Betadex EUROPEAN PHARMACOPOEIA 5.0 01/2005:1070 DEFINITION Betacarotene contains not less than 96.0 per cent and not more than the equivalent of 101.0 per cent of (all-E)-3,7,12,16-Tetramethyl-1,18-bis(2,6,6-trimethylcyclohex1-enyl)octadeca-1,3,5,7,9,11,13,15,17-nonaene, calculated with reference to the dried substance BETADEX Betadexum CHARACTERS A brown-red or brownish-red, crystalline powder, practically insoluble in water, slightly soluble in cyclohexane, practically insoluble in ethanol It is sensitive to air, heat and light, especially in solution Carry out all operations as rapidly as possible avoiding exposure to actinic light ; use freshly prepared solutions IDENTIFICATION Dissolve 50.0 mg in 10 ml of chloroform R and dilute immediately to 100.0 ml with cyclohexane R Dilute 5.0 ml of this solution to 100.0 ml with cyclohexane R (solution A ; use solution A also for the test for related substances) Dilute 5.0 ml of solution A to 50.0 ml with cyclohexane R (Solution B ; use solution B also for the test for related substances and for the assay) Determine the absorbance (2.2.25) of solution B at 455 nm and at 483 nm using cyclohexane R as the compensation liquid The ratio of the absorbance at 455 nm to that at 483 nm is between 1.14 and 1.18 TESTS [C6H10O5]7 Mr 1135 DEFINITION Betadex (betacyclodextrin) contains not less than 98.0 per cent and not more than the equivalent of 101.0 per cent of cyclo-α-(1→4)-D-heptaglucopyranoside, calculated with reference to the dried substance CHARACTERS A white or almost white, amorphous or crystalline powder, sparingly soluble in water, freely soluble in propylene glycol, practically insoluble in ethanol and in methylene chloride Related substances Determine the absorbance (2.2.25) of solution B at 455 nm and that of solution A at 340 nm, used IDENTIFICATION in Identification The ratio of the absorbance at 455 nm to A It complies with the test for specific optical rotation (see that at 340 nm is not less than 1.5 Tests) B Examine the chromatograms obtained in the assay Heavy metals (2.4.8) 2.0 g complies with limit test D for The retention time and size of the principal peak in heavy metals (10 ppm) Prepare the standard using ml of the chromatogram obtained with test solution (b) are lead standard solution (10 ppm Pb) R approximately the same as those of the principal peak in Loss on drying (2.2.32) Not more than 0.2 per cent, the chromatogram obtained with reference solution (c) determined on 1.000 g by drying in vacuo over diphosphorus C Dissolve 0.2 g in ml of iodine solution R4 by warming pentoxide R at 40 °C for h on a water-bath, and allow to stand at room temperature Sulphated ash (2.4.14) Not more than 0.2 per cent, A yellowish-brown precipitate is formed determined on 1.0 g, moistened with a mixture of ml of TESTS dilute sulphuric acid R and ml of alcohol R Solution S Dissolve 1.000 g in carbon dioxide-free water R with heating, allow to cool and dilute to 100.0 ml with the same solvent ASSAY Appearance of solution Solution S is clear (2.2.1) Measure the absorbance (2.2.25) of solution B used pH (2.2.3) To 10 ml of solution S add 0.1 ml of a saturated in Identification at the maximum at 455 nm, using solution of potassium chloride R The pH of the solution cyclohexane R as the compensation liquid is 5.0 to 8.0 Calculate the content of C40H56 taking the specific absorbance Specific optical rotation (2.2.7) : + 160 to + 164, determined on solution S and calculated with reference to the dried to be 2500 substance Reducing sugars Test solution To ml of solution S add ml of cupri-tartaric STORAGE solution R4 Heat on a water-bath for 10 min, cool to room temperature Add 10 ml of ammonium molybdate Store in an airtight container, protected from light, at a reagent R1 and allow to stand for 15 temperature not exceeding 25 °C 1084 See the information section on general monographs (cover pages) Betadex EUROPEAN PHARMACOPOEIA 5.0 Reference solution Prepare a reference solution at the same time and in the same manner as the test solution, using ml of a 0.02 g/l solution of glucose R Measure the absorbance of the test solution and the reference solution (2.2.25) at the maximum at 740 nm using water R as the compensation liquid The absorbance of the test solution is not greater than that of the reference solution (0.2 per cent) Light-absorbing impurities Examine solution S between 230 nm and 750 nm (2.2.25) Between 230 nm and 350 nm, the absorbance is not greater than 0.10 Between 350 nm and 750 nm, the absorbance is not greater than 0.05 Related substances Examine by liquid chromatography (2.2.29), as described under Assay Inject separately test solution (a) and reference solution (b) In the chromatogram obtained with test solution (a) : the areas of any peaks corresponding to gammacyclodextrin and alphacyclodextrin are not greater than half of the area of the corresponding peaks in the chromatogram obtained with reference solution (b) (0.25 per cent) ; the sum of the areas of all the peaks, apart from the principal peak and any peaks corresponding to alphacyclodextrin and gammacyclodextrin, is not greater than half of the area of the peak corresponding to betadex in the chromatogram obtained with reference solution (b) (0.5 per cent) Residual solvents Not more than 10 ppm of trichloroethylene and not more than 10 ppm of toluene Examine by head-space gas chromatography (2.2.28), using the standard additions method and ethylene chloride R as the internal standard Test solutions In each of four identical 20 ml flasks, dissolve 500 mg of the substance to be examined in water R and add 0.10 g of calcium chloride R and 30 µl of α-amylase solution R Add ml of reference solutions (a), (b), (c) and (d), adding a different solution to each flask Dilute to 10 ml with water R Reference solutions Prepare reference solution (a) containing 10 µl of ethylene chloride R per litre From reference solution (a), prepare reference solutions (b), (c) and (d) containing per litre : µl, 10 µl and 15 µl each of trichloroethylene R and of toluene R The chromatographic procedure may be carried out using : — a fused-silica column 25 m long and 0.32 mm in internal diameter coated with a layer about µm thick of macrogol 20 000 R, — helium for chromatography R as the carrier gas, — a flame-ionisation detector, maintaining the temperature of the column at 50 °C, that of the injection port at 140 °C and that of the detector at 280 °C Place the samples in a thermostated chamber at 45 °C for h Inject 200 µl of the head-space of each flask and repeat each test at least three times The retention time of toluene is about 10 The test is not valid unless : the resolutions between the peaks corresponding to trichloroethylene and toluene and between the peaks corresponding to toluene and ethylene chloride are greater than 1.1 and the relative standard deviations of the ratios of the areas of the peaks corresponding to trichloroethylene and toluene to that of the peak corresponding to ethylene chloride are less than per cent Calculate the content of trichloroethylene and of toluene taking their relative densities to be 1.46 and 0.87, respectively Heavy metals (2.4.8) 1.0 g complies with limit test C for heavy metals (10 ppm) Prepare the standard using ml of lead standard solution (10 ppm Pb) R Loss on drying (2.2.32) Not more than 16.0 per cent, determined on 1.000 g by drying in an oven at 120 °C for h Sulphated ash (2.4.14) Not more than 0.1 per cent, determined on 1.0 g General Notices (1) apply to all monographs and other texts 1085 ASSAY Examine by liquid chromatography (2.2.29) Test solution (a) Dissolve 0.25 g of the substance to be examined in water R with heating, cool and dilute to 25.0 ml with the same solvent Test solution (b) Dilute 5.0 ml of test solution (a) to 50.0 ml with water R Reference solution (a) Dissolve 25.0 mg of alfadex CRS, 25.0 mg of gammacyclodextrin CRS and 50.0 mg of betadex CRS in water R and dilute to 50.0 ml with the same solvent Reference solution (b) Dilute 5.0 ml of reference solution (a) to 50.0 ml with water R Reference solution (c) Dissolve 25.0 mg of betadex CRS in water R and dilute to 25.0 ml with the same solvent The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (10 µm), — as mobile phase at a flow rate of 1.5 ml/min a mixture of 10 volumes of methanol R and 90 volumes of water R, — as detector a differential refractometer, — a 50 µl loop injector Equilibrate the column with the mobile phase at a flow rate of 1.5 ml/min for about h Inject each solution Record the chromatograms for 1.5 times the retention time of betadex Adjust the sensitivity of the detector so that the height of the peak corresponding to gammacyclodextrin, in the chromatogram obtained with reference solution (a), is 55 per cent to 75 per cent of the full scale of the recorder The retention time of betadex is about 10 min, the relative retention time of gammacyclodextrin is about 0.3 and that of alfadex is about 0.45 The test is not valid unless the resolution between the peaks corresponding to gammacyclodextrin and alfadex is not less than 1.5, and the relative standard deviation of the area of the peak corresponding to betadex is less than 2.0 per cent If necessary, adjust the concentration of methanol in the mobile phase to achieve the required resolution Calculate the percentage content of [C6H10O5]7 from the area of the principal peak in each of the chromatograms obtained with test solution (b) and reference solution (c) and the declared content of betadex CRS STORAGE Store in an airtight container IMPURITIES A n = : alfadex, B n = : gammacyclodextrin Betahistine mesilate EUROPEAN PHARMACOPOEIA 5.0 01/2005:1071 TESTS Solution S Dissolve 5.0 g in carbon dioxide-free water R prepared from distilled water R, and dilute to 50 ml with BETAHISTINE MESILATE the same solvent Appearance of solution Solution S is clear (2.2.1) and Betahistini mesilas colourless (2.2.2, Method II) pH (2.2.3) The pH of solution S is 2.0 to 3.0 Related substances Examine by liquid chromatography (2.2.29) Test solution Dissolve 50 mg of the substance to be C10H20N2O6S2 Mr 328.4 examined in the mobile phase and dilute to 10.0 ml with the mobile phase DEFINITION Reference solution (a) Dissolve 10 mg of betahistine Betahistine mesilate contains not less than 98.0 per cent mesilate CRS and 10 mg of 2-vinylpyridine R in the mobile and not more than the equivalent of 101.0 per cent of N-methyl-2-(pyridin-2-yl)ethanamine bis(methanesulphonate), phase and dilute to 50.0 ml with the mobile phase Dilute 2.0 ml of the solution to 50.0 ml with the mobile phase calculated with reference to the anhydrous, 2-propanol-free Reference solution (b) Dilute 1.0 ml of the test solution to substance 100.0 ml with the mobile phase PRODUCTION Reference solution (c) Dilute 2.0 ml of reference solution (b) The production method must be evaluated to determine to 10.0 ml with the mobile phase the potential for formation of alkyl mesilates, which is The chromatographic procedure may be carried out using : particularly likely to occur if the reaction medium contains — a stainless steel column 0.25 m long and 4.6 mm in lower alcohols Where necessary, the production method internal diameter packed with octadecylsilyl silica gel for is validated to demonstrate that alkyl mesilates are not chromatography R (5 µm), detectable in the final product — as mobile phase at a flow rate of ml/min a mixture CHARACTERS prepared as follows : dissolve 2.0 g of sodium dodecyl A white, crystalline powder, very hygroscopic, very soluble sulphate R in a mixture of 15 volumes of a 10 per cent V/V in water, freely soluble in alcohol, very slightly soluble in solution of sulphuric acid R, 35 volumes of a 17 g/l 2-propanol solution of tetrabutylammoniumhydrogen sulphate R and 650 volumes of water R ; adjust to pH 3.3 using dilute IDENTIFICATION sodium hydroxide solution R and mix with 300 volumes of acetonitrile R, First identification : B — as detector a spectrophotometer set at 260 nm Second identification : A, C, D Inject 20 µl of reference solution (a) When using a recorder, A Melting point (2.2.14) : 108 °C to 112 °C adjust the sensitivity of the system so that the height of the B Examine by infrared absorption spectrophotometry first peak in the chromatogram obtained with reference (2.2.24), comparing with the spectrum obtained with solution (a) is not less than 70 per cent of the full scale of the betahistine mesilate CRS Examine the substances recorder The test is not valid unless : in the chromatogram prepared as discs obtained with reference solution (a), the resolution between C Examine by thin-layer chromatography (2.2.27), using a the peaks corresponding to 2-vinylpyridine and betahistine suitable silica gel with a fluorescent indicator having an mesilate is at least 3.5 optimal intensity at 254 nm as the coating substance Inject 20 µl of the test solution and of reference solutions (b) Test solution Dissolve 10 mg of the substance to be and (c) Continue the chromatography for times the examined in alcohol R and dilute to ml with the same retention time of betahistine mesilate (which is about min) solvent In the chromatogram obtained with the test solution the Reference solution Dissolve 10 mg of betahistine area of any peak, apart from the principal peak, is not greater mesilate CRS, in alcohol R and dilute to ml with the than the area of the principal peak in the chromatogram same solvent obtained with reference solution (c) (0.2 per cent) ; the sum of the areas of any peaks, apart from the principal peak, is Apply to the plate µl of each solution Develop over not greater than half of the area of the principal peak in the a path of 15 cm using a mixture of 0.75 volumes of concentrated ammonia R, 15 volumes of ethyl acetate R chromatogram obtained with reference solution (b) (0.5 