Chromatographic purity—
LIMIT OF TRIMETHOPRIM DEGRADATION PRODUCT—
Test solution—
Transfer an accurately measured volume of Oral Suspension, equivalent to about 40 mg of trimethoprim, to a separatory funnel. Extract with three 25-mL portions of a mixture of chloroform and methanol (8:2), collecting the extracts in a 125-mL conical flask. Evaporate the combined extracts with the aid of a current of air to dryness on a steam bath. Dissolve the residue in 2.0 mL of the mixture of chloroform and methanol (8:2), then centrifuge.
Standard solution A—
Dissolve an accurately weighed quantity of
USP Trimethoprim RS in a mixture of chloroform and methanol (8:2) to obtain a solution having a known concentration of about 20 mg per mL.
Standard solution B—
Dilute an accurately measured volume of Standard solution A with a mixture of chloroform and methanol (8:2) to obtain a solution having a known concentration of about 0.1 mg per mL.
Procedure—
Apply 5 µL each of the
Test solution, Standard solution A, and
Standard solution B to separate points on a thin-layer chromatographic plate (see
Chromatography 
621
) coated with a 0.25-mm layer of chromatographic silica gel. Place the plate in a saturated chromatographic chamber, and develop the chromatogram in a solvent system consisting of a mixture of chloroform, methanol, and ammonium hydroxide (80:20:3) until the solvent front has moved at least 15 cm. Remove the plate from the chamber, air-dry, and view under short-wavelength UV light: trimethoprim produces a spot at about
RF 0.7, and the trimethoprim degradation product can be seen at
RF 0.3 to 0.5. Any spot from the
Test solution at about
RF 0.3 to 0.5 is not greater in size and intensity than the spot produced by
Standard solution B at about
RF 0.7, corresponding to not more than 0.5%.
LIMIT OF SULFANILAMIDE, SULFANILIC ACID, AND SULFAMETHOXAZOLE N4-GLUCOSIDE)—
Alcohol–methanol mixture—
Mix dehydrated alcohol and methanol (95:5).
Modified Ehrlich's reagent—
Dissolve 100 mg of p-dimethylaminobenzaldehyde in 1 mL of hydrochloric acid, and dilute with alcohol to 100 mL.
Test solution—
Using a syringe, transfer an accurately measured volume of Oral Suspension, equivalent to 200 mg of sulfamethoxazole, to a 100-mL volumetric flask containing 10 mL of ammonium hydroxide; and add 50 mL of methanol. Shake for 3 minutes, and dilute with methanol to volume. Centrifuge a portion of the solution for 3 minutes.
Standard solution A—
Weigh 20 mg of
USP Sulfamethoxazole RS into a 10-mL volumetric flask, dissolve in 1 mL of ammonium hydroxide, dilute with methanol to volume, and mix.
Standard solution B—
Weigh 10 mg of
USP Sulfanilamide RS into a 50-mL volumetric flask, dissolve in 5 mL of ammonium hydroxide, and dilute with methanol to volume. Pipet 5 mL of this solution into a 100-mL volumetric flask, add 10 mL of ammonium hydroxide, and dilute with methanol to volume.
Standard solution C—
Weigh 10 mg of
USP Sulfanilic Acid RS into a 50-mL volumetric flask, dissolve in 5 mL of ammonium hydroxide, and dilute with methanol to volume. Pipet 3 mL of this solution into a 100-mL volumetric flask, add 10 mL of ammonium hydroxide, and dilute with methanol to volume.
Standard solution D—
Weigh 3.0 mg of USP Sulfamethoxazole N4-Glucoside RS into a 50-mL volumetric flask, dissolve in 5 mL of ammonium hydroxide, and dilute with methanol to volume.
Procedure—
Apply 50 µL each of the
Test solution and
Standard solutions A, B, C, and
D to separate points on a thin-layer chromatographic plate (see
Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel. Place the plate in an unsaturated chromatographic chamber, and develop the chromatogram in a solvent system consisting of a mixture of
Alcohol–methanol mixture, heptane, chloroform, and glacial acetic acid (25:25:25:7) until the solvent front has moved at least 12 cm. Remove the plate from the developing chamber, air-dry, spray with
Modified Ehrlich's reagent, and allow the plate to stand for 15 minutes: sulfamethoxazole produces a spot at about
RF 0.7. Any spots from the
Test solution at about
RF 0.5, 0.1, and 0.3 are not greater in size and intensity than spots produced by
Standard solutions B, C, and
D, respectively, corresponding to not more than 0.5% of sulfanilamide, 0.3% of sulfanilic acid, and 3.0% of sulfamethoxazole
N4-glucoside.
Assay—
Mobile phase—
Mix 1400 mL of water, 400 mL of acetonitrile, and 2.0 mL of triethylamine in a 2000-mL volumetric flask. Allow to equilibrate to room temperature, and adjust with 0.2 N sodium hydroxide or dilute glacial acetic acid (1 in 100) to a pH of 5.9 ± 0.1. Dilute with water to volume, and filter through a 0.45-µm membrane, making adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard preparation—
Dissolve accurately weighed quantities of
USP Trimethoprim RS and
USP Sulfamethoxazole RS in methanol, and quantitatively dilute with methanol to obtain a solution containing, in each mL, about 0.32 mg and 0.32
J mg, respectively,
J being the ratio of the labeled amount, in mg, of sulfamethoxazole to the labeled amount, in mg, of trimethoprim in the dosage form. Transfer 5.0 mL of this solution to a 50-mL volumetric flask, dilute with
Mobile phase to volume, and mix to obtain a
Standard preparation having known concentrations of about 0.032 mg of
USP Trimethoprim RS per mL and 0.032
J mg of
USP Sulfamethoxazole RS per mL.
Assay preparation—
Transfer an accurately measured volume of Oral Suspension, equivalent to about 80 mg of Sulfamethoxazole, to a 50-mL volumetric flask with the aid of about 30 mL of methanol. Sonicate the mixture for about 10 minutes with occasional shaking. Allow to equilibrate to room temperature, dilute with methanol to volume, mix, and centrifuge. Transfer 5.0 mL of the supernatant to a second 50-mL volumetric flask, dilute with Mobile phase to volume, mix, and filter.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is about 2 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the relative retention times are about 1.0 for trimethoprim and 1.8 for sulfamethoxazole; the resolution,
R, between sulfamethoxazole and trimethoprim is not less than 5.0; the tailing factor for the trimethoprim and sulfamethoxazole peaks 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 responses for the major peaks. Calculate the quantities, in mg, of trimethoprim (C
14H
18N
4O
3) and sulfamethoxazole (C
10H
11N
3O
3S) in each mL of the Oral Suspension taken by the formula:
(500C/V)(rU / rS),
in which
C is the concentration, in mg per mL, of the appropriate USP Reference Standard in the
Standard preparation; V is the volume, in mL, of Oral Suspension taken; and
rU and
rS are the peak responses of the corresponding analyte obtained from the
Assay preparation and the
Standard preparation, respectively.
