Glipizide

EUROPEAN PHARMACOPOEIA 5.0

D. Dissolve 50 mg in 5 ml of dioxan R. Add 1 ml of a 5 g/l

solution of fluorodinitrobenzene R in dioxan R and boil
for 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.
F. 1-(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-3-[(2Test solution (a). Dissolve 0.20 g of the substance to be
methylphenyl)sulphonyl]urea,
examined in a mixture of equal volumes of methanol R and
of methylene chloride R and dilute to 10 ml with the same
mixture of solvents.
Test solution (b). Dilute 1 ml of test solution (a) to 20 ml with
a mixture of equal volumes of methanol R and methylene
chloride R.
Reference solution (a). Dissolve 10 mg of glipizide CRS in
a mixture of equal volumes of methanol R and methylene
G. N-[(4-methylphenyl)sulphonyl]-1,4a,5,6,7,7a-hexahydrochloride R and dilute to 10 ml with the same mixture of
2H-cyclopenta[d]pyridazine-2-carboxamide.
solvents.
Reference solution (b). Dissolve 5 mg of glipizide
impurity
A CRS in a mixture of equal volumes of methanol R
01/2005:0906
and methylene chloride R and dilute to 50 ml with the same
mixture of solvents.
GLIPIZIDE
Reference solution (c). Dilute 0.5 ml of test solution (a) to
100 ml with a mixture of equal volumes of methanol R and
Glipizidum
methylene chloride R.
Reference solution (d). Dilute 4 ml of reference solution (c)
to 10 ml with a mixture of equal volumes of methanol R and
methylene chloride R.
Reference solution (e). Dilute 5 ml of test solution (a) to
10 ml with reference solution (b).
Apply to the plate 10 l of each solution. Develop over a
path of 15 cm using a mixture of 25 volumes of anhydrous
formic acid R, 25 volumes of ethyl acetate R and 50 volumes
of methylene chloride R. Allow the plate to dry in air and
C21H27N5O4S
Mr 445.5 examine in ultraviolet light at 254 nm. In the chromatogram
obtained with test solution (a) : any spot corresponding to
DEFINITION
glipizide impurity A is not more intense than the spot in the
Glipizide contains not less than 98.0 per cent
chromatogram obtained with reference solution (b) (0.5 per
and not more than the equivalent of 102.0 per
cent) ; any spot apart from the principal spot and the spot
cent of 1-cyclohexyl-3-[[4-[2-[[(5-methylpyrazin-2corresponding to glipizide impurity A is not more intense
yl)carbonyl]amino]ethyl]phenyl]sulphonyl]urea, calculated
than the spot in the chromatogram obtained with reference
with reference to the dried substance.
solution (c) (0.5 per cent) and not more than two such
spots are more intense than the spot in the chromatogram
CHARACTERS
obtained with reference solution (d) (0.2 per cent). The test is
A white or almost white, crystalline powder, practically
insoluble in water, soluble in methylene chloride, sparingly not valid unless the chromatogram obtained with reference
solution (e) shows two clearly separated spots.
soluble in acetone, practically insoluble in alcohol. It
Cyclohexylamine. Not more than 100 ppm, determined by
dissolves in dilute solutions of alkali hydroxides.
gas chromatography (2.2.28), using decane R as internal
IDENTIFICATION
standard.
First identification : B.
Internal standard solution. Dissolve 25 mg of decane R in
hexane R and dilute to 100 ml with the same solvent. Dilute
Second identification : A, C, D.
A. Dissolve about 2 mg in methanol R and dilute to 100 ml 5 ml of this solution to 50 ml with hexane R.
Test solution (a). Dissolve 3.0 g of the substance to
with the same solvent. Examined between 220 nm and
be examined in 50 ml of a 12 g/l solution of sodium
350 nm (2.2.25), the solution shows two absorption
hydroxide R and shake with two quantities, each of 5.0 ml,
maxima, at 226 nm and 274 nm. The ratio of the
absorbance measured at the maximum of 226 nm to that of hexane R. Use the combined upper layers.
measured at the maximum at 274 nm is 2.0 to 2.4.
Test solution (b). Dissolve 3.0 g of the substance to be
examined in 50 ml of a 12 g/l solution of sodium hydroxide R
B. Examine by infrared absorption spectrophotometry
and shake with two quantities, each of 5.0 ml, of the internal
(2.2.24), comparing with the spectrum obtained with
glipizide CRS. Examine the substances prepared as discs. standard solution. Use the combined upper layers.
Reference solution. Dissolve 30.0 mg of cyclohexylamine R
C. Examine the chromatograms obtained in the test for
in a 17.5 g/l solution of hydrochloric acid R and dilute to
related substances in ultraviolet light at 254 nm. The
100.0 ml with the same acid. To 1.0 ml of this solution add
principal spot in the chromatogram obtained with test
solution (b) is similar in position and size to the principal 50 ml of a 12 g/l solution of sodium hydroxide R and shake
with two quantities, each of 5.0 ml, of the internal standard
spot in the chromatogram obtained with reference
solution. Use the combined upper layers.
solution (a).
1662