per cent) and 30 volumes of methanol R Dry the plate at 110 °C for 10 and examine in ultraviolet light at 254 nm Disregard any peak with an area less than 0.025 times that The principal spot in the chromatogram obtained with of the principal peak in the chromatogram obtained with the test solution is similar in position and size to the reference solution (b) principal spot in the chromatogram obtained with the 2-Propanol Not more than 0.5 per cent, determined by the reference solution test for residual solvents (2.4.24) D To 0.1 g add ml of dilute hydrochloric acid R and Chlorides (2.4.4) To 14 ml of solution S add ml of water R shake for about Add ml of barium chloride The solution complies with the limit test for chlorides solution R1 The solution remains clear To a further (35 ppm) 0.1 g add 0.5 g of anhydrous sodium carbonate R, mix and ignite until a white residue is obtained Allow to cool Sulphates (2.4.13) Dilute ml of solution S to 15 ml with and dissolve the residue in ml of water R The solution distilled water R The solution complies with the limit test for sulphates (250 ppm) gives reaction (a) of sulphates (2.3.1) 1086 See the information section on general monographs (cover pages) EUROPEAN PHARMACOPOEIA 5.0 Betamethasone Heavy metals (2.4.8) 12 ml of solution S complies with limit test A for heavy metals (20 ppm) Prepare the standard using lead standard solution (2 ppm Pb) R Water (2.5.12) Not more than 2.0 per cent, determined on C 0.50 g by the semi-micro determination of water examined and the reference substance separately in the smallest necessary quantity of methylene chloride R and evaporate to dryness on a water-bath Using the residues, record the spectra again Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a ASSAY fluorescent indicator having an optimal intensity at 254 nm Dissolve 0.140 g in 50 ml of a mixture of volume of anhydrous acetic acid R and volumes of acetic Test solution Dissolve 10 mg of the substance to be anhydride R Titrate with 0.1 M perchloric acid, determining examined in a mixture of volume of methanol R and the end-point potentiometrically (2.2.20) volumes of methylene chloride R and dilute to 10 ml with the same mixture of solvents ml of 0.1 M perchloric acid is equivalent to 16.42 mg of C10H20N2O6S2 Reference solution (a) Dissolve 20 mg of betamethasone CRS in a mixture of volume of STORAGE methanol R and volumes of methylene chloride R and Store in an airtight container dilute to 20 ml with the same mixture of solvents Reference solution (b) Dissolve 10 mg of IMPURITIES dexamethasone CRS in reference solution (a) and dilute to 10 ml with the same solution Apply separately to the plate µl of each solution Develop over a path of 15 cm using a mixture of volumes of butanol R saturated with water R, 10 volumes of A 2-ethenylpyridine toluene R and 85 volumes of ether R Allow the plate to dry in air and examine in ultraviolet light at 254 nm The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal 01/2005:0312 spot in the chromatogram obtained with reference solution (a) Spray with alcoholic solution of sulphuric BETAMETHASONE acid R Heat at 120 °C for 10 or until the spots appear Allow to cool Examine the chromatograms in daylight and in ultraviolet light at 365 nm The principal Betamethasonum spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (a) The test is not valid unless the chromatogram obtained with reference solution (b) shows two spots which may however not be completely separated D Mix about mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is obtained (usually less than min) Allow to cool, add C22H29FO5 Mr 392.5 ml of water R, 0.05 ml of phenolphthalein solution R1 and about ml of dilute hydrochloric acid R to render DEFINITION the solution colourless Filter Add 1.0 ml of the filtrate Betamethasone contains not less than 97.0 per cent and to a freshly prepared mixture of 0.1 ml of alizarin S not more than the equivalent of 103.0 per cent of 9-fluorosolution R and 0.1 ml of zirconyl nitrate solution R 11β,17,21-trihydroxy-16β-methylpregna-1,4-diene-3,20-dione, Mix, allow to stand for and compare the colour of calculated with reference to the dried substance the solution with that of a blank prepared in the same manner The test solution is yellow and the blank is red CHARACTERS E Add about mg to ml of sulphuric acid R and shake A white or almost white, crystalline powder, practically to dissolve Within min, a deep reddish-brown colour insoluble in water, sparingly soluble in ethanol, very slightly develops Add the solution to 10 ml of water R and mix soluble in methylene chloride The colour is discharged and a clear solution remains IDENTIFICATION TESTS First identification : B, C Specific optical rotation (2.2.7) Dissolve 0.125 g in Second identification : A, C, D, E methanol R and dilute to 25.0 ml with the same solvent The A Dissolve 10.0 mg in ethanol R and dilute to 100.0 ml specific optical rotation is + 118 to + 126, calculated with with the same solvent Place 2.0 ml of the solution in a reference to the dried substance stoppered tube, add 10.0 ml of phenylhydrazine-sulphuric acid solution R, mix and heat in a water-bath at 60 °C for Related substances Examine by liquid chromatography 20 Cool immediately The absorbance (2.2.25) of the (2.2.29) solution measured at 419 nm is not greater than 0.10 Test solution Dissolve 25.0 mg of the substance to be examined in a mixture of equal volumes of acetonitrile R B Examine by infrared absorption spectrophotometry and methanol R and dilute to 10.0 ml with the same solvent (2.2.24), comparing with the spectrum obtained with betamethasone CRS If the spectra obtained in the solid Reference solution (a) Dissolve mg of betamethasone CRS state with the substance to be examined and the reference and mg of methylprednisolone CRS in mobile phase A and substance show differences, dissolve the substance to be dilute to 100.0 ml with the same mobile phase General Notices (1) apply to all monographs and other texts 1087 Betamethasone EUROPEAN PHARMACOPOEIA 5.0 ASSAY Dissolve 0.100 g in alcohol R and dilute to 100.0 ml with the same solvent Dilute 2.0 ml of the solution to 100.0 ml with The chromatographic procedure may be carried out using : alcohol R Measure the absorbance (2.2.25) at the maximum — a stainless steel column 0.25 m long and 4.6 mm in at 238.5 nm internal diameter packed with octadecylsilyl silica gel for Calculate the content of C22H29FO5 taking the specific chromatography R (5 µm), absorbance to be 395 — as mobile phase at a flow rate of 2.5 ml/min, a STORAGE linear-gradient programme using the following Store protected from light conditions : Reference solution (b) Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A Mobile phase A In a 1000 ml volumetric flask mix 250 ml IMPURITIES of acetonitrile R with 700 ml of water R and allow to A dexamethasone, equilibrate ; adjust the volume to 1000 ml with water R and mix again, Mobile phase B Acetonitrile R, Time (min) Mobile phase A (per cent V/V) Mobile phase B (per cent V/V) Comment 100 isocratic 15 100 begin linear gradient 40 100 end chromatogram, return to 100A 41 100 begin equilibration with A 46 = 100 end equilibration, begin next chromatogram B 21-chloro-9-fluoro-11β,17-dihydroxy-16β-methylpregna1,4-diene-3,20-dione, — as detector a spectrophotometer set at 254 nm, maintaining the temperature of the column at 45 °C Equilibrate the column with mobile phase B at a flow rate of 2.5 ml/min for at least 30 and then with mobile phase A for For subsequent chromatograms, use the conditions described from 40 to 46 C 17,21-dihydroxy-16β-methylpregna-1,4,9(11)-triene-3,20dione, Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 20 µl of reference solution (b) is not less than 50 per cent of the full scale of the recorder Inject 20 µl of reference solution (a) When the chromatograms are recorded in the conditions described above, the retention times are : methylprednisolone, about 11.5 minutes and betamethasone, about 12.5 minutes The test is not valid unless the resolution between the peaks corresponding to methylprednisolone and betamethasone is at least 1.5 ; if necessary, adjust the concentration of acetonitrile in mobile phase A D 9-fluoro-11β,17-dihydroxy-16β-methyl-3,20-dioxopregna-1, 4-dien-21-yl ethoxycarboxylate, Inject separately 20 µl of the mixture of equal volumes of acetonitrile R and methanol R as a blank, 20 µl of the test solution and 20 µl of reference solution (b) In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent) and not more than E 9,11β-epoxy-17,21-dihydroxy-16β-methyl-9β-pregna-1,4one such peak has an area greater than half the area of the diene-3,20-dione, principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (2.0 per cent) Disregard any peak due to the blank and any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (b) Loss on drying (2.2.32) Not more than 0.5 per cent, determined on 0.500 g by drying in an oven at 100 °C to F 17,21-dihydroxy-16β-methylpregna-1,4,11-triene-3,20105 °C dione, 1088 See the information section on general monographs (cover pages) EUROPEAN PHARMACOPOEIA 5.0 Betamethasone acetate CHARACTERS A white or almost white, crystalline powder, practically insoluble in water, freely soluble in acetone, soluble in alcohol and in methylene chloride It shows polymorphism IDENTIFICATION First identification : B, C G 11α,17,21-trihydroxy-16β-methylpregna-1,4-diene-3,20Second identification : A, C, D, E, F dione, A Dissolve 10.0 mg in ethanol R and dilute to 100.0 ml with the same solvent Place 2.0 ml of this solution in a ground-glass-stoppered tube, add 10.0 ml of phenylhydrazine-sulphuric acid solution R, mix and heat in a water-bath at 60 °C for 20 Cool immediately The absorbance (2.2.25) of the solution measured at 419 nm is not more than 0.10 B Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with betamethasone acetate CRS If the spectra obtained in H 14-fluoro-11β,17,21-trihydroxy-16β-methyl-8α,9β,14βthe solid state show differences, dissolve the substance to pregna-1,4-diene-3,20-dione, be examined and the reference substance separately in the minimum volume of methanol R, evaporate to dryness on a water-bath and record new spectra using the residues C Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm Test solution Dissolve 10 mg of the substance to be examined in a mixture of volume of methanol R and volumes of methylene chloride R and dilute to 10 ml I 8-fluoro-11β,17,21-trihydroxy-16β-methyl-8α,9β-pregnawith the same mixture of solvents 1,4-diene-3,20-dione, Reference solution (a) Dissolve 20 mg of betamethasone acetate CRS in a mixture of volume of methanol R and volumes of methylene chloride R and dilute to 20 ml with the same mixture of solvents Reference solution (b) Dissolve 10 mg of prednisolone acetate CRS in reference solution (a) and dilute to 10 ml with the same solution Apply to the plate µl of each solution Prepare the mobile phase by adding a mixture of 1.2 volumes of water R and volumes of methanol R to a mixture of J 17,21-dihydroxy-16β-methylpregna-1,4-diene-3,20-dione 15 volumes of ether R and 77 volumes of methylene chloride R Develop over a path of 15 cm Allow the plate to dry in air and examine in ultraviolet light at 254 nm The principal spot in the chromatogram obtained with the 01/2005:0975 test solution is similar in position and size to the principal spot in the chromatogram obtained with reference BETAMETHASONE ACETATE solution (a) Spray with alcoholic solution of sulphuric acid R Heat at 120 °C for 10 or until the spots Betamethasoni acetas appear Allow to cool Examine the plate in daylight and in ultraviolet light at 365 nm The principal spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (a) The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots D Add about mg to ml of sulphuric acid R and shake to dissolve Within min, a deep reddish-brown colour develops Add the solution to 10 ml of water R and mix C24H31FO6 Mr 434.5 The colour is discharged and a clear solution remains DEFINITION E Mix about mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is Betamethasone acetate contains not less than 97.0 per obtained (usually less than min) Allow to cool, add ml cent and not more than the equivalent of 103.0 per cent of water R, 0.05 ml of phenolphthalein solution R1 and of 9-fluoro-11β,17-dihydroxy-16β-methyl-3,20-dioxopregnaabout ml of dilute hydrochloric acid R to render the 1,4-diene-21-yl acetate, calculated with reference to the solution colourless Filter To a freshly prepared mixture anhydrous substance General Notices (1) apply to all monographs and other texts 1089 Betamethasone dipropionate EUROPEAN PHARMACOPOEIA 5.0 of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R, add 1.0 ml of the filtrate Mix, allow to stand for and compare the colour of the solution with that of a blank prepared in the same manner The test solution is yellow and the blank is red F About 10 mg gives the reaction of acetyl (2.3.1) TESTS Specific optical rotation (2.2.7) Dissolve 0.250 g in dioxan R and dilute to 25.0 ml with the same solvent The specific optical rotation is + 120 to + 128, calculated with reference to the anhydrous substance Related substances Examine by liquid chromatography (2.2.29) ASSAY Dissolve 0.100 g in alcohol R and dilute to 100.0 ml with the same solvent Dilute 2.0 ml of the solution to 100.0 ml with alcohol R Measure the absorbance (2.2.25) at the maximum at 240 nm Calculate the content of C24H31FO6 taking the specific absorbance to be 350 STORAGE Store protected from light IMPURITIES A betamethasone, B dexamethasone acetate, Test solution Dissolve 25.0 mg of the substance to be examined in ml of acetonitrile R and dilute to 10.0 ml with the same solvent Reference solution (a) Dissolve mg of betamethasone acetate CRS and mg of dexamethasone