Cinchona barkEUROPEAN PHARMACOPOEIA 5.0 Second identification : A, C, D, E. A. Dissolve 60.0 mg in 1 M hydrochloric acid and dilute to 100 ml with the same acid. Dilute 2 ml of the solution to 100 ml with 1 M hydrochloric acid. Examined between 220 nm and 350 nm (2.2.25), the solution shows two absorption maxima, at 246 nm and 319 nm. The ratio of the absorbance measured at 246 nm to that measured at 319 nm is 2.7 to 3.0. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with cinchocaine hydrochloride CRS. Examine the substances prepared as discs using potassium chloride R. C. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). D. Dissolve 0.5 g in 5 ml of water R. Add 1 ml of dilute ammonia R2. A white precipitate is formed. Filter, wash the precipitate with five quantities, each of 10 ml, of water R and dry in a desiccator. It melts at 64 °C to 66 °C (2.2.14). E. It gives reaction (a) of chlorides (2.3.1). TESTS Solution S. Dissolve 5.0 g in carbon dioxide-free water R prepared from distilled water R, and dilute to 50 ml with the same solvent. Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). pH (2.2.3). Dilute 10 ml of solution S to 50 ml with carbon dioxide-free water R. The pH of the solution is 5.0 to 6.0. Related substances. 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 (a). Dissolve 0.20 g of the substance to be examined in methanol R and dilute to 5 ml with the same solvent. Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with methanol R. Reference solution (a). Dissolve 20 mg of cinchocaine hydrochloride CRS in methanol R and dilute to 5 ml with the same solvent. Reference solution (b). Dilute 1 ml of test solution (b) to 20 ml with methanol R. Reference solution (c). Dilute 1 ml of test solution (b) to 50 ml with methanol R. Reference solution (d). Dissolve 20 mg of benzocaine CRS in methanol R and dilute to 5 ml with the same solvent. Dilute 1 ml of the solution and 1 ml of reference solution (a) to 20 ml with methanol R. Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture of 1 volume of ammonia R, 5 volumes of methanol R, 30 volumes of acetone R and 50 volumes of toluene R. Dry the plate in a current of warm air for 15 min. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not more intense than the principal spot in the chromatogram obtained with reference solution (b) (0.5 per cent) and at most one such spot is more intense than the spot in the chromatogram obtained with reference solution (c) (0.2 per cent). The test is not valid unless the chromatogram obtained with reference solution (d) shows two clearly separated spots. 1292 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. Loss on drying (2.2.32). Not more than 2.0 per cent, determined on 0.500 g by drying in vacuo at 60 °C. Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.300 g in a mixture of 15.0 ml of 0.01 M hydrochloric acid and 50 ml of alcohol R. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the two points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 37.99 mg of C20H30ClN3O2. STORAGE Store in an airtight container, protected from light. IMPURITIES A. R1 = Cl, R2 = NH-[CH2]2-N(C2H5)2 : 2-chloro-N-[2(diethylamino)ethyl]quinoline-4-carboxamide, B. R1 = R2 = OH : 2-hydroxyquinoline-4-carboxylic acid, C. R1 = OH, R2 = NH-[CH2]2-N(C2H5)2 : N-[2(diethylamino)ethyl]-2-hydroxyquinoline-4-carboxamide, D. R1 = O-[CH2]3-CH3, R2 = OH : 2-butoxyquinoline-4carboxylic acid. 01/2005:0174 CINCHONA BARK Cinchonae cortex DEFINITION Whole or cut, dried bark of Cinchona pubescens Vahl (Cinchona succirubra Pavon), of C. calisaya (Weddell), of C. ledgeriana (Moens ex Trimen) or of its varieties or hybrids. Content : minimum 6.5 per cent of total alkaloids, of which 30 per cent to 60 per cent consists of quinine-type alkaloids (dried drug). CHARACTERS Intense bitter, somewhat astringent taste. Macroscopic and microscopic characters described under identification tests A and B. IDENTIFICATION A. The stem and branch bark is supplied in quilled or curved pieces 2 mm to 6 mm thick. The outer surface is dull brownish-grey or grey and frequently bears lichens ; it is usually rough, marked with transverse fissures and longitudinally furrowed or wrinkled ; exfoliation of the outer surface occurs in some varieties. The inner surface is striated and deep reddish-brown ; the fracture is short in the outer part and fibrous in the inner part. See the information section on general monographs (cover pages) 27). Development : twice over a path of 15 cm. parenchymatous idioblasts filled with microprisms of calcium oxalate.1 ml of concentrated ammonia R and 5 ml of methylene chloride R. Thin-layer chromatography (2.10 g of the powdered drug (180) in a test-tube add 0. yellow. add 3 g of powdered tragacanth R and shake until