EUROPEAN PHARMACOPOEIA 5.0 Xanthan gum

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XANTHAN GUM

Xanthani gummiDEFINITIONXanthan gum is a high-molecular-mass anionicpolysaccharide produced by fermentation of carbohydratewith Xanthomonas campestris. It consists of a principalchain of (14)-linked D-glucose units with trisaccharideside chains, on alternating anhydroglucose units,consisting of a glucuronic acid unit included between twomannose units. Most of the terminal units contain a pyruvatemoiety and the mannose unit adjacent to the principal chainmay be acetylated at C-6.Xanthan gum has a molecular mass of approximately 1 106.It contains not less than 1.5 per cent of pyruvoyl groups(C3H3O2 ; relative mass of the group 71.1), calculated withreference to the dried substance. Xanthan gum exists as thesodium, potassium or calcium salt.

CHARACTERSA white or yellowish-white, free-flowing powder, soluble inwater giving a highly viscous solution, practically insolublein organic solvents.

IDENTIFICATIONA. In a flask, suspend 1 g in 15 ml of 0.1 M hydrochloric

acid. Close the flask with a fermentation bulb containingbarium hydroxide solution R and heat carefully for 5 min.The barium hydroxide solution shows a white turbidity.

B. To 300 ml of water R, previously heated to 80 C andstirred rapidly with a mechanical stirrer in a 400 mlbeaker, add, at the point of maximum agitation, a dryblend of 1.5 g of carob bean gum R and 1.5 g of thesubstance to be examined. Stir until the mixture formsa solution, and then continue stirring for 30 min orlonger. Do not allow the water temperature to drop below60 C during stirring. Discontinue stirring and allow themixture to stand for at least 2 h. A firm rubbery gel formsafter the temperature drops below 40 C but no suchgel forms in a 1 per cent control solution of the sampleprepared in the same manner but omitting the carob beangum.

TESTS

pH (2.2.3). The pH of a 10.0 g/l solution is 6.0 to 8.0.Viscosity (2.2.10). The viscosity at 24 1 C is not lessthan 600 mPas. Add 3.0 g within 45 s to 90 s into 250 mlof a 12 g/l solution of potassium chloride R in a 500 mlbeaker stirring with a low-pitch propeller-type stirrer rotatingat 800 r/min. When adding the substance take care thatagglomerates are destroyed. Add an additional quantity of44 ml of water R, to rinse any adhering residue from thewalls of the beaker. Stir the preparation at 800 r/min for 2 hwhilst maintaining the temperature at 24 1 C. Determinethe viscosity within 15 min using a rotating viscosimeter setat 60 r/min and equipped with a rotating spindle 12.7 mmin diameter and 1.6 mm high which is attached to a shaft3.2 mm in diameter. The distance from the top of thecylinder to the lower tip of the shaft being 25.4 mm, and theimmersion depth being 50.0 mm.

2-Propanol. Not more than 750 ppm, determined by gaschromatography (2.2.28) using 2-methyl-2-propanol R asthe internal standard.Internal standard solution. Dilute 0.50 g of2-methyl-2-propanol R to 500 ml with water R.

Test solution. To 200 ml of water R in a 1000 mlround-bottomed flask, add 5.0 g of the substance to beexamined and 1 ml of a 10 g/l emulsion of dimeticone R inliquid paraffin R, stopper the flask and shake for 1 h. Distilabout 90.0 ml, mix the distillate with 4.0 ml of the internalstandard solution and dilute to 100.0 ml with water R.Reference solution. Dilute a suitable quantity of2-propanol R, accurately weighed, with water R to obtaina solution having a known concentration of 2-propanol ofabout 1 mg/ml. To 4.0 ml of this solution add 4.0 ml ofthe internal standard solution and dilute to 100.0 ml withwater R.The chromatographic procedure may be carried out using : a column 1.8 m long and 4.0 mm in internal diameter

packed with styrene-divinylbenzene copolymer R, helium for chromatography R as the carrier gas at a flow

rate of 30 ml/min, a flame-ionisation detector,maintaining the temperature of the column at 165 C, andthat of the injection port and of the detector at 200 C.Inject 5 l of the test solution and 5 l of the referencesolution. The retention time of 2-methyl-2-propanol is about1.5 relative to that of 2-propanol.