acetate CRS in the mobile phase and dilute to 100.0 ml with the mobile phase Reference solution (b) Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase C betamethasone 11,21-diacetate, The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of ml/min a mixture prepared as follows : in a 1000 ml volumetric flask mix 380 ml of acetonitrile R with 550 ml of water R and allow to equilibrate ; dilute to 1000 ml with water R and mix again, D 9,11β-epoxy-17-hydroxy-16β-methyl-3,20-dioxo-9β-pregna1,4-diene-21-yl acetate — as detector a spectrophotometer set at 254 nm Equilibrate the column with the mobile phase at a flow rate of ml/min for about 30 Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 20 µl of reference solution (b) is at least 50 per cent of the full scale of the recorder 01/2005:0809 BETAMETHASONE DIPROPIONATE Betamethasoni dipropionas Inject 20 µl of reference solution (a).When the chromatograms are recorded in the prescribed conditions, the retention times are : betamethasone acetate about 19 and dexamethasone acetate about 22 The test is not valid unless the resolution between the peaks due to betamethasone acetate and dexamethasone acetate is at least 3.3 ; if necessary, adjust slightly the concentration of acetonitrile in the mobile phase Inject 20 µl of the test solution and 20 µl of reference solution (b) Continue the chromatography for 2.5 times the retention time of the principal peak in the chromatogram obtained with the test solution In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than 1.25 times the area of the principal peak in the chromatogram obtained with reference solution (b) (1.25 per cent) Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (b) Water (2.5.12) Not more than 4.0 per cent, determined on 0.100 g by the semi-micro determination of water 1090 C28H37FO7 Mr 504.6 DEFINITION Betamethasone dipropionate contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent of 9-fluoro-11β-hydroxy-16β-methyl-3,20-dioxopregna-1,4-diene17,21-diyl dipropanoate, calculated with reference to the dried substance CHARACTERS A white or almost white, crystalline powder, practically insoluble in water, freely soluble in acetone and in methylene chloride, sparingly soluble in alcohol IDENTIFICATION First identification : B, C See the information section on general monographs (cover pages) EUROPEAN PHARMACOPOEIA 5.0 Betamethasone dipropionate Reference solution (a) Dissolve 25 mg of betamethasone dipropionate CRS in methanol R with gentle heating and Dissolve 10.0 mg in ethanol R and dilute to 100.0 ml dilute to ml with the same solvent This solution is also with the same solvent Place 2.0 ml of this solution used to prepare reference solution (b) Dilute ml of the in a ground-glass-stoppered tube, add 10.0 ml of solution to 10 ml with methylene chloride R phenylhydrazine-sulphuric acid solution R, mix and heat Reference solution (b) Transfer ml of the solution in a water-bath at 60 °C for 20 Cool immediately obtained during preparation of reference solution (a) The absorbance (2.2.25) of the solution measured at to a 15 ml glass tube with a ground-glass stopper or a 419 nm is not more than 0.10 polytetrafluoroethylene cap Add 10 ml of saturated Examine by infrared absorption spectrophotometry methanolic potassium hydrogen carbonate solution R (2.2.24), comparing with the spectrum obtained with and immediately pass a current of nitrogen R briskly betamethasone dipropionate CRS through the solution for Stopper the tube Heat in a water-bath at 45 °C, protected from light, for h Examine by thin-layer chromatography (2.2.27), using Allow to cool as the coating substance a suitable silica gel with a Apply to the plate µl of each solution Prepare the fluorescent indicator having an optimal intensity at mobile phase by adding a mixture of 1.2 volumes of 254 nm water R and volumes of methanol R to a mixture of Test solution Dissolve 10 mg of the substance to be 15 volumes of ether R and 77 volumes of methylene examined in a mixture of volume of methanol R and chloride R Develop over a path of 15 cm Allow the plate volumes of methylene chloride R and dilute to 10 ml to dry in air and examine in ultraviolet light at 254 nm with the same mixture of solvents The principal spot in each of the chromatograms obtained with the test solutions is similar in position and size to Reference solution (a) Dissolve 10 mg of betamethasone the principal spot in the chromatogram obtained with dipropionate CRS in a mixture of volume of methanol R the corresponding reference solution Spray the plate and volumes of methylene chloride R and dilute to with alcoholic solution of sulphuric acid R Heat at 10 ml with the same mixture of solvents 120 °C for 10 or until the spots appear Allow to cool Reference solution (b) Dissolve 10 mg of desoxycortone Examine in daylight and in ultraviolet light at 365 nm acetate CRS in a mixture of volume of methanol R and The principal spot in each of the chromatograms obtained volumes of methylene chloride R and dilute to 10 ml with the test solutions is similar in position, colour in with the same mixture of solvents Dilute ml of this daylight, fluorescence in ultraviolet light at 365 nm and solution to 10 ml with reference solution (a) size to the principal spot in the chromatogram obtained with the corresponding reference solution The principal Apply to the plate µl of each solution Prepare the spot in each of the chromatograms obtained with test mobile phase by adding a mixture of 1.2 volumes of solution (b) and reference solution (b) has an Rf value water R and volumes of methanol R to a mixture of distinctly lower than that of the principal spots in each of 15 volumes of ether R and 77 volumes of methylene the chromatograms obtained with test solution (a) and chloride R Develop over a path of 15 cm Allow the plate reference solution (a) to dry in air and examine in ultraviolet light at 254 nm The principal spot in the chromatogram obtained with the E Add about mg to ml of sulphuric acid R and shake test solution is similar in position and size to the principal to dissolve Within min, a deep reddish-brown colour spot in the chromatogram obtained with reference develops Add the solution to 10 ml of water R and mix solution (a) Spray the plate with alcoholic solution of The colour is discharged and a clear solution remains sulphuric acid R Heat at 120 °C for 10 or until the F Mix about mg with 45 mg of heavy magnesium oxide R spots appear Allow to cool Examine in daylight and and ignite in a crucible until an almost white residue is in ultraviolet light at 365 nm The principal spot in the obtained (usually less than min) Allow to cool, add chromatogram obtained with the test solution is similar ml of water R, 0.05 ml of phenolphthalein solution R1 in position, colour in daylight, fluorescence in ultraviolet and about ml of dilute hydrochloric acid R to render light at 365 nm and size to the principal spot in the the solution colourless Filter Add 1.0 ml of the filtrate chromatogram obtained with reference solution (a) The to a freshly prepared mixture of 0.1 ml of alizarin S test is not valid unless the chromatogram obtained with solution R and 0.1 ml of zirconyl nitrate solution R reference solution (b) shows two clearly separated spots Mix, allow to stand for and compare the colour of the solution with that of a blank prepared in the same Examine by thin-layer chromatography (2.2.27), using manner The test solution is yellow and the blank is red as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at TESTS 254 nm Specific optical rotation (2.2.7) Dissolve 0.250 g in Test solution (a) Dissolve 25 mg of the substance to be dioxan R and dilute to 25.0 ml with the same solvent The examined in methanol R with gentle heating and dilute to ml with the same solvent This solution is also used specific optical rotation is + 63 to + 70, calculated with to prepare test solution (b) Dilute ml of the solution to reference to the dried substance Related substances Examine by liquid chromatography 10 ml with methylene chloride R (2.2.29) Test solution (b) Transfer ml of the solution obtained during preparation of test solution (a) to a 15 ml glass tube Test solution Dissolve 62.5 mg of the substance to be with a ground-glass stopper or a polytetrafluoroethylene examined in the mobile phase and dilute to 25.0 ml with the mobile phase cap Add 10 ml of saturated methanolic potassium hydrogen carbonate solution R and immediately pass a Reference solution (a) Dissolve 2.5 mg of betamethasone current of nitrogen R briskly through the solution for dipropionate CRS and 2.5 mg of beclometasone Stopper the tube Heat in a water-bath at 45 °C, dipropionate CRS in the mobile phase and dilute to 50.0 ml protected from light, for h Allow to cool with the same solvent Second identification : A, D, E, F A B C D General Notices (1) apply to all monographs and other texts 1091 Betamethasone sodium phosphate EUROPEAN PHARMACOPOEIA 5.0 Reference solution (b) Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), 01/2005:0810 BETAMETHASONE SODIUM PHOSPHATE Betamethasoni natrii phosphas — as mobile phase at a flow rate of ml/min a mixture prepared as follows : mix carefully 350 ml of water R with 600 ml of acetonitrile R and allow to equilibrate ; adjust the volume to 1000 ml with water R and mix again, — as detector a spectrophotometer set at 254 nm C H FNa2O8P Mr 516.4 Adjust the sensitivity so that the height of the principal peak 22 28 in the chromatogram obtained with reference solution (b) is DEFINITION 70 per cent to 90 per cent of the full scale of the recorder Betamethasone sodium phosphate contains not less than 96.0 per cent and not more than the equivalent of Equilibrate the column with the mobile phase at a flow rate 103.0 per cent of 9-fluoro-11β,17-dihydroxy-16β-methyl-3,20of ml/min for about 45 Inject 20 µl of reference dioxopregna-1,4-diene-21-yl disodium phosphate, calculated solution (a) When the chromatograms are recorded with reference to the anhydrous substance in the prescribed conditions, the retention times are : CHARACTERS betamethasone dipropionate, about ; beclometasone A white or almost white powder, very hygroscopic, freely dipropionate, about 10.7 The test is not valid soluble in water, slightly soluble in alcohol, practically unless the resolution between the peaks corresponding insoluble in methylene chloride to betamethasone dipropionate and beclometasone dipropionate is at least 2.5 ; if necessary, adjust the IDENTIFICATION concentration of acetonitrile in the mobile phase First identification : B, C Second identification : A, C, D, E, F Inject separately 20 µl of the test solution and 20 µl of reference solution (b) Continue the chromatography for A Dissolve 10.0 mg in ml of water R and dilute to 2.5 times the retention time of the principal peak In 100.0 ml with ethanol R Place 2.0 ml of this solution the chromatogram obtained with the test solution : the in a ground-glass-stoppered tube, add 10.0 ml of area of any peak apart from the principal peak is not phenylhydrazine-sulphuric acid solution R, mix and heat greater than 0.75 times the area of the principal peak in in a water-bath at 60 °C for 20 Cool immediately the chromatogram obtained with reference solution (b) The absorbance (2.2.25) of the solution measured at the (1.5 per cent) and not more than one such peak has an maximum at 450 nm is not more than 0.10 area greater than half the area of the principal peak in the B Examine by infrared absorption spectrophotometry chromatogram obtained with reference solution (b) (1 per (2.2.24), comparing with the spectrum obtained with cent) ; the sum of the areas of all the peaks, apart from the betamethasone sodium phosphate CRS If the spectra principal peak, is not greater than 1.25 times the area of the obtained in the solid state show differences, dissolve the principal peak in the chromatogram obtained with reference substance to be examined and the reference substance solution (b) (2.5 per cent) Disregard any peak with an area separately in the minimum volume of alcohol R, evaporate less than 0.025 times the area of the principal peak in the to dryness on a water-bath and record new spectra using chromatogram obtained with reference solution (b) the residues C Examine by thin-layer chromatography (2.2.27), using Loss on drying (2.2.32) Not more than 1.0 per cent, as the coating substance a suitable silica gel with a determined on 0.500 g by drying in an oven at 100-105 °C fluorescent indicator having an optimal intensity at 254 nm Test solution Dissolve 10 mg of the substance to be ASSAY examined in methanol R and dilute to 10 ml with the same solvent Dissolve 50.0 mg in alcohol R and dilute to 100.0 ml with Reference solution (a) Dissolve 10 mg of betamethasone the same solvent Dilute 2.0 ml of the solution to 50.0 ml sodium phosphate CRS in methanol R and dilute to with alcohol R Measure the absorbance (2.2.25) at the 10 ml with the same solvent maximum at 240 nm Reference solution (b) Dissolve 10 mg of prednisolone sodium phosphate CRS in methanol R and dilute to Calculate the content of C28H37FO7 taking the specific 10 ml with the same solvent Dilute ml of this solution absorbance to be 305 to 10 ml with reference solution (a) Apply to the plate µl of each solution Develop over a path of 15 cm using a mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes STORAGE of butanol R Allow the plate to dry in air and examine in ultraviolet light at 254 nm The principal spot in the chromatogram obtained with the test solution is Store protected from light 1092 See the information section on general monographs (cover pages) EUROPEAN PHARMACOPOEIA 5.0 similar in position and size to the principal spot in the chromatogram obtained with reference solution (a) Spray the plate with alcoholic