EUROPEAN PHARMACOPOEIA 5.

Adenosine

and dilute the filtrate to 15 ml with water R. The solution complies with the limit test for chlorides (100 ppm). When carrying out the test, add 2 ml of dilute nitric acid R instead of 1 ml of dilute nitric acid R. Sulphates (2.4.13). Dilute 10 ml of solution S to 15 ml with IDENTIFICATION distilled water R. The solution complies with the limit test First identification : A. for sulphates (300 ppm). Second identification : B, C. Ammonium. Prepare a cell consisting of two watch-glasses A. Examine by infrared absorption spectrophotometry 60 mm in diameter placed edge to edge. To the inner wall of (2.2.24), comparing with the spectrum obtained with the upper watch-glass stick a piece of red litmus paper R adenine CRS. Examine the substances prepared as discs. 5 mm square and wetted with a few drops of water R. Finely powder the substance to be examined, place 0.5 g in the B. Examine the chromatograms obtained in the test lower watch-glass and suspend in 0.5 ml of water R. To for related substances. The principal spot in the the suspension add 0.30 g of heavy magnesium oxide R. chromatogram obtained with test solution (b) is Briefly triturate with a glass rod. Immediately close the cell similar in position and size to the principal spot in the by putting the two watch-glasses together. Heat at 40 C chromatogram obtained with reference solution (a). C. To 1 g add 3.5 ml of propionic anhydride R and boil for for 15 min. The litmus paper is not more intensely blue coloured than a standard prepared at the same time and in 15 min with stirring. Cool. To the resulting crystalline the same manner using 0.05 ml of ammonium standard mass add 15 ml of light petroleum R and heat to solution (100 ppm NH4) R, 0.5 ml of water R and 0.30 g of boiling with vigorous stirring. Cool and filter. Wash the heavy magnesium oxide R (10 ppm). precipitate with two quantities, each of 5 ml, of light petroleum R. Dissolve the precipitate in 10 ml of water R Heavy metals (2.4.8). 1.0 g complies with limit test C for and boil for 1 min. Filter the mixture at 30 C to 40 C. heavy metals (20 ppm). Prepare the standard using 2 ml of Allow to cool. Filter, and dry the precipitate at 100 C lead standard solution (10 ppm Pb) R. to 105 C for 1 h. The melting point (2.2.14) of the Loss on drying (2.2.32). Not more than 0.5 per cent, precipitate is 237 C to 241 C. determined on 1.000 g by drying in an oven at 100 C to 105 C. TESTS Sulphated ash (2.4.14). Not more than 0.1 per cent, Solution S. Suspend 2.5 g in 50 ml of distilled water R determined on 1.0 g. and boil for 3 min. Cool and dilute to 50 ml with distilled water R. Filter. Use the filtrate as solution S. ASSAY Appearance of solution. Dissolve 0.5 g in dilute hydrochloric Dissolve 0.100 g in a mixture of 20 ml of acetic anhydride R acid R and dilute to 50 ml with the same acid. The solution and 30 ml of anhydrous acetic acid R. Titrate with is clear (2.2.1) and colourless (2.2.2, Method II). 0.1 M perchloric acid, determining the end-point Acidity or alkalinity. To 10 ml of solution S add 0.1 ml of potentiometrically (2.2.20). bromothymol blue solution R1 and 0.2 ml of 0.01 M sodium 1 ml of 0.1 M perchloric acid is equivalent to 13.51 mg of hydroxide. The solution is blue. Add 0.4 ml of 0.01 M C5 H 5 N 5 . hydrochloric acid. The solution is yellow. Related substances. Examine by thin-layer chromatography 01/2005:1486 (2.2.27), using silica gel GF254 R as the coating substance. Test solution (a). Dissolve 0.10 g of the substance to be ADENOSINE examined in dilute acetic acid R, with heating if necessary, and dilute to 10 ml with the same acid. Adenosinum Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with dilute acetic acid R. Reference solution (a). Dissolve 10 mg of adenine CRS in dilute acetic acid R, with heating if necessary, and dilute to 10 ml with the same acid. Reference solution (b). Dilute 1 ml of test solution (b) to 20 ml with dilute acetic acid R. Reference solution (c). Dissolve 10 mg of adenine CRS and 10 mg of adenosine R in dilute acetic acid R, with heating if necessary, and dilute to 10 ml with the same acid. Apply to the plate 5 l of each solution. Develop over a path C H N O Mr 267.2 10 13 5 4 of 12 cm using a mixture of 20 volumes of concentrated ammonia R, 40 volumes of ethyl acetate R and 40 volumes DEFINITION of propanol R. Dry the plate in a current of warm air and Adenosine contains not less than 99.0 per cent and examine in ultraviolet light at 254 nm. Any spot in the not more than the equivalent of 101.0 per cent of chromatogram obtained with test solution (a), apart from 9--D-ribofuranosyl-9H-purin-6-amine, calculated with the principal spot, is not more intense than the spot in the reference to the dried substance. chromatogram obtained with reference solution (b) (0.5 per cent). The test is not valid unless the chromatogram obtained CHARACTERS with reference solution (c) shows two clearly separated spots. A white, crystalline powder slightly soluble in water, soluble in hot water, practically insoluble in alcohol and in methylene Chlorides (2.4.4). To 10 ml of solution S add 1 ml of chloride. It dissolves in dilute mineral acids. concentrated ammonia R and 3 ml of silver nitrate solution R2. Filter. Wash the precipitate with a little water R It melts at about 234 C. General Notices (1) apply to all monographs and other texts 925

CHARACTERS A white powder, very slightly soluble in water and in alcohol. It dissolves in dilute mineral acids and in dilute solutions of alkali hydroxides.

