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Cinchona bark

EUROPEAN 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 l 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.

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EUROPEAN PHARMACOPOEIA 5.0

Cinchona bark

B. Reduce to a powder (355). The powder is reddish-brown. Examine under a microscope using chloral hydrate solution R. The powder shows the following diagnostic characters : thin-walled cork cells filled with reddish-brown contents ; yellow, spindle-shaped striated phloem fibres up to 90 m in diameter and up to 1300 m in length, very thick-walled with an uneven lumen and with conspicuous, funnel-shaped pits ; parenchymatous idioblasts filled with microprisms of calcium oxalate. Examine under a microscope using a 50 per cent V/V solution of glycerol R. The powder shows a few starch granules 6 m to 10 m in diameter ; mostly simple but occasionally with 2 or 3 components. C. Thin-layer chromatography (2.2.27). Test solution. To 0.10 g of the powdered drug (180) in a test-tube add 0.1 ml of concentrated ammonia R and 5 ml of methylene chloride R. Shake vigorously occasionally during 30 min and filter. Evaporate the filtrate to dryness on a water-bath and dissolve the residue in 1 ml of ethanol R. Reference solution. Dissolve 17.5 mg of quinine R, 2.5 mg of quinidine R, 10 mg of cinchonine R and 10 mg of cinchonidine R in 5 ml of ethanol R. Plate : TLC silica gel plate R. Mobile phase : diethylamine R, ethyl acetate R, toluene R (10:20:70 V/V/V). Application : 10 l, as bands. Development : twice over a path of 15 cm. Drying : at 100-105 C then allow to cool. Detection A : spray with anhydrous formic acid R and allow to dry in air. Examine in ultraviolet light at 365 nm. Results A : see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Furthermore, other fluorescent zones are present in the chromatogram obtained with the test solution.
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 (2.8.2) : maximum 2 per cent m/m. Total ash (2.4.16) : maximum 6.0 per cent. Loss on drying (2.2.32) : maximum 10 per cent, determined on 1.000 g of the powdered drug (355) by drying in an oven at 100-105 C for 2 h. ASSAY Test solution. In a 250 ml conical flask mix 1.000 g of the powdered drug (180) with 10 ml of water R and 7 ml of dilute hydrochloric acid R. Heat in a water-bath for 30 min, allow to cool and add 25 ml of methylene chloride R, 50 ml of ether R and 5 ml of a 200 g/l solution of sodium hydroxide R. Shake the mixture repeatedly for 30 min, add 3 g of powdered tragacanth R and shake until the mixture becomes clear. Filter through a plug of absorbent cotton and rinse the flask and the cotton with 5 quantities, each of 20 ml, of a mixture of 1 volume of methylene chloride R and 2 volumes of ether R. Combine the filtrate and washings, evaporate to dryness and dissolve the residue in 10.0 ml of ethanol R. Evaporate 5.0 ml of the solution to dryness, dissolve the residue in 0.1 M hydrochloric acid and dilute to 1000.0 ml with the same acid. Reference solutions. Dissolve separately 30.0 mg of quinine R and 30.0 mg of cinchonine R in 0.1 M hydrochloric acid and dilute each solution to 1000.0 ml with the same acid. Measure the absorbances (2.2.25) of the 3 solutions at 316 nm and 348 nm using 0.1 M hydrochloric acid as the compensation liquid. Calculate the percentage content of alkaloids from the following equations :

m x y

= = =

mass of the drug used, in grams, percentage content of quinine-type alkaloids, percentage content of cinchonine-type alkaloids, absorbance of the test solution at 316 nm, absorbance of the test solution at 348 nm, absorbance of the reference solution containing cinchonine at 316 nm, corrected to a concentration of 1 mg/1000 ml, absorbance of the reference solution containing quinine at 316 nm, corrected to a concentration of 1 mg/1000 ml, absorbance of the reference solution containing cinchonine at 348 nm, corrected to a concentration of 1 mg/1000 ml, absorbance of the reference solution containing quinine at 348 nm, corrected to a concentration of 1 mg/1000 ml.

A316 = A348 = Detection B : spray with iodoplatinate reagent R. Results B : see below the sequence of the zones present in A316c = the chromatograms obtained with the reference solution and the test solution. Furthermore, other zones are A316q = present in the chromatogram obtained with the test solution.
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, which becomes violet-grey (cinchonine) A violet zone, which becomes violet-grey (quinidine) An intense dark blue zone (cinchonidine) _______ A violet zone, which becomes violet-grey (quinine) Test solution

A348c = A348q =

Calculate the content of total alkaloids, (x + y), and the relative content of quinine-type alkaloids, from the following expression :

General Notices (1) apply to all monographs and other texts

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