solution of sulphuric acid R Heat at 120 °C for 10 or until the spots appear Allow to cool Examine in daylight and in ultraviolet light at 365 nm The principal spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (a) The test is not valid unless the chromatogram obtained with reference solution (b) shows two spots which may however not be completely separated Betamethasone sodium phosphate The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of ml/min a mixture prepared as follows : in a 250 ml conical flask, weigh 1.360 g of potassium dihydrogen phosphate R and 0.600 g of hexylamine R, mix and allow to stand for 10 and then dissolve in 185 ml of water R ; add 65 ml of acetonitrile R, mix and filter (0.45 µm), — as detector a spectrophotometer set at 254 nm D Add about mg to ml of sulphuric acid R and shake to dissolve Within min, an intense reddish-brown colour develops Add the solution to 10 ml of water R and mix The colour is discharged and a clear solution remains Equilibrate the column with the mobile phase at a flow rate of ml/min for about 45 F To about 40 mg add ml of sulphuric acid R and heat gently until white fumes are evolved Add nitric acid R dropwise, continue the heating until the solution is almost colourless and cool Add ml of water R, heat until white fumes are again evolved, cool, add 10 ml of water R and neutralise to red litmus paper R with dilute ammonia R1 The solution gives reaction (a) of sodium (2.3.1) and reaction (b) of phosphates (2.3.1) Inject 20 µl of the test solution and 20 µl of reference solution (b) Continue the chromatography for twice the retention time of the principal peak In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (2 per cent) and not more than one such peak has an area greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (3 per cent) Disregard any peak with an area less than 0.025 times the area of the principal peak in the chromatogram obtained with reference solution (b) Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with reference solution (b) is 70 per cent to 90 per cent of the full scale of E Mix about mg with 45 mg of heavy magnesium oxide R the recorder and ignite in a crucible until an almost white residue is obtained (usually less than min) Allow to cool, add Inject 20 µl of reference solution (a) When the ml of water R, 0.05 ml of phenolphthalein solution R1 chromatograms are recorded in the conditions described and about ml of dilute hydrochloric acid R to render above, the retention times are : betamethasone sodium the solution colourless Filter Add 1.0 ml of the filtrate phosphate about 14 ; dexamethasone sodium phosphate to a freshly prepared mixture of 0.1 ml of alizarin S about 15.5 The test is not valid unless the resolution solution R and 0.1 ml of zirconyl nitrate solution R between the peaks corresponding to betamethasone sodium Mix, allow to stand for and compare the colour of phosphate and dexamethasone sodium phosphate is at least the solution with that of a blank prepared in the same 2.0 ; if necessary, increase the concentration of acetonitrile manner The test solution is yellow and the blank is red or increase the concentration of water in the mobile phase TESTS Solution S Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent Appearance of solution Solution S is clear (2.2.1) and not more intensely coloured than reference solution B7 (2.2.2, Method II) Inorganic phosphate Dissolve 50 mg in water R and dilute to 100 ml with the same solvent To 10 ml of this solution add ml of molybdovanadic reagent R, mix and allow to pH (2.2.3) Dilute ml of solution S to ml with carbon stand for Any yellow colour in the solution is not dioxide-free water R The pH of the solution is 7.5 to 9.0 more intense than that in a standard prepared at the same Specific optical rotation(2.2.7) Dissolve 0.250 g in water R time and in the same manner using 10 ml of phosphate and dilute to 25.0 ml with the same solvent The specific standard solution (5 ppm PO4) R (1 per cent) optical rotation is + 98 to + 104, calculated with reference Water (2.5.12) Not more than 8.0 per cent, determined on to the anhydrous substance 0.200 g by the semi-micro determination of water Related substances Examine by liquid chromatography (2.2.29) ASSAY Test solution Dissolve 62.5 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the Dissolve 0.100 g in water R and dilute to 100.0 ml with the same solvent Dilute 5.0 ml of the solution to 250.0 ml with mobile phase water R Measure the absorbance (2.2.25) at the maximum at 241 nm Reference solution (a) Dissolve 25 mg of betamethasone sodium phosphate CRS and 25 mg of dexamethasone Calculate the content of C22H28FNa2O8P taking the specific sodium phosphate CRS in the mobile phase and dilute to absorbance to be 297 25.0 ml with the mobile phase Dilute 1.0 ml of this solution to 25.0 ml with the mobile phase STORAGE Reference solution (b) Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase Store in an airtight container, protected from light General Notices (1) apply to all monographs and other texts 1093 EUROPEAN PHARMACOPOEIA 5.0 Nicotine Related substances Examine by liquid chromatography (2.2.29) Test solution Dissolve 20.0 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the mobile phase Reference solution (a) Dissolve mg of nicotine ditartrate CRS and mg of myosmine R in the mobile phase and dilute to 50.0 ml with the mobile phase Reference solution (b) Dilute 0.4 ml of the test solution to 100.0 ml with the mobile phase The chromatography may be carried out using : — a stainless steel column 0.10 m long and mm in internal diameter, packed with octadecylsilyl silica gel for chromatography R (4 µm), — as mobile phase at a flow rate of 1.5 ml/min a solution prepared as follows : dissolve 2.31 g of sodium dodecyl sulphate R in a mixture of 250 ml of acetonitrile R and ASSAY 750 ml of a 13.6 g/l solution of potassium dihydrogen Dissolve 0.250 g in 20 ml of anhydrous acetic acid R, phosphate R, adjusted to pH 4.5 with sodium hydroxide R heating slightly if necessary, and add ml of acetic or phosphoric acid R, anhydride R Titrate with 0.1 M perchloric acid, using crystal violet solution R as indicator until the colour changes — as detector a spectrophotometer set at 254 nm Inject 25 µl of reference solution (a) When the to greenish-blue chromatograms are recorded in the prescribed conditions the ml of 0.1 M perchloric acid is equivalent to 12.21 mg of retention times are : nicotine about 13 and impurity D C6H6N2O about 11 The test is not valid unless the resolution between the impurity D peak eluting closest to the nicotine peak and the peak due to nicotine is at least 1.5 ; if necessary, 01/2005:1452 adjust the concentration of acetonitrile in the mobile phase Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with 25 µl of NICOTINE reference solution (b) is at least 50 per cent of the full scale of the recorder Nicotinum Inject 25 µl of the test solution and 25 µl of reference solution (b) Continue the chromatography for twice the retention time of the principal peak In the chromatogram obtained with the test solution : the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.4 per cent) ; the sum of the areas of all the C10H14N2 Mr 162.2 peaks, apart from the principal peak, is not greater than twice the area of the principal peak in the chromatogram DEFINITION obtained with reference solution (b) (0.8 per cent) Disregard any peak with an area less than 0.1 times the area of the Nicotine contains not less than 99.0 per cent and principal peak in the chromatogram obtained with reference not more than the equivalent of 101.0 per cent of solution (b) 3-[(2S)-1-methylpyrrolidin-2-yl]pyridine, calculated with reference to the anhydrous substance Water (2.5.12) Not more than 0.5 per cent, determined on 1.00 g by the semi-micro determination of water CHARACTERS A colourless or brownish viscous liquid, volatile, hygroscopic, ASSAY soluble in water, miscible with ethanol Dissolve 60.0 mg in 30 ml of anhydrous acetic acid R Titrate with 0.1 M perchloric acid determining the end-point IDENTIFICATION potentiometrically (2.2.20) A It complies with the test for specific optical rotation (see ml of 0.1 M perchloric acid is equivalent to 8.11 mg of Tests) C10H14N2 B Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph Eur reference spectrum STORAGE of nicotine Store, under nitrogen, in an airtight container, protected from light TESTS Appearance of solution Dissolve 1.0 g in water R and dilute IMPURITIES to 10 ml with the same solvent The solution is clear (2.2.1) and not more intensely coloured than reference solution Y5, BY5 or R5 (2.2.2, Method II) Specific optical rotation (2.2.7) Dissolve 1.00 g in ethanol R and dilute to 50.0 ml with the same solvent The specific A (2S)-1,2,3,6-tetrahydro-2,3′-bipyridyl (anatabine), optical rotation is − 140 to − 152 Apply to the plate µl of each solution Develop over a path of 10 cm using a mixture of volumes of water R, 45 volumes of ethanol R and 48 volumes of chloroform R Allow the plate to dry and examine in ultraviolet light at 254 nm Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution (0.25 per cent) Heavy metals (2.4.8) Dilute 12 ml of solution S to 18 ml with water R 12 ml of the solution complies with limit test A for heavy metals (30 ppm) Prepare the standard using lead standard solution (1 ppm Pb) R Loss on drying (2.2.32) Not more than 0.5 per cent, determined on 1.00 g by drying in vacuo for 18 h Sulphated ash (2.4.14) Not more than 0.1 per cent, determined on 1.0 g General Notices (1) apply to all monographs and other texts 2095 Nicotine resinate EUROPEAN PHARMACOPOEIA 5.0 Transfer 1.0 ml of the filtrate to a 20 ml volumetric flask, dilute to volume with 0.1 M hydrochloric acid and mix Determine the absorbance (2.2.25) at the minima at about 236 nm and 282 nm and at the maximum at 259 nm using 1.0 ml of a g/l solution of sodium chloride R diluted to 20 ml with 0.1 M hydrochloric acid as compensation liquid Calculate the percentage of nicotine release from the expression : B 3-(1-methyl-1H-pyrrol-2-yl)pyridine (β-nicotyrine), C (5S)-1-methyl-5-(pyridin-3-yl)pyrrolidin-2-one (cotinine), D 3-(4,5-dihydro-3H-pyrrol-2-yl)pyridine (myosmine), E (1RS,2S)-1-methyl-2-(pyridin-3-yl)pyrrolidine 1-oxide (nicotine N-oxide) 01/2005:1792 NICOTINE RESINATE Nicotini resinas DEFINITION Complex of nicotine (3-[(2S)-1-methylpyrrolidin-2-yl]pyridine) with a weak cationic exchange resin Content : 95.0 per cent to 115.0 per cent of the declared content of nicotine stated on the label (anhydrous susbtance) It may contain glycerol CHARACTERS Appearance : white or slightly yellowish powder Solubility : practically insoluble in water IDENTIFICATION A Infrared absorption spectrophotometry (2.2.24) Preparation : shake a quantity of the substance to be examined equivalent to 100 mg of nicotine with a mixture of 10 ml of dilute ammonia R2, 10 ml of water R, ml of strong sodium hydroxide solution R and 20 ml of hexane R for Transfer the upper layer to a beaker and evaporate to produce an oily residue Record the spectrum of the oily residue as a thin film between sodium chloride R plates Comparison : Ph Eur reference spectrum of nicotine B It complies with the test for nicotine release (see Tests) TESTS Nicotine release : minimum 70 per cent of the content determined under Assay in 10 Transfer an accurately weighed quantity of the substance to be examined equivalent to about mg of nicotine, to a glass-stoppered test-tube, add 10.0 ml of a g/l solution of sodium chloride R previously heated to 37 °C and shake vigorously for 10 Immediately filter the liquid through a dry filter paper discarding the first millilitre of filtrate 2096 323 = specific absorbance of nicotine at 259 nm, C = percentage of nicotine in the substance to be examined on the basis of the amount determined in the assay, m = mass of the substance to be examined, in milligrams, A259, A236, A282 = absorbances of the solution at the wavelength indicated by the subscript Related substances Liquid chromatography (2.2.29) Test solution Accurately weigh a quantity of the substance to be examined equivalent to 20 mg of nicotine into a glass-stoppered test-tube, add 5.0 ml of dilute ammonia R2, 5.0 ml of water R and shake vigorously for 10 Centrifuge at about 2000 r/min for 10 and dilute 3.0 ml of the clear solution to 10.0 ml with a 39.5 g/l solution of phosphoric acid R Reference solution (a) Weigh 60.0 mg of nicotine ditartrate CRS into a glass-stoppered test-tube, add 5.0 ml of dilute ammonia R2, 5.0 ml of water R and shake until dissolution is complete Dilute 3.0 ml of the clear solution to 10.0 ml with a 39.5 g/l solution of phosphoric acid R Reference solution (b) Dissolve mg of myosmine R in acetonitrile R and dilute to 5.0 ml with the same solvent To 2.0 ml add 1.0 ml of the clear solution obtained during preparation of reference solution (a) and dilute to 10.0 ml with a 39.5 g/l solution of phosphoric acid R Reference solution (c) Dilute 1.0 ml of the test solution to 10.0 ml with a 39.5 g/l solution of phosphoric acid R Dilute 1.0 ml to 20.0 ml with the mobile phase Column : — size : l = 0.10 m, Ø = mm, — stationary phase : octadecylsilyl silica gel for chromatography R (4 µm) Mobile phase : dissolve 2.31 g of sodium dodecyl sulphate R in a mixture of 250 ml of acetonitrile R and 750 ml of a 13.6 g/l solution of potassium dihydrogen phosphate R adjusted to pH 4.5 with dilute sodium hydroxide R or dilute phosphoric acid R Flow rate : 1.5 ml/min Detection : spectrophotometer at 254 nm Injection : 50 µl Run time : twice the retention time of nicotine System suitability : reference solution (b) : — resolution : minimum 1.5 between the peaks due to impurity D and nicotine Limits : — any impurity : not more than the area of the principal peak in