IP 2010 RIFAMPICIN, ISONIAZIDN AND ETHAMBUTOL TABLETS

Rifampicin, Isoniazid and Ethambutol 0.19 per cent w/v of sodium perchlorate, 0.59 per cent
w/v of citric acid and 2.09 per cent w/v of potassium
Tablets dihydrogen phosphate and 35 volumes of acetonitrile,
Rifampin, Isonicotinylhydrazid and Ethambutol - flow rate. 1.5 ml per minute,
spectrophotometer set at 254 nm,
Hydrochloride Tablets
injection volume. 20 fll.
Rifampicin, Isoniazid and Ethambutol Tablets contain not less
Inject reference solution (b). The test is not valid unless the
than 90.0 per cent and not more than 110.0 per cent of the
resolution between rifampicin and rifampicin quinone is not
stated amounts ofrifampicin, C43HssN4012' isoniazid, C6H7N30
less than 4, the tailing factor is not more than 2.0 and the
and ethambutol hydrochloride CIQH24N 20 2.2HCl.
column efficiency is not less than 2000 theoretical plates.
Usual strength. Rifampicin 300 mg, Isoniazid 200 mg and
Inject the test solution and reference solution (a). The relative
Ethambutol 200 mg.
retention times are 1.0,0.55, 1.25, 1.51 and 2.61 forrifampicin,
Identification rifampicin quinone, rifampicin N-oxide, 3-formylrifamycin SV
Isonicotinyl hydrazone and 3-formyhifamycin SV respectively.
A. In the Assay for rifampicin and isoniazid, the principal Multiply the areas of each known impurity by its response
peaks in the chromatogram obtained with the test solution factor. The response factors are 1.00, 1.19, 1.03, 1.22 and 1.25
correspond to the peaks in the chromatogram obtained with for rifampicin, rifampicin quinone, rifampicin N-oxide,
the reference solution. Isonicotinyl hydrazone and 3-formylrifamycin SV respectively.
B. In the Assay for ethambutol hydrochloride the principal In the chromatogram obtained with the test solution the area
peak in the chromatogram obtained with the test solution of any peak due to rifampicin quinone should not be more
corresponds to the peak in the chromatogram obtained with than 4 times the area ofthe principal peak in the chromatogram
the reference solution. obtained with the reference solution (4 per cent), the area of
Tests any peak due to rifampicin N-oxide should not be more than
1.5 times the area of the principal peak in the chromatogram
Related substances. Determine by liquid chromatography obtained with the reference solution (1.5 per cent), the area of
(2.4.14). any peak due to 3-formylrifamycin SV Isonicotinyl hydrazone
NOTE - Prepare the solutions immediately before use. should not be more than 5 times the area ofthe principal peak
in the chromatogram obtained with the reference solution
Solvent mixture. A mixture of 10 volumes of a 21.01 per cent
( 5 per cent) and the area ofany peak due to 3-formylrifamycin
w/v solution of citric acid, 23 volumes of a 13.61 per cent
SV should not be more than the area ofthe principal peak in
w/v solution ofpotassium dihydrogen phosphate, 77 volumes
the chromatogram obtained with the reference solution (I per
of a 17.42 per cent w/v solution of dipotassium hydrogen
cent ). In the chromatogram obtained with the test solution
phosphate, 640 volumes of water and 250 volumes of
the area of any unknown peak should not be more than
acetonitrile.
1.5 times the area ofthe principal peak in the chromatogram
Test solution. Weigh accurately a quantity of the powdered obtained with the reference solution (1.5 per cent).
tablets containing 200 mg ofRifampicin, dissolve in 100 ml of
Dissolution (2.5.2).
acetonitrile and filter. Dilute 5 ml ofthis solution to 50 ml with
the solvent mixture. Apparatus No.2,
Medium. 900 ml of 0.1 M hydrochloric acid,
Reference solution (a). A solution containing 0.02 per cent
Speed and time. 100 rpm and 45 minutes.
w/v of rifampicin RS in acetonitrile. Dilute 1 ml ofthis solution
to 100 ml with the solvent mixture. Withdraw a suitable volume ofthe medium and filter promptly
through a membrane filter disc with an average pore diameter
Reference solution (b). A solution containing 0.01 per cent
not greater than 0.8 flm. Reject the first few ml ofthe filtrate.
w/v each of rifampicin RS and rifampicin quinone RS in
acetonitrile. Dilute 5 ml of this solution to' 50 ml with the Reference stock solution. A solution containing 0.0165 per
solvent mixture. cent w/v of rifampicin RS, 0.00825 per cent w/v of isoniazid
Chromatographic system RS and 0.031 per cent w/v of ethambutol hydrochloride RS in
a stainless steel column 10 cm x 4.6 mm, packed with the dissolution medium. Keep this reference stock solution in
octylsilane bonded to porous silica (5 flm), the dissolution bath during the test run.
column temperature. 30°, For rifampicin - Dilute the filtrate, if necessary, with the
mobile phase: a mixture of 65 volumes of a solution same solvent. Measure the absorbance (2.4.7) ofthe resulting
containing 0.1 per cent v/v of orthophosphoric acid, solution at the maximum at about 475 nm. Calculate the content

