Identification
A:
The retention times of the rifampin, isoniazid, and pyrazinamide peaks in the chromatogram of the Assay preparation correspond to those in the chromatogram of the Standard preparation, as obtained in the Assay for rifampin, isoniazid, and pyrazinamide.
B:
The retention time of the ethambutol peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay for ethambutol hydrochloride.
Dissolution 711
Medium:
10 mM pH 6.8 sodium phosphate buffer, prepared by dissolving 7 g of anhydrous dibasic sodium phosphate in 5 L of water, and adjusting with phosphoric acid to a pH of 6.8; 900 mL.
Apparatus 2:
100 rpm.
Time:
45 minutes.
Procedure
Determine the amounts of rifampin (C43H58N4O12), isoniazid (C6H7N3O), pyrazinamide (C5H5N3O), and ethambutol hydrochloride (C10H24N2O2·2HCl) dissolved using filtered portions of the solution under test and by employing the procedures set forth in the Assay for rifampin, isoniazid, and pyrazinamide and the Assay for ethambutol hydrochloride.
Tolerances
Not less than 75% (Q) of the labeled amounts of C43H58N4O12, C6H7N3O, C5H5N3O, and C10H24N2O2·2HCl is dissolved in 45 minutes.
Assay for rifampin, isoniazid, and pyrazinamide
Buffer solution
Dissolve 1.4 g of anhydrous dibasic sodium phosphate in 1 L of water, and adjust with phosphoric acid to a pH of 6.8.
Solution A
Prepare a filtered and degassed mixture of Buffer solution and acetonitrile (96:4).
Solution B
Prepare a filtered and degassed mixture of acetonitrile and Buffer solution (55:45).
Mobile phase
Use variable mixtures of
Solution A and
Solution B as directed for
Chromatographic system. Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard preparation
Dissolve accurately weighed quantities of
USP Rifampin RS,
USP Isoniazid RS, and
USP Pyrazinamide RS in a mixture of
Buffer solution and methanol (96:4) to obtain a solution having known concentrations of about 0.16 mg per mL, 0.08 mg per mL, and 0.43 mg per mL, respectively.
[NOTEUse this solution within 10 minutes.
]
Assay preparation
Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed quantity of the powder, equivalent to about 8 mg of isoniazid, to a 100-mL volumetric flask, and add about 90 mL of Buffer solution. Sonicate for about 10 minutes, allow to equilibrate to room temperature, dilute with Buffer solution to volume, and mix. [NOTEUse this solution within 2 hours.]
Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a 238-nm detector and a 4.6-mm × 25-cm column that contains a 5-µm base-deactivated packing L1. The flow rate is about 1.5 mL per minute. The chromatograph is programmed as follows.
Time (minutes) |
Solution A
(%) |
Solution B
(%) |
Elution |
0 |
100 |
0 |
equilibration |
05 |
100 |
0 |
isocratic |
56 |
100®0 |
0®100 |
linear gradient |
615 |
0 |
100 |
isocratic |
Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the relative retention times for rifampin, isoniazid, and pyrazinamide are about 1.8, 0.7, and 1.0, respectively; the resolution,
R, between isoniazid and pyrazinamide is not less than 4; the column efficiencies, determined from the rifampin, isoniazid, and pyrazinamide peaks are not less than 50,000 theoretical plates, 6000 theoretical plates, and 10,000 theoretical plates, respectively; the tailing factor is not more than 2.0; and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure
Separately inject equal volumes (about 20 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the quantities, in mg, of rifampin (C
43H
58N
4O
12), isoniazid (C
6H
7N
3O), and pyrazinamide (C
5H
5N
3O) in the portion of Tablets taken by the formula:
100C(rU / rS),
in which
C is the concentration, in mg per mL, of the appropriate USP Reference Standard in the
Standard preparation; and
rU and
rS are the peak responses of the corresponding analyte obtained from the
Standard preparation and the
Assay preparation, respectively.
Assay for ethambutol hydrochloride
Diluent
Dissolve 1.4 g of anhydrous dibasic sodium phosphate in 1 L of water, and adjust with phosphoric acid to a pH of 6.8.
Buffer solution
Mix 1.0 mL of triethylamine and 1 L of water, and adjust with phosphoric acid to a pH of 7.0.
Mobile phase
Prepare a filtered and degassed mixture of acetonitrile and
Buffer solution (50:50). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard preparation
Dissolve an accurately weighed quantity of
USP Ethambutol Hydrochloride RS in
Diluent to obtain a solution having a known concentration of about 0.3 mg per mL.
Assay preparation
Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed quantity of the powder, equivalent to about 30 mg of ethambutol hydrochloride, to a 100-mL volumetric flask, and add about 90 mL of Diluent. Sonicate for about 10 minutes, allow to equilibrate to room temperature, dilute with Diluent to volume, and mix. Pass a portion of this solution through a filter, discarding the first 10 mL of the filtrate.
Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a 200-nm detector and a 4.6-mm × 15-cm column that contains a 5-µm base-deactivated packing L10. The flow rate is about 1.0 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the tailing factor is not more than 3; and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure
Separately inject equal volumes (about 100 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the quantity, in mg, of ethambutol hydrochloride (C
10H
24N
2O
2·2HCl) in the portion of Tablets taken by the formula:
100C(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Ethambutol Hydrochloride RS in the
Standard preparation; and
rU and
rS are the ethambutol peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.