the chromatogram obtained with reference solution (c) (0.5 per cent), See the information section on general monographs (cover pages) Nicotinic acid EUROPEAN PHARMACOPOEIA 5.0 01/2005:0459 — total: not more than twice the area of the principal peak in the chromatogram obtained with reference solution (c) (1.0 per cent), — disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.05 per cent) Water (2.5.12) : maximum 5.0 per cent Suspend 1.0 g in 20.0 ml of methanol R, shake for 30 and allow to stand for 30 Use 10 ml of the methanol layer for the titration Carry out a blank titration ASSAY Liquid chromatography (2.2.29) as described in the test for related substances Calculate the percentage content of nicotine using the chromatograms obtained with the test solution and reference solution (a) taking into account the declared content of nicotine ditartrate CRS STORAGE In an airtight container, protected from light LABELLING The label states the content of nicotine IMPURITIES A (2S)-1,2,3,6-tetrahydro-2,3′-bipyridyl (anatabine), B 3-(1-methyl-1H-pyrrol-2-yl)pyridine (β-nicotyrine), C (5S)-1-methyl-5-(pyridin-3-yl)pyrrolidin-2-one (cotinine), D 3-(4,5-dihydro-3H-pyrrol-2-yl)pyridine (myosmine), E (1RS,2S)-1-methyl-2-(pyridin-3-yl)pyrrolidine 1-oxide (nicotine N-oxide) General Notices (1) apply to all monographs and other texts NICOTINIC ACID Acidum nicotinicum C6H5NO2 Mr 123.1 DEFINITION Nicotinic acid contains not less than 99.5 per cent and not more than the equivalent of 100.5 per cent of pyridine-3-carboxylic acid, calculated with reference to the dried substance CHARACTERS A white, crystalline powder, soluble in boiling water and in boiling alcohol, sparingly soluble in water It dissolves in dilute solutions of the alkali hydroxides and carbonates IDENTIFICATION First identification : A, B Second identification : A, C A Melting point (2.2.14) : 234 °C to 240 °C B Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with nicotinic acid CRS C Dissolve about 10 mg in 10 ml of water R To ml of the solution add ml of cyanogen bromide solution R and ml of a 25 g/l solution of aniline R and shake A yellow colour develops TESTS Related substances Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance Test solution Dissolve 0.5 g of the substance to be examined in water R, warming slightly if necessary, and dilute to 25 ml with the same solvent Reference solution Dilute 0.5 ml of the test solution to 100 ml with water R Apply separately to the plate µl of each solution Develop over a path of 15 cm using a mixture of volumes of water R, 10 volumes of anhydrous formic acid R and 85 volumes of propanol R Dry the plate at 100 °C to 105 °C for 10 and examine in ultraviolet light at 254 nm Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution (0.5 per cent) Chlorides (2.4.4) Dissolve 0.25 g in water R, heating on a water-bath, and dilute to 15 ml with the same solvent The solution complies with the limit test for chlorides (200 ppm) Heavy metals (2.4.8) 1.0 g complies with limit test C for heavy metals (20 ppm) Prepare the standard using ml of lead standard solution (10 ppm Pb) R Loss on drying (2.2.32) Not more than 1.0 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C for h Sulphated ash (2.4.14) Not more than 0.1 per cent, determined on 1.0 g 2097 Nifedipine EUROPEAN PHARMACOPOEIA 5.0 ASSAY Dissolve 0.250 g in 50 ml of water R Titrate with 0.1 M sodium hydroxide, using 0.25 ml of phenolphthalein solution R as indicator, until a pink colour is obtained Carry out a blank titration ml of 0.1 M sodium hydroxide is equivalent to 12.31 mg of C6H5NO2 STORAGE Store protected from light NIFEDIPINE Nifedipinum Detection : examine in ultraviolet light at 254 nm Results : the principal spot in the chromatogram obtained with the test solution is similar in position, appearance at 254 nm and size to the principal spot in the chromatogram obtained with the reference solution D To 25 mg in a test tube, add 10 ml of a mixture of 1.5 volumes of hydrochloric acid R, 3.5 volumes of water R and volumes of alcohol R and dissolve with gentle heating Add 0.5 g of zinc R in granules and allow to stand for with occasional swirling Filter into a second test tube, add ml of a 10 g/l solution of sodium nitrite R to the filtrate and allow to stand for Add 01/2005:0627 ml of a 50 g/l solution of ammonium sulphamate R, corrected shake vigorously with care and add ml of a g/l solution of naphthylethylenediamine dihydrochloride R An intense red colour develops which persists for not less than TESTS Impurity D and other basic impurities Transfer g to a 250 ml conical flask and dissolve in 160 ml of glacial acetic acid R using an ultrasonic bath Titrate with 0.1 M perchloric acid using 0.25 ml of naphtholbenzein solution R as indicator until the colour changes from brownish-yellow to green Not more than 0.48 ml of 0.1 M perchloric acid is required (0.14 per cent) Related substances Liquid chromatography (2.2.29) Test solution Dissolve 0.200 g of the substance to be C17H18N2O6 Mr 346.3 examined in 20 ml of methanol R and dilute to 50.0 ml with the mobile phase DEFINITION Reference solution (a) Dissolve 10 mg of nifedipine Dimethyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine- impurity A CRS in methanol R and dilute to 25.0 ml with 3,5-dicarboxylate the same solvent Content : 98.0 per cent to 102.0 per cent (dried substance) Reference solution (b) Dissolve 10 mg of nifedipine impurity B CRS in methanol R and dilute to 25.0 ml with CHARACTERS the same solvent Appearance : yellow, crystalline powder Reference solution (c) Mix 1.0 ml of reference solution (a), Solubility : practically insoluble in water, freely soluble in 1.0 ml of reference solution (b) and 0.1 ml of the test solution acetone, sparingly soluble in ethanol and dilute to 20.0 ml with the mobile phase Dilute 2.0 ml of When exposed to daylight and to artificial light of certain this solution to 10.0 ml with the mobile phase wavelengths, it readily converts to a nitrosophenylpyridine Column : derivative Exposure to ultraviolet light leads to the — size : l = 0.15 m, Ø = 4.6 mm, formation of a nitrophenylpyridine derivative — stationary phase : octadecylsilyl silica gel for Prepare solutions immediately before use in the dark or chromatography R (5 µm) under long-wavelength light (> 420 nm) and protect them Mobile phase : acetonitrile R, methanol R, water R from light (9:36:55 V/V/V) IDENTIFICATION Flow rate : 1.0 ml/min First identification : B Detection : spectrophotometer at 235 nm Second identification : A, C, D Injection : 20 µl ; inject the test solution and reference A Melting point (2.2.14) : 171 °C to 175 °C solution (c) B Infrared absorption spectrophotometry (2.2.24) Run time : twice the retention time of nifedipine Comparison : nifedipine CRS Elution order : impurity A, impurity B, nifedipine C Thin-layer chromatography (2.2.27) Retention time : nifedipine = about 15.5 Test solution Dissolve 10 mg of the substance to be System suitability : reference solution (c) : examined in methanol R and dilute to 10 ml with the — resolution : minimum 1.5 between the peaks due to same solvent impurity A and impurity B and minimum 1.5 between the Reference solution Dissolve 10 mg of nifedipine CRS in peaks due to impurity B and nifedipine methanol R and dilute to 10 ml with the same solvent Limits : Plate : TLC silica gel F254 plate R — impurity A : not more than the area of the corresponding Mobile phase : ethyl acetate R, cyclohexane R peak in the chromatogram obtained with reference (40:60 V/V) solution (c) (0.1 per cent), Application : µl — impurity B : not more than the area of the corresponding Development : over 3/4 of the plate peak in the chromatogram obtained with reference Drying : in air solution (c) (0.1 per cent), 2098 See the information section on general monographs (cover pages) Nifuroxazide EUROPEAN PHARMACOPOEIA 5.0 01/2005:1999 corrected — any other impurity : not more than the area of the peak due to nifedipine in the chromatogram obtained with reference solution (c) (0.1 per cent), NIFUROXAZIDE — total: not more than 0.3 per cent, — disregard limit : 0.1 times the area of the peak due to nifedipine in the chromatogram obtained with reference solution (c) (0.01 per cent) Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for h Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g Nifuroxazidum ASSAY C12H9N3O5 Dissolve 0.1300 g in a mixture of 25 ml of 2-methyl-2-propanol R and 25 ml of perchloric acid solution R Titrate with 0.1 M cerium sulphate using 0.1 ml of ferroin R as indicator, until the pink colour disappears Titrate slowly towards the end of the titration Carry out a blank titration DEFINITION 1-(4-Hydroxybenzoyl)-2-[(5-nitrofuran-2-yl)methylene]diazane Content : 98.5 per cent to 101.5 per cent (dried substance) ml of 0.1 M cerium sulphate is equivalent to 17.32 mg of C17H18N2O6 STORAGE Protected from light IMPURITIES Specified impurities : A, B, C, D Mr 275.2 CHARACTERS Appearance : bright yellow, crystalline powder Solubility : practically insoluble in water, slightly soluble in alcohol, practically insoluble in methylene chloride IDENTIFICATION Infrared absorption spectrophotometry (2.2.24) Comparison : Ph Eur reference spectrum of nifuroxazide TESTS Specific absorbance (2.2.25) : 940 to 1000 at the absorption maximum at 367 nm Protected from light, dissolve 10.0 mg in 10 ml of ethylene glycol monomethyl ether R and dilute to 100.0 ml with methanol R Dilute 5.0 ml of this solution to 100.0 ml with methanol R Impurity A : maximum 0.05 per cent Test solution (a) Dissolve 1.0 g of the substance to be examined in dimethyl sulphoxide R and dilute to 10.0 ml A R = NO2 : dimethyl 2,6-dimethyl-4-(2-nitrophenyl)pyridine- with the same solvent 3,5-dicarboxylate (nitrophenylpyridine analogue), Test solution (b) To 5.5 ml of test solution (a) add 50.0 ml of water R while stirring Allow to stand for 15 and filter Reference solution To 0.5 ml of test solution (a) add 5.0 ml B R = NO : dimethyl 2,6-dimethyl-4-(2-nitrosophenyl)pyridine- of a 50 mg/l solution of 4-hydroxybenzohydrazide R in 3,5-dicarboxylate (nitrosophenylpyridine analogue), dimethyl sulphoxide R Add 50.0 ml of water R while stirring Allow to stand for 15 and filter Add 0.5 ml of phosphomolybdotungstic reagent R and 10.0 ml of sodium carbonate solution R separately to 10.0 ml of test solution (b) and to 10.0 ml of the reference solution Allow to stand for h Examine the solutions at 750 nm The absorbance (2.2.25) of the solution obtained with test solution (b) is not greater than that obtained with the reference solution Related substances Liquid chromatography (2.2.29) Use freshly prepared solutions, protected from light C methyl 2-(2-nitrobenzylidene)-3-oxobutanoate, Test solution Dissolve 0.100 g of the substance to be examined in 15.0 ml of dimethylformamide R and dilute to 100.0 ml with the mobile phase If precipitation occurs, use the supernatant liquid Reference solution (a) Dissolve 10.0 mg of methyl parahydroxybenzoate R (impurity B) in 2.0 ml of dimethylformamide R and dilute to 20.0 ml with the mobile phase Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase D methyl 3-aminobut-2-enoate General Notices (1) apply to all monographs and other texts 2099 Nikethamide EUROPEAN PHARMACOPOEIA 5.0 Reference solution (b) Dissolve mg of the substance to be examined and 10 mg of methyl parahydroxybenzoate R in ml of dimethylformamide R and dilute to 20 ml with the mobile phase Dilute ml of this solution to 100 ml with the mobile phase Column : — size : l = 0.25 m, Ø = 4.6 mm, — stationary phase: spherical octadecylsilyl silica gel for chromatography R (5 µm) with a specific surface area of 340 m2/g, a pore size of 10 nm and a carbon loading of 19 per cent Mobile phase : acetonitrile R, water R (35:65 V/V) Flow rate : ml/min Detection : spectrophotometer at 280 nm Injection : 20 µl Run time : times the retention time of nifuroxazide Relative retention with reference to nifuroxazide (retention time = about 6.5 min) : impurity A = about 0.4 ; impurity B = about 1.2 ; impurity C = about 2.8 ; impurity D = about 5.2 System suitability : reference solution (b) : — resolution : minimum 3.0 between the peaks due to nifuroxazide and impurity B Limits : — any impurity : not more than 0.6 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.3 per cent), and not more than such peak has an area greater than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), — total: not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent), — disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent) Heavy metals (2.4.8) : maximum 20 ppm 1.0 g complies with limit test D Prepare the standard using ml of lead standard solution (10 ppm Pb) R Loss on drying (2.2.32) : maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C for h Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g ASSAY Dissolve 0.200 g, if necessary with heating, in 30 ml of dimethylformamide R and add 20 ml of water R Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20) ml of 0.1 M sodium hydroxide is equivalent to 27.52 mg of C12H9N3O5 STORAGE Protected from light IMPURITIES Specified impurities : A, B, C, D A R = NH-NH2 : (4-hydroxybenzoyl)diazane (p-hydroxybenzohydrazide), 2100 B R = OCH3 : methyl 4-hydroxybenzoate, C (5-nitrofuran-2-yl)methylene diacetate, D 1,2-bis[(5-nitrofuran-2-yl)methylene]diazane (5-nitrofurfural azine) 01/2005:0233 NIKETHAMIDE Nicethamidum C10H14N2O Mr 178.2 DEFINITION Nikethamide contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of N,N-diethylpyridine-3-carboxamide, calculated with reference to the anhydrous substance CHARACTERS An oily liquid or a crystalline mass, colourless or slightly yellowish, miscible with water and with alcohol IDENTIFICATION First identification : A, B Second identification : A, C, D A Dissolve 