Magnesium Stearate
Change the statement to:
Magnesium Stearate contains not less than 3.8 per cent and not more than 5.0 per cent of Mg, calculate on
the dried basis. The fatty acid fraction contains not less than 40.0 per cent of stearic acid and the sum of
stearic acid and palmitic acid is not less than 90.0 per cent.
Change Identification test to:
To 5.0 g add 50 ml of peroxide-free ether, 20 ml of dilute nitric acid and 20 ml of water and heat under a
reflux condenser until dissolution is complete. Allow to cool. In a separating funnel, separate the aqueous
layer and shake the ether layer with 2 quantities, each of 4 ml, of water. Combine the aqueous layers,
wash with 15 ml of peroxide-free ether and dilute to 50 ml with water (solution A). Evaporate the organic
layer to dryness and dry the residue at 105°.
A. The residue obtained in the preparation of solution A has a freezing point (2.4.11) not less than
53º.
B. The acid value of the fatty acids is 195 to 210, determined on 0.2 g of the residue obtained in the
preparation of solution A, dissolved in 25 ml of the prescribed mixture of solvents (2.3.23).
C. In the test for fatty acid composition, the principle peaks in the chromatogram obtained with the
test solution corresponds to the peak in the chromatogram obtained with the reference solution.
D. 1 ml of solution A gives the reaction of magnesium (2.3.1).
Change Assay to:
Fatty acid composition. Determine by gas chromatography (2.4.13).
Test solution. Dissolve 100 mg of the substance under examination in 5 ml of boron trifluoride-methanol
solution. Boil under a reflux condenser for 10 minutes, add 4.0 ml of heptane through the condenser for 10
minutes, and add 20.0 ml of a saturated sodium chloride solution. Shake and allow the layers to separate.
Remove about 2.0 ml of the organic layer and dry 20 mg of anhydrous sodium sulphate. Dilute 1.0 ml of
the solution to 10.0 ml with heptane.
Reference solution. Dissolve 50 mg each of palmitic acid RS and stearic acid RS in 5.0 ml of boron
trifluoride-methanol solution. Boil under a reflux condenser for 10 minutes, add 4.0 ml of heptane through
the condenser for 10 minutes, and add 20.0 ml of a saturated sodium chloride solution. Shake and allow the
layers to separate. Remove about 2.0 ml of the organic layer and dry 20 mg of anhydrous sodium sulphate.
Dilute 1.0 ml of this solution to 10.0 ml with heptane.
Chromatographic system
- a stainless steel column 30 m x 0.32 mm, packed with fused silica coated with macrogol 20000
(film thickness 0.5 µm);
temperature:
time
temperature
(min)
(º)
Column
0-2
70
2-36
70-240
36-41
240
- Inlet port at 220º and detector at 260º,
- flame ionization detector,
- flow rate. 2.4 ml per minute, nitrogen as the carrier gas.
Inject 1µl of the reference solution. The relative retention with reference to methyl stearate for methyl
palmitate is about 0.8. The test is not valid unless the resolution between the peaks due to methyl stearate
and methyl palmitate is not less than 5.0.
Inject 1µl of the test solution and the reference solution.
Calculate the percentage content of stearic acid and palmitic acid.
Mannitol
Identification
Add the following test
A. Determine by infrared absorption spectrophotometery (2.4.6). Compare the spectrum with that obtained
with mannitol RS or with the reference spectrum of mannitol.
Mannitol Injection
Change Identification to:
A. Evaporate to dryness on a water-bath a volume containing 0.2 g of Mannitol. The residue melts at 165°
to 170° (2.4.21).
B. Determine by thin layer chromatography (2.4.17), coating the plate with the silica gel G.
Mobile phase. A mixture of 10 volumes of water, 70 volumes of propan-1-ol and 20 volumes of ethyl
acetate.
Test solution. Dilute a volume of injection containing 0.25 g of Mannitol to 10 ml with water.
Reference solution. A 0.25 per cent w/v solution of mannitol RS in water.
Apply to the plate 2 µl of each solution. After development, dry the plate in air and spray with the 0.2 per
cent w/v solution of sodium periodate. Dry the plate in air for 15 minutes and spray with a 2.0 per cent w/v
solution of 4,4’-methylenebis-N,N-dimethylanaline in a mixture of 1 volume of glacial acetic acid and 4
volumes of acetone, heat at 105º for 30 minutes and examine in ultraviolet light at 365 nm. The principal
spot in chromatogram obtained with the test solution corresponds to that in the chromatogram obtained
with the reference solution.