Adipic acid

EUROPEAN PHARMACOPOEIA 5.0

1 ml of 0.1 M perchloric acid is equivalent to 26.72 mg of IDENTIFICATION Examine by infrared absorption spectrophotometry (2.2.24), C10H13N5O4. comparing with the spectrum obtained with adenosine CRS. IMPURITIES TESTS A. adenine, Solution S. Suspend 5.0 g in 100 ml of distilled water R and heat to boiling. Allow to cool, filter with the aid of vacuum and dilute to 100 ml with distilled water R. Appearance of solution. Solution S is colourless (2.2.2, Method II). Acidity or alkalinity. To 10 ml of solution S, add 0.1 ml of bromocresol purple solution R and 0.1 ml of 0.01 M B. D-ribose, hydrochloric acid. The solution is yellow. Add 0.4 ml of 0.01 M sodium hydroxide, the solution is violet-blue. Specific optical rotation (2.2.7). Dissolve 1.25 g in 1 M hydrochloric acid and dilute to 50.0 ml with the same acid. Determined within 10 min and calculated with reference to the dried substance, the specific optical rotation is 45 to 49. Related substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F254 plate R. Test solution. Dissolve 0.20 g of the substance to be examined in dilute acetic acid R with slight heating and dilute to 5 ml with the same acid. C. R = H : adenosine 3-(dihydrogen phosphate), Reference solution (a). Dilute 1 ml of the test solution to D. R = PO3H2 : adenosine 3-(trihydrogen diphosphate), 100 ml with water R. Reference solution (b). Dissolve 10 mg of adenosine CRS E. R = PO2H-O-PO3H2 : adenosine 3-(tetrahydrogen and 10 mg of adenine CRS in dilute acetic acid R, with triphosphate). heating if necessary, and dilute to 10 ml with the same acid. Apply to the plate 5 l of each solution. Develop over a 01/2005:1586 path of 12 cm using a mixture of 10 volumes of water R, 30 volumes of concentrated ammonia R and 60 volumes ADIPIC ACID of propanol R. Allow the plate to dry in a current of warm air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart Acidum adipicum from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (a) (1 per cent). Spray with a 5 g/l solution of potassium permanganate R in 1 M sodium hydroxide. Allow the plate C6H10O4 Mr 146.1 to dry in a current of warm air and examine in daylight. Any spot in the chromatogram obtained with the test solution, DEFINITION apart from the principal spot, is not more intense than Hexanedioic acid. the spot in the chromatogram obtained with reference Content : 99.0 per cent to 101.0 per cent (dried substance). solution (a) (1 per cent). The test is not valid unless the chromatogram obtained with reference solution (b) shows CHARACTERS two clearly separated spots. Appearance : white, crystalline powder. Chlorides (2.4.4). Dilute 10 ml of solution S to 15 ml Solubility : sparingly soluble in water, soluble in boiling with water R. The solution complies with the limit test for water, freely soluble in alcohol and in methanol, soluble in chlorides (100 ppm). acetone. Sulphates (2.4.13). 15 ml of solution S complies with the IDENTIFICATION limit test for sulphates (200 ppm). A. Melting point (2.2.14) : 151 C to 154 C. Ammonium (2.4.1). 0.5 g complies with limit test B for B. Infrared absorption spectrophotometry (2.2.24). ammonium (10 ppm). Prepare the standard using 5 ml of ammonium standard solution (1 ppm NH4) R. Comparison : adipic acid CRS. Loss on drying (2.2.32). Not more than 0.5 per cent, TESTS determined on 1.000 g by drying in an oven at 100 C to Solution S. Dissolve 5.0 g with heating in distilled water R 105 C. and dilute to 50 ml with the same solvent. Allow to cool Sulphated ash (2.4.14). Not more than 0.1 per cent, and to crystallise. Filter through a sintered-glass filter (40). determined on 1.0 g. Wash the filter with distilled water R. Collect the filtrate and the washings until a volume of 50 ml is obtained. ASSAY Dissolve 0.200 g, warming slightly if necessary, in a mixture Appearance of solution. The solution is clear (2.2.1) and of 20 ml of acetic anhydride R and 30 ml of anhydrous acetic colourless (2.2.2, Method II). acid R. Titrate with 0.1 M perchloric acid, determining the Dissolve 1.0 g in methanol R and dilute to 20 ml with the end-point potentiometrically (2.2.20). same solvent. 926 See the information section on general monographs (cover pages)