0.15 g in 0.01 M hydrochloric acid and dilute to 100.0 ml with the same acid Dilute 1.0 ml of this solution to 100.0 ml with 0.01 M hydrochloric acid Examined between 230 nm and 350 nm (2.2.25) in a cm cell, the solution shows a single absorption maximum, at 263 nm The specific absorbance at the maximum is about 285 B Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with nikethamide CRS C Heat 0.1 g with ml of dilute sodium hydroxide solution R Diethylamine is evolved progressively and is recognisable by its characteristic odour and by its turning red litmus paper R blue D Dilute ml of solution S (see Tests) to 250 ml with water R To ml of this solution add ml of cyanogen bromide solution R Add ml of a 25 g/l solution of aniline R and shake A yellow colour develops TESTS Solution S Dissolve 2.5 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent See the information section on general monographs (cover pages) EUROPEAN PHARMACOPOEIA 5.0 Appearance The substance to be examined, in liquid form or liquefied by slight heating, is clear (2.2.1) and not more intensely coloured than reference solution Y5 (2.2.2, Method II) pH (2.2.3) The pH of solution S is 6.0 to 7.8 Refractive index (2.2.6) 1.524 to 1.526 Related substances Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance Test solution Dissolve 0.4 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent Reference solution (a) Dissolve 40 mg of ethylnicotinamide CRS in methanol R and dilute to 100 ml with the same solvent Reference solution (b) Dilute ml of reference solution (a) to 10 ml with methanol R Apply separately to the plate 10 µl of each solution Develop over a path of 15 cm using a mixture of 25 volumes of propanol R and 75 volumes of chloroform R Allow the plate to dry in air and examine in ultraviolet light at 254 nm In the chromatogram obtained with the test solution, any spot corresponding to ethylnicotinamide is not more intense than the spot in the chromatogram obtained with reference solution (a) (1.0 per cent) and any spot, apart from the principal spot and the spot corresponding to ethylnicotinamide, is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.1 per cent) Heavy metals (2.4.8) Dilute 10 ml of solution S to 25 ml with water R 12 ml of this solution complies with limit test A for heavy metals (10 ppm) Prepare the standard using lead standard solution (1 ppm Pb) R Water (2.5.12) Not more than 0.3 per cent, determined on 2.00 g by the semi-micro determination of water Sulphated ash (2.4.14) Not more than 0.1 per cent, determined on 1.0 g Nimesulide CHARACTERS Appearance : yellowish crystalline powder Solubility : practically insoluble in water, freely soluble in acetone, slightly soluble in ethanol mp : about 149 °C It shows polymorphism IDENTIFICATION Infrared absorption spectrophotometry (2.2.24) Preparation : discs Comparison : nimesulide CRS If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separately in acetone R, evaporate to dryness and record new spectra using the residues TESTS Absorbance (2.2.25) : maximum 0.50 at 450 nm Dissolve 1.0 g in acetone R and dilute to 10.0 ml with the same solvent Related substances Liquid chromatography (2.2.29) Test solution Dissolve 20 mg of the substance to be examined in ml of acetonitrile R and dilute to 20.0 ml with water R Reference solution (a) Dissolve 10 mg of nimesulide impurity C CRS and 10 mg of nimesulide impurity D CRS in 20 ml of acetonitrile R and dilute to 50.0 ml with water R Dilute 1.0 ml of the solution to 50.0 ml with the mobile phase Reference solution (b) Dilute 1.0 ml of the test solution to 10.0 ml with the mobile phase Dilute 1.0 ml of the solution to 100.0 ml with the mobile phase Column : — dimensions : l = 0.125 m, Ø = 4.0 mm, — stationary phase : octadecylsilyl silica gel for chromatography R ASSAY Mobile phase : a mixture of 35 volumes of acetonitrile R and Dissolve 0.150 g in a mixture of ml of acetic anhydride R 65 volumes of a 1.15 g/l solution of ammonium dihydrogen and 20 ml of anhydrous acetic acid R Titrate with phosphate R adjusted to pH 7.0 with ammonia R 0.1 M perchloric acid, determining the end-point Flow rate : 1.3 ml/min potentiometrically (2.2.20) Detection : spectrophotometer at 230 nm ml of 0.1 M perchloric acid is equivalent to 17.82 mg of Injection : 20 µl C10H14N2O Run time : times the retention time of nimesulide System suitability : 01/2005:1548 — resolution : minimum of 2.0 between the principal peaks in the chromatogram obtained with reference solution (a) Limits : NIMESULIDE — any impurity : not more than the area of the principal peak in the chromatogram obtained with reference Nimesulidum solution (b) (0.1 per cent), — total : not more than times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent), — disregard limit : 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.01 per cent) Heavy metals (2.4.8) : maximum 20 ppm 1.0 g complies with limit test D Prepare the standard using C13H12N2O5S Mr 308.3 ml of lead standard solution (10 ppm Pb) R Loss on drying (2.2.32) : maximum 0.5 per cent, determined DEFINITION on 1.000 g by drying in an oven at 100-105 °C for h N-(4-Nitro-2-phenoxyphenyl)methanesulphonamide Sulphated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g Content : 98.5 per cent to 101.5 per cent (dried substance) General Notices (1) apply to all monographs and other texts 2101 Nimodipine EUROPEAN PHARMACOPOEIA 5.0 ASSAY Dissolve 0.240 g in 30 ml of previously neutralised acetone R and add 20 ml of water R Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20) ml of 0.1 M sodium hydroxide is equivalent to 30.83 mg of C13H12N2O5S IMPURITIES A R1 = SO2-CH3, R2 = H, R3 = R4 = NO2 : N-(2,4-dinitro-6-phenoxyphenyl)methanesulphonamide, B R1 = SO2-CH3, R2 = R3 = R4 = H : N-(2phenoxyphenyl)methanesulphonamide, C R1 = R2 = R3 = R4 = H : 2-phenoxyaniline, D R1 = R2 = R4 = H, R3 = NO2 : 4-nitro-2-phenoxyaniline, E R1 = R2 = SO2-CH3, R3 = R4 = H : N,N-bis(methylsulphonyl)2-phenoxyaniline, F R1 = R2 = SO2-CH3, R3 = NO2, R4 = H : N,N-bis(methylsulphonyl)-4-nitro-2-phenoxyaniline, G 4-nitro-2-phenoxyphenol 01/2005:1245 NIMODIPINE Nimodipinum C21H26N2O7 Mr 418.4 DEFINITION Nimodipine contains not less than 98.5 per cent and not more than the equivalent of 101.5 per cent of 2-methoxyethyl 1-methylethyl (4RS)-2,6-dimethyl-4-(3-nitrophenyl)-1,4dihydropyridine-3,5-dicarboxylate, calculated with reference to the dried substance CHARACTERS A light yellow or yellow, crystalline powder, practically insoluble in water, freely soluble in ethyl acetate, sparingly soluble in ethanol 2102 It shows polymorphism Exposure to ultraviolet light leads to the formation of a nitrophenylpyridine derivative Prepare solutions immediately before use either protected from light or under long-wavelength light (> 420 nm) IDENTIFICATION Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with nimodipine CRS If the spectra obtained in the solid state show differences, record further spectra using 20 g/l solutions in methylene chloride R and a 0.2 mm cell TESTS Solution S Dissolve 1.0 g in acetone R and dilute to 20.0 ml with acetone R Appearance of solution Solution S is clear (2.2.1) Optical rotation (2.2.7) The angle of optical rotation, determined on solution S, is − 0.10° to + 0.10° Related substances Examine by liquid chromatography (2.2.29) Test solution Dissolve 40.0 mg of the substance to be examined in 2.5 ml of tetrahydrofuran R and dilute to 25.0 ml with the mobile phase Reference solution (a) Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase Dilute 2.0 ml of the solution to 10.0 ml with the mobile phase Reference solution (b) Dissolve 20.0 mg of nimodipine impurity A CRS in 2.5 ml of tetrahydrofuran R and dilute to 25.0 ml with the mobile phase Dilute 1.0 ml of the solution to 20.0 ml with the mobile phase Reference solution (c) Dilute 0.5 ml of the test solution to 20.0 ml with the mobile phase Reference solution (d) Mix 1.0 ml of reference solution (b) and 1.0 ml of reference solution (c) and dilute to 25.0 ml with the mobile phase The chromatographic procedure may be carried out using : — a stainless steel column 0.125 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 2.0 ml/min a mixture of 20 volumes of methanol R, 20 volumes of tetrahydrofuran R and 60 volumes of water R, — as detector a spectrophotometer set at 235 nm, maintaining the temperature of the column at 40 °C Adjust the sensitivity of the system so that the height of the peak corresponding to nimodipine in the chromatogram obtained with 20 µl of reference solution (d) is at least 50 per cent of the full scale of the recorder Inject 20 µl of reference solution (d) When the chromatograms are recorded in the prescribed conditions, the retention times are : impurity A about and nimodipine about The test is not valid unless the resolution between the peaks corresponding to impurity A and nimodipine is at least 1.5 Inject separately 20 µl of the test solution and 20 µl of reference solution (a) Record the chromatogram of the test solution for four times the retention time of nimodipine In the chromatogram obtained with the test solution : the area of the peak due to impurity A is not greater than the corresponding peak in the chromatogram obtained with reference solution (d) (0.1 per cent) ; none of the peaks, apart from the principal peak and the peak due to impurity A, has an area greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per See the information section on general monographs (cover pages) EUROPEAN PHARMACOPOEIA 5.0 cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent) Disregard any peak due to the solvent and any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (d) Loss on drying (2.2.32) Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C Sulphated ash (2.4.14) Not more than 0.1 per cent, determined on 1.0 g ASSAY Dissolve 0.180 g with gentle heating in a mixture of 25 ml of 2-methyl-2-propanol R and 25 ml of perchloric acid solution R Add 0.1 ml of ferroin R Titrate with 0.1 M cerium sulphate Titrate slowly towards the end of the titration Carry out a blank titration ml of 0.1 M cerium sulphate is equivalent to 20.92 mg of C21H26N2O7 STORAGE Store protected from light IMPURITIES A 2-methoxyethyl 1-methylethyl 2,6-dimethyl-4-(3nitrophenyl)pyridine-3,5-dicarboxylate, Nitrazepam DEFINITION Nitrazepam contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 7-nitro-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one, calculated with reference to the dried substance CHARACTERS A yellow, crystalline powder, practically insoluble in water, slightly soluble in alcohol IDENTIFICATION First identification : A, C Second identification : A, B, D, E A Melting point (2.2.14) : 226 °C to 230 °C B Protect the solutions from light and measure the absorbances immediately Dissolve 25.0 mg in a g/l solution of sulphuric acid R in methanol R and dilute to 250.0 ml with the same solvent Dilute 5.0 ml of the solution to 100.0 ml with a g/l solution of sulphuric acid R in methanol R Examined between 230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 280 nm The specific absorbance at the maximum is 890 to 950 C Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with nitrazepam CRS D Dissolve about 20 mg in a mixture of ml of hydrochloric acid R and 10 ml of water R Boil for min, cool and add ml of a g/l solution of sodium nitrite R Allow to stand for and add ml of a g/l solution of sulphamic acid R and mix Allow to stand for and add ml of a g/l solution of naphthylethylenediamine dihydrochloride R A red colour is produced E Dissolve about 10 mg in ml of methanol R, warming if necessary, and add 0.05 ml of dilute sodium hydroxide solution R An intense yellow colour is produced TESTS Related substances Carry out the test protected from light Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance Test solution Dissolve 0.2 g of the substance to be examined in acetone R and dilute to 10 ml with the same solvent Prepare the solution immediately before use Reference solution (a) Dissolve 10 mg of B R = CH(CH3)2 : bis(1-methylethyl) 2,6-dimethyl-4-(3aminonitrobenzophenone R in acetone R and nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate, dilute to 100 ml with the same solvent Dilute 10 ml of the C R = CH2-CH2-OCH3 : bis(2-methoxyethyl) 2,6-dimethyl-4-(3- solution to 50 ml with acetone R nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate Reference solution (b) Dissolve 10 mg of nitrazepam impurity A CRS in acetone R and dilute to 100 ml with the same solvent Dilute 10 ml of the solution to 50 ml with 01/2005:0415 acetone R Reference solution (c) Dilute ml of the test solution to NITRAZEPAM 20 ml with acetone R Dilute ml of this solution to 50 ml with acetone R Nitrazepamum Apply separately to the plate 10 µl of each solution Develop over a path of 12 cm using a mixture of 15 volumes of ethyl acetate R and 85 volumes of nitromethane R Allow the plate to dry in air and examine in ultraviolet light at 254 nm In the chromatogram obtained with the test solution : any spot corresponding to aminonitrobenzophenone is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.1 per cent) ; any spot corresponding to nitrazepam impurity A is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.1 per cent) ; any spot apart from the principal spot and C15H11N3O3 Mr 281.3 the spots corresponding to aminonitrobenzophenone and General Notices (1) apply to all monographs and other texts 2103 Nitrendipine EUROPEAN PHARMACOPOEIA 5.0 nitrazepam impurity A is not more intense than the spot in the chromatogram obtained with reference solution (c) (0.1 per cent) Heavy metals (2.4.8) 1.0 g complies with limit test D for heavy metals (20 ppm) Prepare the standard using ml of lead standard solution (10 ppm Pb) R Loss on drying (2.2.32) Not more than 0.5 per cent, determined on 