TESTS. 35 CARBENICILLIN SODIUM Identity tests Description. A white or almost white powder; odourless; hygroscopic. Melting behaviour. About 207°C with strong decomposition and discoloration. Colour and other reactions 1. Dissolve 5 mg in 3 ml of water, add 0.10g of hydroxylamine hydrochloride R and 1.0 ml of sodium hydroxide (~ 80 g/l) TS and allow to stand for 5 minutes. Then add 1.3 ml of hydrochloric acid (~ 70 g/l) TS and 10 drops of ferric chloride (25 g/l) TS; a violet-red colour is produced. 2. Dissolve 10mg in 1.0ml of water and add 2.0ml of a mixture composed of 2.0 ml of potassio-cupric tartrate TS and 6 ml of water; a light blue solution is produced. 3. Dissolve 5 mg in 1.0 ml of water and add 1-2 drops of ferric chloride (25 gf) TS; a yellowish precipitate is produced. 4. (a) Add about I mg to a solution of 10 mg of paraformaldehyde R in 1 ml of sulfuric acid (~ 1760 g/l) TS; a colourless solution is produced (keep this solution for test 4(6)). (6) Heat the solution from test 4(a) in a water-bath for 2 minutes and cool; a light brown colour is produced. 5. Dissolve 0.05 g in 5 ml of water, acidify with about 0.5 ml of glacial acetic acid R and filter. To 2 ml of the filtrate add 1.0 ml of magnesium uranyl acetate TS. Scratch the inside of the tube with a glass rod to induce crystallization; a yellow, crystalline precipitate is produced. Degradation test Discoloration and a change in the physical aspect of the test substance usually indicate gross degradation; in particular, no hard, sticky, yellow mass, or gel, or yellow-orange liquid should be observed. CARBIDOPA Identity tests Description. A white to creamy white powder; odourless or almost odourless.36 BASIC TESTS FOR PHARMACEUTICAL SUBSTANCES Melting behaviour. About 185°C with foaming and decomposition. Eutectic temperature. With dicyandiamide R, about 159°C. Colour and other reactions 1. Dissolve 5 mg in 1.0 ml of water and add 3-4 drops of ferric chloride (25 g/l) TS; a greenish brown colour develops that changes to brown-red. Add 2-3 drops of hydrochloric acid (~ 70 g/l) TS; the colour changes to light greenish yellow. 2. Dissolve 25 mg in 4 ml of water by warming, cool and add 2,0 ml of copper(II) acetate (45 g/l) TS; a green precipitate is obtained. Heat for a few minutes on a water-bath; an evolution of gas is observed and the colour of the precipitate changes to brown-red. CEFALEXIN Identity tests Description. A white or cream-coloured powder; odour, characteristic. Melting behaviour. About 190°C with decomposition and discoloration. Eutectic temperature. With dicyandiamide R, not sharp, starting at about 156°C. Colour and other reactions 1. Dissolve Smg in 3ml of water, add 0.10g of hydroxylamine hydrochloride R and 1.0 ml of sodium hydroxide (~ 80 g/l) TS and allow to stand for 5 minutes. Then add 1.3 ml of hydrochloric acid (~ 70 g/l) TS and 10 drops of ferric chloride (25 g/l) TS; a violet-red colour is produced. 2. Dissolve 10 mg in 1.0 ml of water and add 2.0 ml of a mixture composed of 2.0 mi of potassio-cupric tartrate TS and 6 ml of water; an olive-green colour is immediately produced which changes to yellow- brown after 30 seconds. 3. Dissolve 5 mg in 1.0 ml of water and add 1-2 drops of ferric chloride (25 g/l) TS; a colourless solution is produced. 4, (a) Add about 1 mg to a solution of 10 mg of paraformaldehyde R in ml of sulfuric acid (~ 1760 g/l) TS; a yellow colour is produced. (6) Heat the solution from test 4(a) in a water-bath for 2 minutes and cool; the yellow colour of the solution remains. Degradation test Discoloration of the test substance usually indicates gross degradation.3. TESTS: 37 CEFALOTIN SODIUM Identity tests Description. A white or almost white powder; odour, faint. Melting behaviour. About 213°C with strong decomposition (turns brown). Eutectic temperature. With dicyandiamide R, starting at about 169°C; with phenolphthalein R, starting at about 204°C. Colour and other reactions 1. Dissolve Smg in 3ml of water, add 0.10g of hydroxylamine hydrochloride R and 1.0 ml of sodium hydroxide (~ 80 g/l) TS and allow to stand for 5 minutes. Then add 1.3 ml of hydrochloric acid (~ 70 g/l) TS and 10 drops of ferric chloride (25 g/l) TS; a violet-red colour is produced. 2. Dissolve 10 mg in 1.0 ml of water, add 2.0 ml of a mixture composed of 2.0 ml of potassio-cupric tartrate TS and 6 ml of water and allow to stand for 30 seconds; a green colour is produced which changes to yellow-brown after 2 minutes. 3. Dissolve 5 mg in a few drops of ethanol (~ 750 g/l) TS and add 1.0 ml of water and 1-2 drops of ferric chloride (25 g/l) TS; a yellow precipitate is produced, 4. (a) Add about 1 mg to a solution of 10 mg of paraformaldehyde R in 1 ml of sulfuric acid (~ 1760 g/l) TS; a red to brown-black solution is produced. (6) Heat the solution from test 4(a) in a water-bath for 2 minutes and allow to cool; the colour of the solution changes to dark brown-black. 5. Dissolve 0.05 g in 5 ml of water, acidify with about 0.5 ml of glacial acetic acid R and filter. To 2 ml of the filtrate add 1.0 ml of magnesium uranyl acetate TS. Scratch the inside of the tube with a glass rod to induce crystallization; a yellow, crystalline precipitate is produced. CETRIMIDE Identity tests Description. A white to creamy white, voluminous, free-flowing powder; odour, characteristic and faint. Melting point. About 240°C. Colour and other reactions 1. Dissolve 1.0 g in 50 mi of water and shake; a lot of foam is produced. {Keep the solution for tests 2 and 4.)