C. Dissolve 0.5 g of the residue obtained in test A in sufficient carbon dioxide-free water prepared from
distilled water to produce 5 ml (solution A). Add 0.3 ml of solution A to 3 ml of a cooled mixture prepared
by adding 6 ml of sulphuric acid to 3 ml of a freshly prepared 10 per cent w/v solution of catechol while
cooling in ice. Heat gently over a naked flame for about 30 seconds; a pink colour is produced.
Mebendazole
Change Identification to:
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that
obtained with mebendazole RS or with the reference spectrum of mebendazole.
B. In the test for Related substances, the principal peak in the chromatogram obtained with the test
solution corresponds to the principal peak in the chromatogram obtained with the reference solution.
C. To about 10 mg add 5 ml of ethanol (95 per cent), 1 ml of dinitrobenzene solution and 1 ml of sodium
hydroxide solution; an intense yellow colour is produced.
Related substances change to:
Related substances. Determine by liquid chromatography (2.4.14).
Test solution. Dissolve 25 mg of the substance under examination in 25 ml of dimethylformamide.
Reference solution. Dilute 1.0 ml of the test solution to 100.0 ml with of dimethylformamide. Dilute 5.0 ml
of this solution to 20.0 ml with dimethylformamide.
Chromatographic system
- a stainless steel column 10 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (3
µm),
- column temperature. 40º,
mobile phase: A. a mixture of 0.75 per cent w/v solution of ammonium acetate,
B. acetonitrile,
- a linear gradient programme using the conditions given below,
- flow rate. 1.2 ml per minute,
- spectrophotometer set at 250 nm,
- a 10 µl loop injector.
Time
( in min.)
Mobile phase A
( per cent v/v)
0-15
15-20
20-25
25-26
26-30
)
80-70
70-10
10
10-80
80
Mobile phase B
(per cent v/v)
20-30
30-90
90
90-20
20
Inject the test solution and the reference solution. In the chromatogram obtained with the test solution the
area of any secondary peak is not more than twice the area of the principal peak in the chromatogram
obtained with the reference solution (0.5 per cent), the sum of all the secondary peaks is not more than 4
times the area of the principal peak in the chromatogram obtained with the reference solution (1.0 per cent)
Ignore any peak with an area less than 0.2 times the area of the principal peak in the chromatogram
obtained with the reference solution (0.05 per cent).
Mebendazole Tablets
Change Identification B to:
B. In the test for Related substances, the chromatogram obtained with the test solution corresponds to the
peak in the chromatogram obtained with the reference solution.
Related substances change to:
Related substances. Determine by liquid chromatography (2.4.14).
Test solution. Disperse a quantity of powdered tablets containing about 25 mg of Mebendazole in 25 ml of
dimethylformamide.
Reference solution. Dilute 1.0 ml of the test solution to 100.0 ml with dimethylformamide. Dilute 5.0 ml of
this solution to 20.0 ml with dimethylformamide.
Chromatographic system
-
a stainless steel column 10 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica
(3µm),
column temperature 40º,
mobile phase: A. a mixture of 0.75 per cent w/v solution of ammonium acetate,
o
B. acetonitrile,
a linear gradient programme using the conditions given below,
flow rate. 1.2 ml per minute,
spectrophotometer set at 250 nm,
a 10 µl loop injector.
Time
( in min.)
Mobile phase A
( per cent v/v)
0-15
15-20
20-25
25-26
26-30
)
80-70
70-10
10
10-80
80
Mobile phase B
( per cent v/v)
20-30
30-90
90
90-20
20
Inject the test solution and the reference solution. In the chromatogram obtained with the test solution the
area of any secondary peak is not more than twice the area of the principal peak in the chromatogram
obtained with the reference solution (0.5 per cent), the sum of all the secondary peaks is not more than 4
times the area of the principal peak in the chromatogram obtained with the reference solution (1.0 per cent)
Ignore any peak with an area less than 0.2 times the area of the principal peak in the chromatogram
obtained with the reference solution (0.05 per cent).
Megestrol Acetate
Change Related foreign steroids test to:
Related substances. Determine by liquid chromatography (2.4.14).
Solvent mixture. 145 volumes of tetrahydrofuron and 255 volumes of acetonitrile.
Test solution. Dissolve 25 mg of the substance under examination in 20 ml of the solvent mixture, dilute to
50 ml with water.
Reference solution (a). Dissolve 25 mg of medroxyprogesterone acetate RS (megestrol acetate impurity A
RS) in 20 ml of the solvent mixture, dilute to 50 ml with water.
Reference solution (b). Dilute 1.0 ml of reference solution (a) to 200.0 ml with the mobile phase.
Reference solution (c). To 3.0 ml of the test solution, add 1 ml of reference solution (a) and dilute to 50 ml
with the mobile phase.