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RIFAMPICIN, ISONIAZID AND ETHAMBUTOL TABLETS IP 2010

ofC43H58N40lz in the medium from the absorbance obtained Reference solution. A solution containing 0.08 per cent w/v of
from suitably diluted reference stock solution. rifampicin RS and 0.04 per cent w/v of isoniazid RS in
For isoniazid- Determine by liquid chromatography (2.4.14). methanol. Dilute 10.0 ml of this solution to 50.0 ml with the
diluent.
Test solution. Suitably dilute the filtered medium with 0.05 M
Chromatographic system
potassium dihydrogen orthophosphate with the pH adjusted
to 6.2 with 0.1 M sodium hydroxide. - a stainless steel column 25 cm x 4.6 mm, packed with
octadecy1si1ane bonded to porous silica (5 11m),
Reference solution. Suitably dilute the reference stock solution -column temperature. 30°,
with 0.05 M potassium dihydrogen orthophosphate with the mobile phase: A. a mixture of 96 volumes of buffer
pH adjusted to 6.2 with 0.1 M sodium hydroxide. solution pH 6.8 (diluent) prepared by dissolving 1.4 g of
Chromatographic system disodium hydrogen orthophosphate anhydrous in
a stainless steel column 30 cm x 4.6 rom, packed with 1000 ml of water, and adjusting the pH to 6.8 ± 0.05 with
octadecylsilane bonded to porous silica (5 11m), dilute phosphoroic acid, and 4 volumes of acetonitrile,
- mobile phase: a mixture of99 volumes of0.05 M potassium B: a mixture of 45 volumes of the buffer
dihydrogen phosphate and 1 volume of acetonitrile solution and 55 volumes of acetonitrile,
previously adjusted to pH 4.0 with a 2 per cent w/v flow rate. 1.5 ml per minute,
solution of phosphoric acid, a linear gradient programme using the conditions given
flow rate. 1 ml per minute, below,
spectrophotometer set at 254 nm, spectrophotometer set at 238 nm,
injection volume. 20 Ill. injection volume. 20 Ill.
Inject the reference solution. The test is not valid unless the Time Mobile phase A Mobile phase B
tailing factor is not more than 2.0, the column efficiency in not (in min.) (per cent v/v) (per cent v/v)
less than 1500 theoretical plates and the relative standard o 100 o
deviation for replicate injections is not more than 2.0 per cent.
5 100 o
Inject the test solution and the reference solution.
6 0 100
Calculate the content ofC 6H 7N 30 in the dissolution medium. 15 0 100
For ethambutol hydrochloride - Determine by liquid 16 100 o
chromatography (2.4.14).
22 100 o
Test solution. Suitably dilute the filtered medium with 0.05 M
NOTE - Saturate the column with mobile phase B for about
potassium dihydrogen orthophosphate adjusted to pH 6.2
1 hour.
with 0.1 M sodium hydroxide.
Inject the reference solution. The test is not valid unless the
Reference solution. Suitably dilute the reference stock solution
tailing factor is not less than 2.0 for rifampicin and isoniazid,
with 0.05 M potassium dihydrogen orthophosphate with the
the column efficiency determined from isoniazid peak is not
pH adjusted to 6.2 with 0.1 M sodium hydroxide.
less than 3000 and that from rifampicin is not less than 25000
Use the chromatographic system described under the Assay theoretical plates respectively, and the relative standard
of ethambutol hydrochloride. deviation for replicate injections is not more than 2.0 per cent.
Calculate the content of CIOHz4NzOz.2HCl in the dissolution The relative retention times are about 1.0 for rifampicin and
medium. about 0.3 for isoniazid.

D. Not less than 75 per cent of the stated amounts of Calculate the contents of C43H58N40lZ and C 6H 7N30 in the
C43H58N401Z, C6H 7N 30 and CIOHz4NzOz.2HCl. tablets.
Other tests. Comply with the tests stated under Tablets. For ethambutol hydrpchloride Determine by liquid
chromatography (2.4.14).
Assay. For rifampicin and isoniazid - Determine by liquid
chromatography (2.4.14). Test solution. Weigh accurately a quantity of the powdered
tablets containing about 60mg of Ethambutol Hydrochloride
Test solution. Weigh and powder 20 tablets. Weigh accurately
and dissolve in 100.0 ml ofthe diluent.
a quantity ofthe powdered tablets containing about 40 mg of
Isoniazid dissolve in 100.0 ml of methanol, dilute to 500.0 ml Reference solution. A 0.06 per cent w/v solution of ethambutol
with the diluent and mix. hydrochloride RS in the diluent.