See the information section on general monographs (cover pages)

Glucagon

EUROPEAN PHARMACOPOEIA 5.0

01/2005:0612

The chromatographic procedure may be carried out using :

a glass column 1.5 m long and 4 mm in internal diameter
packed with silanised diatomaceous earth for gas
chromatography R impregnated with 10 per cent m/m
of macrogol 20 000 R and 4 per cent m/m of potassium
hydroxide R,

GLUCAGON
Glucagonum

nitrogen 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 80 C and
that of the injection port and of the detector at 120 C.
Inject 1 l of the reference solution and adjust the sensitivity
of the detector so that the heights of the first peak (decane)
and the second peak (cyclohexylamine) are not less than
50 per cent of the full scale of the recorder. The test is not
valid unless the resolution between these two peaks is at
least 2. Inject 1 l of test solution (a). In the chromatogram
obtained, verify that there is no peak with the same retention
time as that of the internal standard. Inject separately
1 l of test solution (b) and 1 l of the reference solution.
From the chromatogram obtained with the reference
solution, calculate the ratio (R) of the area of the peak
due to cyclohexylamine to the area of the peak due to the
internal standard. From the chromatogram obtained with
test solution (b), calculate the ratio of the area of any peak
corresponding to cyclohexylamine to the area of the peak
due to the internal standard : this ratio is not greater than R.
Heavy metals (2.4.8). 2.0 g complies with limit test C for
heavy metals (20 ppm). Prepare the standard using 4 ml of
lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32). Not more than 0.5 per cent,
determined on 1.000 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. Titrate
with 0.1 M lithium methoxide using 0.2 ml of quinaldine
red solution R as indicator until the colour changes from
red to colourless.
1 ml of 0.1 M lithium methoxide is equivalent to 44.55 mg
of C21H27N5O4S.
IMPURITIES

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

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

C153H225N43O49S

Mr 3482

DEFINITION
Glucagon is a polypeptide hormone obtained from beef
or pork pancreas and which increases the blood-glucose
concentration by promoting rapid breakdown of liver
glycogen. The potency is not less than 1 IU/mg, calculated
with reference to the dried substance. Glucagon is prepared
in conditions designed to minimise microbial contamination.
CHARACTERS
A white or almost white powder, practically insoluble in
water and in most organic solvents. It dissolves in dilute
mineral acids and in dilute solutions of the alkali hydroxides.
IDENTIFICATION
A. 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 the
electropherogram obtained with test solution (a)
corresponds in position to the principal band in the
electropherogram obtained with reference solution (a).
TESTS
Absorbance. Dissolve 2.5 mg in 0.01 M hydrochloric acid
and dilute to 10.0 ml with the same acid. The specific
absorbance (2.2.25) determined at the maximum at 276 nm
is 21 to 25, calculated with reference to the dried substance.
Related substances. Examine by polyacrylamide gel
electrophoresis (2.2.31), using rod gels 75 mm long and
5 mm in diameter and, as buffer, tris-glycine buffer solution
pH 8.3 R. The electrode in the upper reservoir is the cathode
and that in the lower reservoir the anode.
Use the following gel mixture : mix 1 volume of
a solution containing in 100 ml 36.6 mg of
tris(hydroxymethyl)aminomethane R, 0.23 ml of
tetramethylethylenediamine R and 48.0 ml of 1 M
hydrochloric acid and 2 volumes of a solution containing in
100 ml 0.735 g of methylene-bisacrylamide R and 30.0 g of
acrylamide R. Add sufficient urea R to give a concentration
of 480 g/l in the final solution and dilute to 7 volumes
with water R. If necessary, heat to not more than 40 C to
dissolve the urea. Degas the solution and add 1 volume of a
5.6 g/l solution of ammonium persulphate R.
Test solution (a). Dissolve 10 mg of the substance to be
examined in 0.5 ml of 0.01 M sodium hydroxide.
Test solution (b). Dilute 0.25 ml of test solution (a) to 5 ml
with 0.01 M sodium hydroxide.
Reference solution (a). Dissolve a quantity of glucagon CRS
equivalent to 5 IU in 0.01 M sodium hydroxide and dilute to
25 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.
1663