Chromatographic system
- a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica
(5 µm),
- mobile phase: a mixture of 14.5 volumes of tetrahydrofuran, 22.5 volumes of acetonitrile and
60 volumes of water,
- flow rate. 1.5 ml per minute,
- spectrophotometer set at 254 nm,
- a 20 µl loop injector.
Inject reference solution (c). The test is not valid unless the resolution between the peaks due to megestrol
acetate and megestrol acetate impurity A is not less than 4.0.
Inject the test solution and reference solution (b). Run the chromatogram 1.5 times the retention time of the
principal peak. In the chromatogram obtained with the test solution, the area of the peak corresponding to
megestrol acetate impurity A is not more than the area of the principal peak in the chromatogram obtained
with reference solution (b) (0.5 per cent). The sum of areas of all the secondary peaks other than megestrol
acetate impurity A is not more than twice the area of the principal peak in the chromatogram obtained with
reference solution (b) (1.0 per cent). Ignore any peak with an area less than 0.1 times the area of the
principle peak in the chromatogram obtained with reference solution (b) (0.05 per cent).
Mercaptopurine
Change Hypoxanthine test to:
Hypoxanthine. Determine by thin layer chromatography (2.4.17), coating the plate with the silica gel
GF254.
Mobile phase. A mixture of 3 volumes of concentrated ammonia, 7 volumes of water and 90 volumes of
acetone.
Test solution. Dissolve 50 mg of the substance under examination in 1 ml of dimethyl sulphoxide and dilute
to 10 ml with methanol.
Reference solution. Dilute 10 mg of hypoxanthine in 10 ml of dimethyl sulphoxide and diluted to 100 ml
with methanol.
Apply separately to the plate 5 µl of each solution. Allow the mobile phase to rise 10 cm. After
development, dry the plate in air and examine at 254 nm. Any secondary spot corresponding to
hypoxathine in the chromatogram obtained with the test solution is not more intense than the spot in the
chromatogram obtained with the reference solution (2.0 per cent).
Metronidazole
Change Related substances to:
Related substances. Determine by liquid chromatography (2.4.14).
Note- Prepare the solutions protected from light.
Test solution. Dissolve 50 mg of the substance under examination in 100 ml of the mobile phase.
Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of
this solution to 10.0 ml with the mobile phase.
Reference solution (b). Dissolve 5 mg of 2-methyl-4-nitromidazole RS (metronidazole impurity A RS) in the
mobile phase, add 10.0 ml of the test solution and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml
of this solution to 100.0 ml with the mobile phase.
Chromatographic system
- a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica
(5 µm),
- mobile phase: a mixture 30 volumes of methanol and 70 volumes of a 0.14 per cent w/v solution
of potassium dihydrogen phosphate,
- flow rate. 1 ml per minute,
- spectrophotometer set at 315 nm,
- a 10 µl loop injector.
Inject reference solution (b). The test is not valid unless the resolution between the peak due to
metronidazole and metronidazole impurity A is not less than 2.0.
Inject the test solution and reference solution (a). In the chromatogram obtained with the test solution the
area of any secondary peak is not more than the area of principal peak in the chromatogram obtained with
reference solution (a) (0.1 per cent), the sum of areas of all the secondary peaks is not more than twice the
area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent). Ignore
any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with
reference solution (a) (0.01 per cent).
Metronidazole Tablets
Change Related substances to:
Related substances. Determine by liquid chromatography (2.4.14).
Test solution. Shake a quantity of powdered tablets containing about 100 mg of Metronidazole in 100 ml of
the mobile phase,
Reference solution (a). A 0.0005 per cent w/v solution of 2-methyl-5-nitroimidazole RS in the mobile
phase.
Reference solution (b). A 0.0005 per cent w/v solution of 2-methyl-5-nitroimidazole RS in the test solution.
Chromatographic system
- a stainless steel column 20 cm x 4.6 mm packed with octadecylsilane bonded to porous silica
(10 µm) (such as Spherisorb ODS1),
- mobile phase: a mixture of 30 volumes of methanol and 70 volumes of a 0.1 M potassium
dihydrogen orthophosphate prepared by dissolving 1.4 g of potassium dihydrogen orthophosphate
with 1000 ml of water,
- flow rate. 1 ml per minute,
- spectrophotometer set at 315 nm,
- a 20 µl loop injector.
Inject reference solution (b). Adjust the sensitivity so that the height of the peak due to 2-methyl-5nitroimidazole is about 50 per cent of full scale deflection. Measure the height (a) of the peak due to 2methyl-5-nitroimidazole and the height (b) of the lowest part of the curve separating this peak from the
principal peak. The test is not valid unless a is greater than 10b.