1.00 g by drying in an oven at 100 °C to 105 °C for h Sulphated ash (2.4.14) Not more than 0.1 per cent, determined on 1.0 g ASSAY Dissolve 0.250 g in 25 ml of acetic anhydride R Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20) ml of 0.1 M perchloric acid is equivalent to 28.13 mg of C15H11N3O3 CHARACTERS A yellow, crystalline powder, practically insoluble in water, freely soluble in ethyl acetate, sparingly soluble in ethanol and in methanol It shows polymorphism Exposure to ultraviolet light leads to the formation of a nitrophenylpyridine derivative Prepare solutions immediately before use either protected from light or under long-wavelength light (> 420 nm) IDENTIFICATION Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with nitrendipine CRS If the spectra obtained in the solid state show differences, record further spectra using 20 g/l solutions in methylene chloride R and a 0.2 mm cell TESTS Optical rotation (2.2.7) Dissolve 0.2 g in acetone R and dilute to 10.0 ml with the same solvent The angle of optical STORAGE rotation is − 0.10° to + 0.10° Store protected from light Related substances Examine by liquid chromatography IMPURITIES (2.2.29) Test solution Dissolve 40.0 mg of the substance to be examined in 2.5 ml of tetrahydrofuran R and dilute to 25.0 ml with the mobile phase Reference solution (a) Dilute 2.0 ml of the test solution to 10.0 ml with the mobile phase Dilute 1.0 ml of this solution to 25.0 ml with the mobile phase Reference solution (b) Dissolve 20.0 mg of nitrendipine impurity A CRS in 2.5 ml of tetrahydrofuran R and dilute to 25.0 ml with the mobile phase Dilute 1.0 ml of this solution A 3-amino-6-nitro-4-phenylquinolin-2(1H)-one, to 20.0 ml with the mobile phase Reference solution (c) Dilute 0.5 ml of the test solution to 20.0 ml with the mobile phase Reference solution (d) Mix 1.0 ml of reference solution (b) and 1.0 ml of reference solution (c) and dilute to 25.0 ml with the mobile phase The chromatographic procedure may be carried out using : — a stainless steel column 0.125 m long and mm in internal diameter packed with octadecylsilyl silica gel for B (2-amino-5-nitrophenyl)phenylmethanone chromatography R (5 µm), — as mobile phase at a flow rate of ml/min a mixture of 14 volumes of acetonitrile R, 22 volumes of 01/2005:1246 tetrahydrofuran R and 64 volumes of water R, — as detector a spectrophotometer set at 235 nm, NITRENDIPINE maintaining the temperature of the column at 40 °C Adjust the sensitivity of the system so that the height of the Nitrendipinum nitrendipine peak in the chromatogram obtained with 20 µl of reference solution (d) is at least 50 per cent of the full scale of the recorder Inject 20 µl of reference solution (d) When the chromatogram is recorded in the prescribed conditions, the retention times are : nitrendipine impurity A about and nitrendipine about The test is not valid unless the resolution between the peaks corresponding to nitrendipine impurity A and nitrendipine is at least 2.0 Inject separately 20 µl of the test solution and 20 µl of C18H20N2O6 Mr 360.4 reference solution (a) Record the chromatogram of the test solution for five times the retention time of the principal DEFINITION peak In the chromatogram obtained with the test solution : Nitrendipine contains not less than 98.5 per cent and not the area of the peak corresponding to nitrendipine impurity A more than the equivalent of 101.5 per cent of ethyl methyl is not greater than the area of the corresponding peak in the (4RS)-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridinechromatogram obtained with reference solution (d) (0.1 per 3,5-dicarboxylate, calculated with reference to the dried cent) ; none of the peaks, apart from the principal peak substance and the peaks corresponding to nitrendipine impurity A 2104 See the information section on general monographs (cover pages) Nitric oxide EUROPEAN PHARMACOPOEIA 5.0 has an area greater than that of the peak corresponding to nitrendipine in the chromatogram obtained with reference solution (a) (0.8 per cent) ; the sum of the areas of all the peaks, apart from the principal peak, is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (1.2 per cent) Disregard any peak with an area less than 0.5 times the area of the nitrendipine peak in the chromatogram obtained with reference solution (d) Loss on drying (2.2.32) Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 100-105 °C Sulphated ash (2.4.14) Not more than 0.1 per cent, determined on 1.0 g IDENTIFICATION A Dilute ml to 100 ml with water R The solution is strongly acid (2.2.4) B 0.2 ml of the solution obtained in identification test A gives the reaction of nitrates (2.3.1) TESTS Appearance of solution Dilute ml to 10 ml with water R The solution is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II) Chlorides (2.4.4) To g add 10 ml of water R and 0.3 ml of silver nitrate solution R2 and allow to stand for protected from light Any opalescence is not more intense than that of a standard prepared in the same manner using ASSAY 13 ml of water R, 0.5 ml of nitric acid R, 0.5 ml of chloride Dissolve 0.160 g with gentle heating if necessary in a mixture standard solution (5 ppm Cl) R and 0.3 ml of silver nitrate of 25 ml of 2-methyl-2-propanol R and 25 ml of perchloric solution R2 (0.5 ppm) acid solution R Titrate with 0.1 M cerium sulphate, using Sulphates (2.4.13) To 15 g add 0.2 g of sodium carbonate R 0.1 ml of ferroin R as indicator Titrate slowly towards the After carbon dioxide has evolved, evaporate to dryness end of the titration Carry out a blank titration Dissolve the residue in 15 ml of distilled water R The ml of 0.1 M cerium sulphate is equivalent to 18.02 mg of solution complies with the limit test for sulphates (10 ppm) C18H20N2O6 Iron (2.4.9) Dissolve the residue obtained in the test for sulphated ash in ml of dilute hydrochloric acid R and STORAGE dilute to 20 ml with water R Dilute ml to 10 ml with Store protected from light water R The solution complies with the limit test for iron IMPURITIES (10 ppm) Heavy metals (2.4.8) Carefully evaporate 10.0 g to dryness on a water-bath Moisten the residue with a few drops of dilute hydrochloric acid R and dilute to 20 ml with water R 12 ml of the solution complies with limit test A for heavy metals (2 ppm) Prepare the standard using lead standard solution (2 ppm Pb) R Sulphated ash Carefully evaporate 20.00 g to dryness Moisten the residue with a few drops of sulphuric acid R and A ethyl methyl 2,6-dimethyl-4-(3-nitrophenyl)pyridine-3,5ignite to dull red The residue does not exceed 0.01 per cent dicarboxylate, ASSAY To 0.750 g add 50 ml of water R and titrate with M sodium hydroxide, determining the end-point potentiometrically (2.2.20) ml of M sodium hydroxide is equivalent to 63.0 mg of HNO3 STORAGE Store protected from light B R = CH3 : dimethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4dihydropyridine-3,5-dicarboxylate, 01/2005:1550 C R = CH2-CH3 : diethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4dihydropyridine-3,5-dicarboxylate NITRIC OXIDE Nitrogenii oxidum 01/2005:1549 NO NITRIC ACID DEFINITION Nitric oxide contains not less than 99.0 per cent V/V of NO This monograph applies to nitric oxide for medicinal use Acidum nitricum HNO3 Mr 30.01 Mr 63.0 CHARACTERS DEFINITION A colourless gas which turns brown when exposed to air At Nitric acid contains not less than 68.0 per cent m/m and not 20 °C and at a pressure of 101 kPa, volume dissolves in about 21 volumes of water more than 70.0 per cent m/m of HNO3 CHARACTERS A clear, colourless to almost colourless liquid, miscible with water It has a relative density of about 1.41 General Notices (1) apply to all monographs and other texts PRODUCTION Carbon dioxide Not more than 3000 ppm V/V, determined by gas chromatography (2.2.28) Gas to be examined The substance to be examined 2105 Nitrofural EUROPEAN PHARMACOPOEIA 5.0 Reference gas Use a mixture containing 3000 ppm V/V of carbon dioxide R1 in nitrogen R The chromatography may be carried out using : — a stainless steel column 3.5 m long and mm in internal diameter packed with ethylvinylbenzene-divinylbenzene copolymer R, — helium for chromatography R as the carrier gas at a flow rate of 15 ml/min, — a thermal conductivity detector, — a loop injector, maintaining the temperature of the column at 50 °C Inject the gas to be examined and the reference gas The test is not valid unless the chromatograms obtained show a clear separation of carbon dioxide from nitric oxide Calculate the carbon dioxide content in the gas to be examined from the area of the carbon dioxide peak in the chromatogram obtained with the reference gas Nitrogen Not more than 3000 ppm V/V, determined by gas chromatography (2.2.28) Gas to be examined The substance to be examined Reference gas Use a mixture containing 3000 ppm V/V of nitrogen R in helium for chromatography R The chromatography may be carried out using : — a stainless steel column 3.5 m long and mm in internal diameter packed with molecular sieve for chromatography R (0.5 nm), — helium for chromatography R as the carrier gas at a flow rate of 15 ml/min, — a thermal conductivity detector, — a loop injector, maintaining the temperature of the column at 50 °C Inject the gas to be examined and the reference gas The test is not valid unless the chromatograms obtained show a clear separation of nitrogen from nitric oxide Calculate the nitrogen content in the gas to be examined from the area of the peak due to nitrogen in the chromatogram obtained with the reference gas Nitrogen dioxide Not more than 400 ppm V/V, determined using an ultraviolet absorption spectrophotometry analyser Gas to be examined The substance to be examined Reference gas (a) Use nitrogen R1 Reference gas (b) Use a mixture containing 400 ppm V/V of nitrogen dioxide R in nitrogen R The apparatus consists of the following : — an ultraviolet-visible light source (analytical wavelength about 400 nm), — a sample gas cell through which the feed gas flows, — a closed reference gas cell containing nitrogen R1 in parallel with the sample gas cell, — a rotating chopper which feeds light alternately through the reference gas cell and the sample gas cell, — a semiconductor detector which generates a frequency modulated output whose amplitude is a measure of the difference of absorption of the sample gas and the reference gas Carry out the analysis in the following way : — set the zero of the instrument using reference gas (a) through the sample gas cell at a flow rate of litre/min, — adjust the span while feeding reference gas (b) through the sample gas cell at a flow rate of litre/min, 2106 — feed the gas to be examined through the sample gas cell at a flow rate of litre/min, read the value from the instrument output and calculate if necessary the concentration of nitrogen dioxide Nitrous oxide Not more than 3000 ppm V/V, determined by gas chromatography (2.2.28) Gas to be examined The substance to be examined Reference gas Use a mixture containing 3000 ppm V/V of nitrous oxide R in nitrogen R The chromatography may be carried out using : — a stainless steel column 3.5 m long and mm in internal diameter packed with ethylvinylbenzene-divinylbenzene copolymer R, — helium for chromatography R as the carrier gas at a flow rate of 15 ml/min, — a thermal conductivity detector, — a loop injector, maintaining the temperature of the column at 50 °C Inject the gas to be examined and the reference gas The test is not valid unless the chromatograms obtained show a clear separation of nitrous oxide from nitric oxide Calculate the nitrous oxide content in the gas to be examined from the area of the peak due to nitrous oxide in the chromatogram obtained with the reference gas Water Not more than 100 ppm V/V, determined using an electrolytic hygrometer (2.5.28) Assay Determine the content of nitric oxide by difference using the mass balance equation after determining the sum of the impurities described under Production IDENTIFICATION Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph Eur reference spectrum of nitric oxide STORAGE Store compressed at a pressure not exceeding 2.5 MPa (25 bars) measured at 15 °C, in suitable containers complying with the legal regulations IMPURITIES A carbon dioxide, B nitrogen, C nitrogen dioxide, D nitrous oxide, E water 01/2005:1135 NITROFURAL Nitrofuralum C6 H N O Mr 198.1 DEFINITION Nitrofural contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent of 2-[(5-nitrofuran-2-yl)methylene]diazanecarboxamide, calculated with reference to the dried substance See the information section on general monographs (cover pages) Nitrofurantoin EUROPEAN PHARMACOPOEIA 5.0 CHARACTERS the resolution between the peaks due to nitrofurantoin and A yellow or brownish-yellow, crystalline powder, very slightly nitrofural is at least 2.0 Inject 20 µl of the test solution and 20 µl of reference solution (a) Continue the chromatography soluble in water, slightly soluble in alcohol for ten times the retention time of nitrofural, which is IDENTIFICATION about In the chromatogram obtained with the test solution : the area of any peak, apart from the principal First identification : B peak, is not greater than the area of the principal peak in Second identification : A, C, D the chromatogram obtained with reference solution (a) A Carry out the test protected from bright light Use the (0.5 per cent) ; the sum of the areas of all the peaks, apart solution prepared for the assay Examined between from the principal peak, is not greater than twice the area 220 nm and 400 nm (2.2.25), the solution shows two of the principal peak in the chromatogram obtained with absorption maxima, at 260 nm and 375 nm The ratio of reference solution (a) (1.0 per cent) Disregard any peak with the absorbance measured at the maximum at 375 nm to an area less than 