Glipizide EUROPEAN PHARMACOPOEIA 5.0

F. 1-(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-3-[(2-methylphenyl)sulphonyl]urea,

G. N-[(4-methylphenyl)sulphonyl]-1,4a,5,6,7,7a-hexahydro-2H-cyclopenta[d]pyridazine-2-carboxamide.

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GLIPIZIDE

Glipizidum

C21H27N5O4S Mr 445.5

DEFINITIONGlipizide contains not less than 98.0 per centand not more than the equivalent of 102.0 percent of 1-cyclohexyl-3-[[4-[2-[[(5-methylpyrazin-2-yl)carbonyl]amino]ethyl]phenyl]sulphonyl]urea, calculatedwith reference to the dried substance.

CHARACTERSA white or almost white, crystalline powder, practicallyinsoluble in water, soluble in methylene chloride, sparinglysoluble in acetone, practically insoluble in alcohol. Itdissolves in dilute solutions of alkali hydroxides.

IDENTIFICATIONFirst identification: B.Second identification : A, C, D.A. Dissolve about 2 mg in methanol R and dilute to 100 ml

with the same solvent. Examined between 220 nm and350 nm (2.2.25), the solution shows two absorptionmaxima, at 226 nm and 274 nm. The ratio of theabsorbance measured at the maximum of 226 nm to thatmeasured at the maximum at 274 nm is 2.0 to 2.4.

B. Examine by infrared absorption spectrophotometry(2.2.24), comparing with the spectrum obtained withglipizide CRS. Examine the substances prepared as discs.

C. Examine the chromatograms obtained in the test forrelated substances in ultraviolet light at 254 nm. Theprincipal spot in the chromatogram obtained with testsolution (b) is similar in position and size to the principalspot in the chromatogram obtained with referencesolution (a).

D. Dissolve 50 mg in 5 ml of dioxan R. Add 1 ml of a 5 g/lsolution of fluorodinitrobenzene R in dioxan R and boilfor 2-3 min. A yellow colour is produced.

TESTS

Related substances. Examine by thin-layer chromatography(2.2.27), using silica gel GF254 R as the coating substance.Test solution (a). Dissolve 0.20 g of the substance to beexamined in a mixture of equal volumes of methanol R andof methylene chloride R and dilute to 10 ml with the samemixture of solvents.Test solution (b). Dilute 1 ml of test solution (a) to 20 ml witha mixture of equal volumes of methanol R and methylenechloride R.Reference solution (a). Dissolve 10 mg of glipizide CRS ina mixture of equal volumes of methanol R and methylenechloride R and dilute to 10 ml with the same mixture ofsolvents.Reference solution (b). Dissolve 5 mg of glipizideimpurity A CRS in a mixture of equal volumes ofmethanol Rand methylene chloride R and dilute to 50 ml with the samemixture of solvents.Reference solution (c). Dilute 0.5 ml of test solution (a) to100 ml with a mixture of equal volumes of methanol R andmethylene chloride R.Reference solution (d). Dilute 4 ml of reference solution (c)to 10 ml with a mixture of equal volumes of methanol R andmethylene chloride R.Reference solution (e). Dilute 5 ml of test solution (a) to10 ml with reference solution (b).Apply to the plate 10 l of each solution. Develop over apath of 15 cm using a mixture of 25 volumes of anhydrousformic acid R, 25 volumes of ethyl acetate R and 50 volumesof methylene chloride R. Allow the plate to dry in air andexamine in ultraviolet light at 254 nm. In the chromatogramobtained with test solution (a) : any spot corresponding toglipizide impurity A is not more intense than the spot in thechromatogram obtained with reference solution (b) (0.5 percent) ; any spot apart from the principal spot and the spotcorresponding to glipizide impurity A is not more intensethan the spot in the chromatogram obtained with referencesolution (c) (0.5 per cent) and not more than two suchspots are more intense than the spot in the chromatogramobtained with reference solution (d) (0.2 per cent). The test isnot valid unless the chromatogram obtained with referencesolution (e) shows two clearly separated spots.

Cyclohexylamine. Not more than 100 ppm, determined bygas chromatography (2.2.28), using decane R as internalstandard.Internal standard solution. Dissolve 25 mg of decane R inhexane R and dilute to 100 ml with the same solvent. Dilute5 ml of this solution to 50 ml with hexane R.Test solution (a). Dissolve 3.0 g of the substance tobe examined in 50 ml of a 12 g/l solution of sodiumhydroxide R and shake with two quantities, each of 5.0 ml,of hexane R. Use the combined upper layers.Test solution (b). Dissolve 3.0 g of the substance to beexamined in 50 ml of a 12 g/l solution of sodium hydroxide Rand shake with two quantities, each of 5.0 ml, of the internalstandard solution. Use the combined upper layers.Reference solution. Dissolve 30.0 mg of cyclohexylamine Rin a 17.5 g/l solution of hydrochloric acid R and dilute to100.0 ml with the same acid. To 1.0 ml of this solution add50 ml of a 12 g/l solution of sodium hydroxide R and shakewith two quantities, each of 5.0 ml, of the internal standardsolution. Use the combined upper layers.