Inject the test solution and reference solution (a). Run the chromatogram 3 times the retention time of the
principal peak. In the chromatogram obtained with the test solution the area of any secondary peak is not
more than the area of the peak due to 2-methyl-5-nitroimidazole in the chromatogram obtained with
reference solution (a).
Metronidazole Injection
Change Identification test to:
A. Shake a volume of the injection containing about 0.1 g of Metronidazole with 9 g of sodium chloride for
5 minutes. Add 20 ml of acetone, shake for further 5 minutes and allow to separate. Evaporate the upper
layer to dryness. On the residue, determine by infrared absorption spectrophotometery (2.4.6). Compare the
spectrum with that obtained with metronidazole RS or with the reference spectrum of metronidazole.
B. Heat 2 ml of the injection in a water-bath for 5 minutes with 10 mg of zinc powder and 0.25 ml of 2 M
hydrochloric acid for 5 minutes and cool in ice. The solution gives the reaction of primary aromatic amines
(2.3.1).
Change Related substances to:
Related substances. Determine by liquid chromatography (2.4.14).
Test solution. Dilute a volume of injection containing about 100 mg of metronidazole in 100 ml with the
mobile phase.
Reference solution (a). A 0.0005 per cent w/v solution of 2-methyl-5-nitroimidazole RS in the mobile
phase.
Reference solution (b). A 0.0005 per cent w/v solution of 2-methyl-5-nitroimidazole RS in the test solution.
Chromatographic system
- a stainless steel column 20 cm x 4.6 mm packed with octadecylsilane bonded to porous silica
(10 µm) (such as Spherisorb ODS1),
- mobile phase: a mixture of 30 volumes of methanol and 70 volumes of a 0.1 M potassium
dihydrogen orthophosphate prepared by dissolving 1.4 g of potassium dihydrogen orthophosphate
with 1000 ml of water,
- flow rate. 1 ml per minute,
- spectrophotometer set at 315 nm,
- a 20 µl loop injector.
Inject reference solution (b). Adjust the sensitivity so that the height of the peak due to 2-methyl-5nitroimidazole is about 50 per cent of full scale deflection. Measure the height (a) of the peak due to 2methyl-5-nitroimidazole and the height (b) of the lowest part of the curve separating this peak from the
principal peak. The test is not valid unless a is greater than 10b.
Inject the test solution and reference solution (a). Run the chromatogram 3 times the retention time of the
principal peak. In the chromatogram obtained with the test solution the area of any secondary peak is not
more than the area of the peak due to 2-methyl-5-nitroimidazole in the chromatogram obtained with
reference solution (a) (0.5 per cent).
Morphine sulphate
Change Other alkaloids to:
Related substances. Determine by liquid chromatography (2.4.14).
Solvent mixture. 1.0 per cent v/v solution of acetic acid.
Test solution. Dissolve 125 mg of the substance under examination in 50 ml of the solvent mixture.
Reference solution (a). Dilute 1.0 ml of the test solution to 100 ml with the solvent mixture. Dilute 2.0 ml
of this solution to 10.0 ml with the solvent mixture.
Reference solution (b). Dissolve 5 mg of morphine for system suitability RS (containing morphine
impurities B, C, E and F) in 2 ml of the solvent mixture.
Chromatographic system
-
a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5
µm),
column temperature. 35º,
mobile phase: A. a 0.1 per cent w/v solution of sodium heptanesulphonate adjusted to pH 2.6 with
orthophosphoric acid,
B. methanol,
a linear gradient programme using the conditions given below,
flow rate. 1.5 ml per minute,
spectrophotometer set at 230 nm,
a 10 µl loop injector.
Time
( in min.)
)0-2
2-35
35-40
Mobile phase A
(per cent v/v)
85
85-50
50
Mobile phase B
(per cent v/v)
15
15-50
50
Inject reference solution (b). The test is not valid unless the peak-to-valley ratio, where Hp is height above
the baseline of the peak due to morphine impurity F, Hv is the height above the baseline of the lowest point
of the curve separating this peak from the peak due to morphine is not less than 2.0.
Inject the test solution and reference solution (a). In the chromatogram obtained with the test solution, the
area of the peak corresponding to 2,2′-bimorphine (morphine impurity B) is not more than twice the area of
the principal peak in the chromatogram obtained with reference solution (a) (0.4 per cent). The area of the
each peak corresponding to oripavine (morphine impurity C) and morphinone (morphine impurity E) is not
more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per
cent). The area of any other secondary peak is not more than the area of the principal peak in the
chromatogram obtained with reference solution (a) (0.2 per cent) and sum of all the secondary peaks is not
more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (a)
(1.0 per cent). Ignore any peak with an area less than 0.25 times the area of the principle peak in the
chromatogram obtained with reference solution (a) (0.05 per cent).