0.05 times that of the principal peak in the that measured at the maximum at 260 nm is 1.15 to 1.30 chromatogram obtained with reference solution (a) B Examine by infrared absorption spectrophotometry Loss on drying (2.2.32) Not more than 0.5 per cent, (2.2.24), comparing with the spectrum obtained with determined on 1.000 g by drying in an oven at 100-105 °C nitrofural CRS Examine the substances prepared as Sulphated ash (2.4.14) Not more than 0.1 per cent, discs determined on 1.0 g C Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance Test solution Dissolve 10 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent Reference solution Dissolve 10 mg of nitrofural CRS in methanol R and dilute to 10 ml with the same solvent Apply to the plate µl of each solution Develop over a path of 15 cm using a mixture of 10 volumes of methanol R and 90 volumes of nitromethane R Allow the plate to dry in air and spray with phenylhydrazine hydrochloride solution R The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution D Dissolve about mg in ml of dimethylformamide R and add 0.1 ml of alcoholic potassium hydroxide solution R A violet-red colour is produced ASSAY Carry out the assay protected from bright light Dissolve 60.0 mg in 20 ml of dimethylformamide R and dilute to 500.0 ml with water R Dilute 5.0 ml to 100.0 ml with water R Prepare a reference solution in the same manner using 60.0 mg of nitrofural CRS Measure the absorbances (2.2.25) of the two solutions at the maximum at 375 nm Calculate the content of C6H6N4O4 from the absorbances measured and the concentrations of the solutions TESTS pH (2.2.3) To 1.0 g add 100 ml of carbon dioxide-free water R Shake and filter The pH of the filtrate is 5.0 to 7.0 Related substances Examine by liquid chromatography (2.2.29) Test solution Dissolve 0.10 g of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase Reference solution (a) Dissolve 10.0 mg of (5-nitro-2-furyl)methylene diacetate R in the mobile phase and dilute to 20.0 ml with the mobile phase Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase Reference solution (b) Dissolve 10 mg of nitrofural CRS and 10 mg of nitrofurantoin R in the mobile phase and dilute to 100 ml with the mobile phase Dilute ml of this solution to 100 ml with the mobile phase The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of ml per minute a mixture of 40 volumes of acetonitrile R and 60 volumes of water R, — as detector a spectrophotometer set at 310 nm Inject 20 µl of reference solution (a) Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is not less than 50 per cent of the full scale of the recorder Inject 20 µl of reference solution (b) The test is not valid unless, in the chromatogram obtained, A bis[(5-nitrofuran-2-yl)methylene]diazane, General Notices (1) apply to all monographs and other texts STORAGE Store protected from light IMPURITIES B (5-nitrofuran-2-yl)methylene diacetate 01/2005:0101 NITROFURANTOIN Nitrofurantoinum C8 H N O Mr 238.2 DEFINITION Nitrofurantoin contains not less than 98.0 per cent and not more than the equivalent of 102.0 per cent of 1-[[(5nitrofuran-2-yl)methylene]amino]imidazolidine-2,4-dione, calculated with reference to the dried substance 2107 Nitrogen EUROPEAN PHARMACOPOEIA 5.0 CHARACTERS A yellow, crystalline powder or yellow crystals, odourless or almost odourless, very slightly soluble in water and in alcohol, soluble in dimethylformamide IDENTIFICATION A Carry out the test protected from bright light Use the solution prepared for the assay Examined between 220 nm and 400 nm (2.2.25), the solution shows two absorption maxima, at 266 nm and 367 nm The ratio of the absorbance at the maximum at 367 nm to that at the maximum at 266 nm is 1.36 to 1.42 B Dissolve about 10 mg in 10 ml of dimethylformamide R To ml of the solution add 0.1 ml of 0.5 M alcoholic potassium hydroxide A brown colour develops TESTS Related substances Examine by thin-layer chromatography (2.2.27), using silica gel HF254 R as the coating substance Test solution Dissolve 0.25 g of the substance to be examined in a minimum of dimethylformamide R and dilute to 10 ml with acetone R Reference solution Dilute ml of the test solution to 100 ml with acetone R Apply separately to the plate 10 µl of each solution Develop over a path of 15 cm using a mixture of 10 volumes of methanol R and 90 volumes of nitromethane R Allow the plate to dry in air and heat at 100 °C to 105 °C for Examine in ultraviolet light at 254 nm Spray with phenylhydrazine hydrochloride solution R Heat the plate at 100 °C to 105 °C for a further 10 When examined in ultraviolet light and after spraying, any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution (1.0 per cent) Loss on drying (2.2.32) Not more than 1.0 per cent, determined on 1.00 g by drying in an oven at 100 °C to 105 °C Sulphated ash (2.4.14) Not more than 0.1 per cent, determined on 1.0 g ASSAY Carry out the assay protected from bright light Dissolve 0.120 g in 50 ml of dimethylformamide R and dilute to 1000.0 ml with water R Dilute 5.0 ml of the solution to 100.0 ml with a solution containing 18 g/l of sodium acetate R and 0.14 per cent V/V of glacial acetic acid R Measure the absorbance (2.2.25) at the absorption maximum at 367 nm, using the sodium acetate solution described above as compensation liquid Calculate the content of C8H6N4O5, taking the specific absorbance to be 765 STORAGE Store protected from light, at a temperature below 25 °C 01/2005:1247 NITROGEN Nitrogenium N2 2108 Mr 28.01 DEFINITION Nitrogen contains not less than 99.5 per cent V/V of N2 This monograph applies to nitrogen for medicinal use CHARACTERS A colourless, odourless gas At 20 °C and at a pressure of 101 kPa, volume dissolves in about 62 volumes of water and about 10 volumes of alcohol PRODUCTION Carbon dioxide Not more than 300 ppm V/V, determined using an infrared analyser (2.5.24) Gas to be examined The substance to be examined It must be filtered to avoid stray light phenomena Reference gas (a) Use nitrogen R1 Reference gas (b) Use a mixture containing 300 ppm V/V of carbon dioxide R1 in nitrogen R1 Calibrate the apparatus and set the sensitivity using reference gases (a) and (b) Measure the content of carbon dioxide in the gas to be examined Carbon monoxide Not more than ppm V/V, determined using an infrared analyser (2.5.25) Gas to be examined The substance to be examined It must be filtered to avoid stray light phenomena Reference gas (a) Use nitrogen R1 Reference gas (b) Use a mixture containing ppm V/V of carbon monoxide R in nitrogen R1 Calibrate the apparatus and set the sensitivity using reference gases (a) and (b) Measure the content of carbon monoxide in the gas to be examined Oxygen Not more than 50 ppm V/V, determined using an oxygen analyser with a detector scale ranging from ppm V/V to 100 ppm V/V and equipped with an electrochemical cell The gas to be examined passes through a detection cell containing an aqueous solution of an electrolyte, generally potassium hydroxide The presence of oxygen in the gas to be examined produces variation in the electric signal recorded at the outlet of the cell that is proportional to the oxygen content Calibrate the analyser according to the instructions of the manufacturer Pass the gas to be examined through the analyser using a suitable pressure regulator and airtight metal tubes and operating at the prescribed flow-rates until constant readings are obtained Water Not more than 67 ppm V/V, determined using an electrolytic hygrometer (2.5.28) Assay Examine by gas chromatography (2.2.28) Gas to be examined The substance to be examined Reference gas (a) Use ambient air Reference gas (b) Use nitrogen R1 The chromatographic procedure may be carried out using : — a stainless steel column m long and mm in internal diameter packed with an appropriate molecular sieve for chromatography (0.5 nm), — helium for chromatography R as the carrier gas at a flow rate of 40 ml/min, — a thermal conductivity detector, — a loop injector, maintaining the temperature of the column at 50 °C and that of the detector at 130 °C Inject reference gas (a) Adjust the injected volumes and operating conditions so that the height of the peak due to nitrogen in the chromatogram obtained with the reference See the information section on general monographs (cover pages) EUROPEAN PHARMACOPOEIA 5.0 gas is at least 35 per cent of the full scale of the recorder The assay is not valid unless the chromatograms obtained show a clear separation of oxygen and nitrogen Inject the gas to be examined and reference gas (b) Calculate the content of N2 in the gas to be examined Nitrogen, low-oxygen Solubility : at 20 °C and at a pressure of 101 kPa, volume dissolves in about 62 volumes of water and about 10 volumes of alcohol PRODUCTION Oxygen : maximum ppm V/V, determined using an oxygen IDENTIFICATION analyser with a detector scale ranging from ppm V/V to 100 ppm V/V and equipped with an electrochemical cell First identification : A The gas to be examined passes through a detection cell Second identification : B, C containing an aqueous solution of an electrolyte, generally A Examine the chromatograms obtained in the Assay The potassium hydroxide The presence of oxygen in the gas retention time of the principal peak in the chromatogram to be examined produces variation in the electric signal obtained with the substance to be examined is recorded at the outlet of the cell that is proportional to the approximately the same as that of the principal peak in oxygen content the chromatogram obtained with reference gas (b) B In a 250 ml conical flask replace the air by the substance Calibrate the analyser according to the manufacturer’s instructions Pass the gas to be examined through the to be examined Place a burning or glowing splinter of analyser using a suitable pressure regulator and airtight wood in the flask The splinter is extinguished metal tubes and operating at the prescribed flow rates until C In a suitable test tube, place 0.1 g of magnesium R in constant readings are obtained turnings Close the tube with a two-hole stopper fitted with a glass tube reaching about cm above the turnings Impurities Gas chromatography (2.2.28) Gas to be examined The substance to be examined Pass the substance to be examined through the glass tube for without heating, then for 15 while Reference gas (a) Use ambient air heating the test tube to a red glow After cooling, add Reference gas (b) Use nitrogen R1 ml of dilute sodium hydroxide solution R The evolving Column : vapours change the colour of moistened red litmus — material : stainless steel, paper R blue — size : l = m, Ø = mm, TESTS — stationary phase : appropriate molecular sieve for Carbon dioxide Not more than 300 ppm V/V, determined chromatography (0.5 nm) using a carbon dioxide detector tube (2.1.6) Carrier gas : helium for chromatography R Carbon monoxide Not more than ppm V/V, determined Flow rate : 40 ml/min using a carbon monoxide detector tube (2.1.6) Temperature : Water vapour Not more than 67 ppm V/V, determined — column : 50 °C, using a water vapour detector tube (2.1.6) — detector : 130 °C STORAGE Detection : thermal conductivity Store as a compressed gas or a liquid in appropriate System suitability : reference gas (a) : adjust the injected containers complying with the legal regulations volumes and operating conditions so that the height of the peak due to nitrogen in the chromatogram obtained is at IMPURITIES least 35 per cent of the full scale of the recorder : A carbon dioxide, — the chromatogram obtained shows a clear separation of oxygen and nitrogen B carbon monoxide, Limit : C oxygen, — total : not more than 0.5 per cent of the sum of the areas of all the peaks (0.5 per cent V/V) D water IDENTIFICATION 01/2005:1685 First identification : A Second identification : B, C NITROGEN, LOW-OXYGEN A Examine the chromatograms obtained in the test for impurities (see Production) Nitrogenium oxygenio depletum Results : the principal peak in the chromatogram obtained with the gas to be examined is similar in retention time to the principal peak in the chromatogram obtained with N2 Mr 28.01 reference gas (b) DEFINITION B In a 250 ml conical flask replace the air by the gas to be This monograph applies to nitrogen which is used for examined Place a burning or glowing splinter of wood in inerting finished medicinal products which are particularly the flask The splinter is extinguished sensitive to degradation by oxygen It does not necessarily C In a suitable test tube, place 0.1 g of magnesium R in apply to nitrogen used in earlier production steps turnings Close the tube with a two-hole stopper fitted Content : minimum 99.5 per cent V/V of N2, calculated by with a glass tube reaching about cm above the turnings deduction of the sum of impurities found when performing Pass the gas to be examined through the glass tube for the test for impurities without heating, then for 15 while heating the test tube to a red glow After cooling, add ml of dilute CHARACTERS sodium hydroxide solution R The evolving vapours turn the colour of moistened red litmus paper R blue Colourless and odourless gas General Notices (1) apply to all monographs and other texts 2109 ... 60 – 26 60 → 170 26 – 41 Relative density (2. 2 .5) : 0.961 to 0.9 75 Refractive index (2. 2.6) : 1 . 52 8 to 1 .53 9 Optical rotation (2. 2.7) : + 10.0° to + 24 .0° 170 Injection port 22 0 Detector 27 0 Detection... solution Solution S is clear (2. 2.1) and methanol R colourless (2. 2 .2, Method II) Measure the absorbance (2. 2. 25 ) of the solution at 52 8 nm, Specific optical rotation (2. 2.7) The specific optical... to 25 . 0 ml with the Dissolve 0.100 g in water R and dilute to 100.0 ml with the same solvent Dilute 5. 0 ml of the solution to 25 0 .0 ml with mobile phase water R Measure the absorbance (2. 2. 25 )