Other polysaccharides. Examine by thin-layerchromatography (2.2.27), using a TLC silica gel plate R.Test solution. To 10 mg in a thick-walled centrifuge test tubeadd 2 ml of a 230 g/l solution of trifluoroacetic acid R,shake vigorously to dissolve the forming gel, stopper the testtube, and heat the mixture at 120 C for 1 h. Centrifuge thehydrolysate, transfer the clear supernatant liquid carefullyinto a 50 ml flask, add 10 ml of water R and evaporate thesolution to dryness under reduced pressure. Take up theresidue thus obtained in 10 ml of water R and evaporate todryness under reduced pressure. Wash three times with20 ml of methanol R and evaporate under reduced pressure.To the resulting clear film which has no odour of acetic acid,add 0.1 ml of water R and 1 ml of methanol R. Centrifuge toseparate the amorphous precipitate. Dilute the supernatantliquid, if necessary, to 1 ml with methanol R.Reference solution. Dissolve 10 mg of glucose R and 10 mgof mannose R in 2 ml of water R and dilute to 10 ml withmethanol R.Apply separately to the plate, as bands, 5 l of each solution.Develop over a path of 15 cm using a mixture of 10 volumesof a 16 g/l solution of sodium dihydrogen phosphate R,40 volumes of butanol R and 50 volumes of acetone R.Spray evenly with a solution of 0.5 g of diphenylamine R in25 ml of methanol R to which 0.5 ml of aniline R and 2.5 mlof phosphoric acid R have been added. Heat for 5 min at120 C. Examine in daylight. The test is not valid unless thechromatogram obtained with the reference solution showstwo clearly separated greyish-brown zones due to glucoseand mannose in the middle third. The chromatogramobtained with the test solution shows corresponding zones.In addition, one weak reddish and two faint bluish-greybands may be visible just above the starting line. One or twobluish-grey bands may also be seen in the upper quarter ofthe chromatogram. No other bands are visible.

Loss on drying (2.2.32). Not more than 15.0 per cent,determined on 1.000 g by drying in an oven at 100 C to105 C for 2.5 h.

Total ash (2.4.16) : 6.5 per cent to 16.0 per cent.Microbial contamination. Total viable aerobic count(2.6.12) not more than 103 bacteria and 102 fungi per gram,determined by plate count. It complies with the test forEscherichia coli (2.6.13).

General Notices (1) apply to all monographs and other texts 2715

Xylazine hydrochloride for veterinary use EUROPEAN PHARMACOPOEIA 5.0

ASSAYTest solution. Dissolve a quantity of the substance to beexamined corresponding to 120.0 mg of the dried substancein water R and dilute to 20.0 ml with the same solvent.Reference solution. Dissolve 45.0 mg of pyruvic acid R inwater R and dilute to 500.0 ml with the same solvent.Place 10.0 ml of the test solution in a 50 ml round-bottomedflask, add 20.0 ml of 0.1 M hydrochloric acid and weigh.Boil on a water-bath under a reflux condenser for 3 h.Weigh and adjust to the initial mass with water R. In aseparating funnel mix 2.0 ml of the solution with 1.0 ml ofdinitrophenylhydrazine-hydrochloric solution R. Allow tostand for 5 min and add 5.0 ml of ethyl acetate R. Shake andallow the solids to settle. Collect the upper layer and shakewith three quantities, each of 5.0 ml, of sodium carbonatesolution R. Combine the aqueous layers and dilute to 50.0 mlwith sodium carbonate solution R. Mix. Treat 10.0 ml of thereference solution at the same time and in the same manneras for the test solution.Immediately measure the absorbance of the two solutions(2.2.25) at 375 nm, using sodium carbonate solution R asthe compensation liquid.The absorbance of the test solution is not less than that ofthe reference solution, which corresponds to a content ofpyruvic acid of not less than 1.5 per cent.