1662 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 5.0 Glucagon

The chromatographic procedure may be carried out using :

a glass column 1.5 m long and 4 mm in internal diameterpacked with silanised diatomaceous earth for gaschromatography R impregnated with 10 per cent m/mof macrogol 20 000 R and 4 per cent m/m of potassiumhydroxide R,

nitrogen for chromatography R as the carrier gas at aflow rate of 30 ml/min,

a flame-ionisation detector,

maintaining the temperature of the column at 80 C andthat of the injection port and of the detector at 120 C.

Inject 1 l of the reference solution and adjust the sensitivityof the detector so that the heights of the first peak (decane)and the second peak (cyclohexylamine) are not less than50 per cent of the full scale of the recorder. The test is notvalid unless the resolution between these two peaks is atleast 2. Inject 1 l of test solution (a). In the chromatogramobtained, verify that there is no peak with the same retentiontime as that of the internal standard. Inject separately1 l of test solution (b) and 1 l of the reference solution.From the chromatogram obtained with the referencesolution, calculate the ratio (R) of the area of the peakdue to cyclohexylamine to the area of the peak due to theinternal standard. From the chromatogram obtained withtest solution (b), calculate the ratio of the area of any peakcorresponding to cyclohexylamine to the area of the peakdue to the internal standard : this ratio is not greater than R.

Heavy metals (2.4.8). 2.0 g complies with limit test C forheavy metals (20 ppm). Prepare the standard using 4 ml oflead standard solution (10 ppm Pb) R.

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 0.2 per cent,determined on 1.0 g.

ASSAY

Dissolve 0.400 g in 50 ml of dimethylformamide R. Titratewith 0.1 M lithium methoxide using 0.2 ml of quinaldinered solution R as indicator until the colour changes fromred to colourless.

1 ml of 0.1 M lithium methoxide is equivalent to 44.55 mgof C21H27N5O4S.

IMPURITIES

A. 5-methyl-N-[2-(4-sulphamoylphenyl)ethyl]pyrazine-2-carboxamide,

B. cyclohexanamine.

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GLUCAGON

Glucagonum

C153H225N43O49S Mr 3482

DEFINITIONGlucagon is a polypeptide hormone obtained from beefor pork pancreas and which increases the blood-glucoseconcentration by promoting rapid breakdown of liverglycogen. The potency is not less than 1 IU/mg, calculatedwith reference to the dried substance. Glucagon is preparedin conditions designed to minimise microbial contamination.

CHARACTERSA white or almost white powder, practically insoluble inwater and in most organic solvents. It dissolves in dilutemineral acids and in dilute solutions of the alkali hydroxides.

IDENTIFICATIONA. It causes a rise of blood-glucose concentration in the test

animals when injected as prescribed in the assay.B. Examine the electropherograms obtained in the test

for related substances. The principal band in theelectropherogram obtained with test solution (a)corresponds in position to the principal band in theelectropherogram obtained with reference solution (a).

TESTS

Absorbance. Dissolve 2.5 mg in 0.01 M hydrochloric acidand dilute to 10.0 ml with the same acid. The specificabsorbance (2.2.25) determined at the maximum at 276 nmis 21 to 25, calculated with reference to the dried substance.

Related substances. Examine by polyacrylamide gelelectrophoresis (2.2.31), using rod gels 75 mm long and5 mm in diameter and, as buffer, tris-glycine buffer solutionpH 8.3 R. The electrode in the upper reservoir is the cathodeand that in the lower reservoir the anode.Use the following gel mixture : mix 1 volume ofa solution containing in 100 ml 36.6 mg oftris(hydroxymethyl)aminomethane R, 0.23 ml oftetramethylethylenediamine R and 48.0 ml of 1 Mhydrochloric acid and 2 volumes of a solution containing in100 ml 0.735 g of methylene-bisacrylamide R and 30.0 g ofacrylamide R. Add sufficient urea R to give a concentrationof 480 g/l in the final solution and dilute to 7 volumeswith water R. If necessary, heat to not more than 40 C todissolve the urea. Degas the solution and add 1 volume of a5.6 g/l solution of ammonium persulphate R.Test solution (a). Dissolve 10 mg of the substance to beexamined in 0.5 ml of 0.01 M sodium hydroxide.Test solution (b). Dilute 0.25 ml of test solution (a) to 5 mlwith 0.01 M sodium hydroxide.Reference solution (a). Dissolve a quantity of glucagon CRSequivalent to 5 IU in 0.01 M sodium hydroxide and dilute to25 ml with the same solvent.Reference solution (b). Dilute 8 ml of reference solution (a)to 10 ml with 0.01 M sodium hydroxide.Reference solution (c). Dilute 6 ml of reference solution (a)to 10 ml with 0.01 M sodium hydroxide.

General Notices (1) apply to all monographs and other texts 1663