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IP 2010 RIFAMPICIN, ISONIAZID AND PYRAZINAMIDE TABLETS

Chromatographic system Test solution. Weigh accurately a quantity of the powdered

- a stainless steel column 15 cm x 4.6 mm, packed with tablets containing about 200 mg of Rifampicin, dissolve in
nitrile groups chemically bonded to porous silica 100 ml of acetonitrile and filter. Dilute 5 ml ofthis solution to
particles 5 (/-lm) (such as Zorbax SB CN), 50 ml with the solvent mixture.
mobile phase: a mixture of 50 volumes of acetonitrile,
Reference solution (a). A solution containing 0.02 per cent
and 50 volumes ofa buffer solution pH 7.0 prepared by
w/v of rifampicin RS in acetonitrile. Dilute Iml ofthis solution
dissolving 1 ml of triethylamine in 1000 ml of water and
to a 100 ml with the solvent mixture.
adjusting the pH to 7.0 with dilute phosphoric acid,
flow rate. 1 ml per minute, , Reference solution (b). A solution containing 0.01 per cent
spectrophotometer set at 200 urn, w/v each of rifampicin RS and rifampicin qUinone RS in
injection volume. 20 /-ll. acetonitrile. Dilute 5 ml of this solution to 50 ml with the
solvent mixture.
Inject the reference solution. The test is not valid unless the
relative standard deviation for replicate injections is not more Chromatographic system
than 2.0 per cent, the tailing factor is not more than 3.0 and the - a stainless steel column 10 cm x 4.6 mm, packed with
column efficiency determined from Ethambutol hydrochloride octylsilane bonded to porous silica (5 /-lm),
peak is not less than 1500 theoretical plates. column temperature. 30°,
Inject the test solution and the reference solution. mobile phase: a mixture of 65 volumes of a solution
containing 0.1 per cent v/v of orthophosphoric acid,
Calculate the content ofCIOH24N202.2HCl in the tablets. 0.19 per cent w/v of sodium perchlorate, 0.59 per cent
Storage. Store protected from moisture. w/v of citric acid and 2.09 per cent w/v of potassium
dihydrogen phosphate, 35 volumes of acetonitrile,
flow rate. 1.5 ml per minute,
spectrophotometer set at 254 nm,
Rifampicin, Isoniazid and - injection volume. 20 /-ll.
Pyrazinamide Tablets Inject reference solution (b). The test is not valid unless the
resolution between rifampicin and rifampicin quinone is not
Rifampin, Isonicotinylhydrazid and Pyrazinamide Tablets
less than 4, the tailing factor is not more than 2.0 and the
Rifampicin, .Isoniazid and Pyrazinamide Tablets contain not column efficiency is not less than 2000 theoretical plates;
less than 90.0 per cent and not more than 110.0 per cent ofthe
Inject the test solution and reference solution (a). The relative
stated amounts ofrifampicin, C43HssN4012' isoniazid, C6 H7N30
and pyrazinamide CsHsN30. retention times are 1.0,0.55, 1.25, 1.51 and 2.61 for rifampicin,
rifampicin quinone, rifampicin N-oxide, 3-formylrifamycin SV
Usual strength. Rifampicin 120 mg, Isoniazid 50 mg and Isonicotinyl hydrazone and 3-formylrifamycin SV respectively.
Pyrazinamide 300 mg. Multiply the area of each known impurity by its response
factor. The response factors are 1.00, 1.19, 1.03, 1.22 and 1.25
Identification for rifampicin, rifampicin quinone, rifampicin N-oxide,
Isonicotinyl hydrazone and 3-formylrifamycin SV respectively.
In the Assay of rifampicin, isoniazid and pyrazinamide, the
principal peaks in the chromatogram obtained with the test In the chromatogram obtained with the test solution the area
solution correspond to the peaks in the chromatogram of any peak due to rifampicin quinone should not be more
obtained with the reference solution. than 4 times the area ofthe principal peak in the chromatogram
obtained with the reference solution (4 per cent), the area of
Tests any peak due to rifampicin N-oxide should not be more than
Related substances. Determine by liquid chromatography 1.5 times the area of the principal peak in the chromatogram
(2.4.14). obtained with the reference solution (1.5 per cent), the area of
any peak due to 3-formylrifamycin SV Isonicotinyl hydrazone
NOTE - Prepare the solutions immediately before use.
should not be more than 5 times the area ofthe principal peak
Solvent mixture. A mixture of 10 volumes ofa 21.01 per cent in the chromatogram obtained with the reference solution
w/v solution of citric acid, 23 volumes of a 13.61 per cent (5 per cent) and the area ofany peak due to 3-formylrifamycin
w/v solution ofpotassium dihydrogen phosphate, 77 volumes SV should not be more than the area of the principal peak in
of 17.42 per cent w/v solution of dipotassium hydrogen the chromatogram obtained with the reference solution ( 1 per
phosphate, 640 volumes of water and 250 volume,S of cent ). In the chromatogram obtained with the test solution
acetonitrile. the area of any unknown peak should not be more than

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