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XYLAZINE HYDROCHLORIDEFOR VETERINARY USE

Xylazini hydrochloridum ad usumveterinarium

C12H17ClN2S Mr 256.8

DEFINITIONN-(2,6-Dimethylphenyl)-5,6-dihydro-4H-1,3-thiazin-2-aminehydrochloride.Content : 98.0 per cent to 102.0 per cent (dried substance).

CHARACTERSAppearance : white or almost white, crystalline powder,hygroscopic.Solubility : freely soluble in water, very soluble in methanol,freely soluble in methylene chloride.

IDENTIFICATIONA. Infrared absorption spectrophotometry (2.2.24).

Preparation : discs.Comparison : xylazine hydrochloride CRS.

B. It gives reaction (b) of chlorides (2.3.1).

TESTS

Solution S. Dissolve 5.0 g in carbon dioxide-free water Rprepared from distilled water R, heating at 60 C ifnecessary ; allow to cool and dilute to 50.0 ml with the samesolvent.Appearance of solution. Solution S is not more opalescentthan reference suspension II (2.2.1) and is colourless (2.2.2,Method II).

pH (2.2.3) : 4.0 to 5.5 for solution S.

Impurity A : maximum 100 ppm.Solution A. Dissolve 0.25 g of the substance to be examinedin methanol R and dilute to 10 ml with the same solvent.This solution is used to prepare the test solution.Solution B. Dissolve 50 mg of 2,6-dimethylaniline R inmethanol R and dilute to 100 ml with the same solvent.Dilute 1 ml of the solution to 100 ml with methanol R. Thissolution is used to prepare the reference solution.Using 2 flat-bottomed tubes with an inner diameter of about10 mm, place in the first tube 2 ml of solution A, and in thesecond tube 1 ml of solution B and 1 ml of methanol R. Toeach tube add 1 ml of a freshly prepared 10 g/l solution ofdimethylaminobenzaldehyde R in methanol R and 2 ml ofglacial acetic acid R and allow to stand at room temperaturefor 10 min. Compare the colours in diffused daylight, viewingvertically against a white background. Any yellow colourin the test solution is not more intense than that in thereference solution.

Related substances. Liquid chromatography (2.2.29).Prepare the solutions immediately before use.Solvent mixture. Mix 8 volumes of acetonitrile R, 30 volumesof methanol R and 62 volumes of a 2.72 g/l solution ofpotassium dihydrogen phosphate R adjusted to pH 7.2 withdilute sodium hydroxide solution R.Test solution. Dissolve 0.100 g of the substance to beexamined in the solvent mixture and dilute to 20.0 ml withthe solvent mixture.Reference solution. Dissolve 5.0 mg of the substance tobe examined, 5.0 mg of 2,6-dimethylaniline R, 5.0 mgof xylazine impurity C CRS and 5.0 mg of xylazineimpurity E CRS in acetonitrile R and dilute to 100.0 ml withthe same solvent. Dilute 1.0 ml of this solution to 10.0 mlwith the solvent mixture.Column : size : l = 0.15 m, = 3.9 mm, stationary phase : end-capped octylsilyl silica gel for

chromatography with polar incorporated groups R(5 m),

temperature : 40 C.Mobile phase : mobile phase A : mix 30 volumes of methanol R

and 70 volumes of a 2.72 g/l solution of potassiumdihydrogen phosphate R adjusted to pH 7.2 with dilutesodium hydroxide solution R,

mobile phase B : methanol R, acetonitrile R (30:70 V/V),

Time(min)

Mobile phase A(per cent V/V)

Mobile phase B(per cent V/V)

0 - 15 89 28 11 72

15 - 21 28 72

21 - 22 28 89 72 11

22 - 33 89 11

Flow rate : 1.0 ml/min.Detection : spectrophotometer at 230 nm.Equilibration : for at least 30 min with a mixture of28 volumes of mobile phase A and 72 volumes of mobilephase B.Injection : 20 l.Relative retention with reference to xylazine (retentiontime = about 7.5 min) : impurity A = about 0.8 ;impurity E = about 1.6 ; impurity C = about 2.2.

2716 See the information section on general monographs (cover pages)