the mixture becomes clear.0 ml with the same acid. (x + y). Reduce to a powder (355). and the relative content of quinine-type alkaloids. absorbance of the reference solution containing quinine at 348 nm.0 mg of quinine R and 30.5 mg of quinidine R. of a mixture of 1 volume of methylene chloride R and 2 volumes of ether R. determined on 1.32) : maximum 10 per cent. from the following expression : General Notices (1) apply to all monographs and other texts 1293 . absorbance of the test solution at 316 nm. The powder shows a few starch granules 6 µm to 10 µm in diameter . corrected to a concentration of 1 mg/1000 ml. ethyl acetate R. Application : 10 µl. each of 20 ml. funnel-shaped pits . Calculate the percentage content of alkaloids from the following equations : m x y = = = mass of the drug used. absorbance of the reference solution containing quinine at 316 nm. Reference solution. Total ash (2.0 Cinchona bark B. Reference solutions. Furthermore. spindle-shaped striated phloem fibres up to 90 µm in diameter and up to 1300 µm in length. Filter through a plug of absorbent cotton and rinse the flask and the cotton with 5 quantities. which becomes violet-grey (cinchonine) A violet zone. Drying : at 100-105 °C then allow to cool. 50 ml of ether R and 5 ml of a 200 g/l solution of sodium hydroxide R. other zones are A316q = present in the chromatogram obtained with the test solution. corrected to a concentration of 1 mg/1000 ml. A316 = A348 = Detection B : spray with iodoplatinate reagent R.1 M hydrochloric acid and dilute each solution to 1000. dissolve the residue in 0. toluene R (10:20:70 V/V/V). Detection A : spray with anhydrous formic acid R and allow to dry in air. Examine under a microscope using chloral hydrate solution R. which becomes violet-grey (quinidine) An intense dark blue zone (cinchonidine) _______ A violet zone. Evaporate the filtrate to dryness on a water-bath and dissolve the residue in 1 ml of ethanol R. The powder is reddish-brown.EUROPEAN PHARMACOPOEIA 5. percentage content of cinchonine-type alkaloids.2. absorbance of the test solution at 348 nm. allow to cool and add 25 ml of methylene chloride R. Measure the absorbances (2. corrected to a concentration of 1 mg/1000 ml. Results B : see below the sequence of the zones present in A316c = the chromatograms obtained with the reference solution and the test solution. other fluorescent zones are present in the chromatogram obtained with the test solution. absorbance of the reference solution containing cinchonine at 316 nm. To 0. which becomes violet-grey (quinine) Test solution A348c = A348q = Calculate the content of total alkaloids. very thick-walled with an uneven lumen and with conspicuous. percentage content of quinine-type alkaloids. Test solution.0 ml of the solution to dryness. Shake vigorously occasionally during 30 min and filter. Combine the filtrate and washings. mostly simple but occasionally with 2 or 3 components. Furthermore. Reference solution Top of the plate _______ Cinchonine : a violet zone which becomes violet-grey Quinidine : a violet zone which becomes violet-grey Cinchonidine : an intense dark blue zone _______ Quinine : a violet zone which becomes violet-grey Reference solution _______ A violet zone. Shake the mixture repeatedly for 30 min.2) : maximum 2 per cent m/m. in grams.000 g of the powdered drug (355) by drying in an oven at 100-105 °C for 2 h.0 ml of ethanol R. Loss on drying (2. Plate : TLC silica gel plate R.1 M hydrochloric acid and dilute to 1000. Mobile phase : diethylamine R. Top of the plate _______ Quinidine : a distinct blue fluorescent zone _______ Quinine : a distinct blue fluorescent zone _______ A distinct blue fluorescent zone (quinidine) _______ A distinct blue fluorescent zone (quinine) Test solution TESTS Foreign matter (2.1 M hydrochloric acid as the compensation liquid. corrected to a concentration of 1 mg/1000 ml. absorbance of the reference solution containing cinchonine at 348 nm.0 per cent. Evaporate 5. In a 250 ml conical flask mix 1.25) of the 3 solutions at 316 nm and 348 nm using 0. The powder shows the following diagnostic characters : thin-walled cork cells filled with reddish-brown contents . Dissolve separately 30.0 mg of cinchonine R in 0.2. Dissolve 17.000 g of the powdered drug (180) with 10 ml of water R and 7 ml of dilute hydrochloric acid R. evaporate to dryness and dissolve the residue in 10. C. Heat in a water-bath for 30 min.8. ASSAY Test solution. Examine under a microscope using a 50 per cent V/V solution of glycerol R. 2. Results A : see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. 10 mg of cinchonine R and 10 mg of cinchonidine R in 5 ml of ethanol R.2.4. as bands. Examine in ultraviolet light at 365 nm.5 mg of quinine R.16) : maximum 6.0 ml with the same acid.