Other name. Co-trimoxazole oral suspension

Category. Antibacterials.

Storage. The oral suspension should be kept in a tightly-closed container, protected from light.

Additional information. Strength in the current WHO Model list of essential medicines: 200 mg Sulfamethoxazole, 40 mg Trimethoprim per 5 mL.

Strength in the current WHO Model list of essential medicines for children: 200 mg Sulfamethoxazole, 40 mg Trimethoprim per 5 mL.

Requirements

Comply with the monograph for Liquid preparations for oral use.

Definition. Sulfamethoxazole and Trimethoprim oral suspension is a suspension containing Sulfamethoxazole and Trimethoprim in a suitable vehicle which may be flavoured. The oral suspension contains not less than 90.0% and not more than 110.0% of the amounts of Sulfamethoxazole (C₁₀H₁₁N₃O₃S) and Trimethoprim (C₁₄H₁₈N₄O₃) stated on the label.

Identity tests

• Either test A or B may be applied.

A. Carry out test A.1 or, where UV detection is not available, test A.2.

A.1 Carry out the test as described under 1.14.1. Thin-layer chromatography using silica gel R6 as the coating substance and a mixture of 100 volumes of dichloromethane R, 10 volumes of methanol R and 5 volumes of dimethylformamide R as the mobile phase. Apply separately to the plate 10 µl of each of the following two solutions. For solution (A) add 20 mL of methanol R to 5 mL of the oral suspension, mix, shake with 10 g of anhydrous sodium sulfate R, centrifuge and use the supernatant liquid. For solution (B) use 10 mg of sulfamethoxazole RS and 2 mg of trimethoprim RS per mL methanol R. After removing the plate from the chromatographic chamber allow it to dry exhaustively in air or in a current of cool air. Examine the chromatogram in ultraviolet light (254 nm).

The principal spots obtained with solution (A) correspond in position, appearance and intensity to those obtained with solution (B).

A.2 Carry out the test as described under 1.14.1 Chromatography, Thin-layer chromatography using silica gel R5 as the coating substance and the conditions described above under test A.1. Spray the plate with dilute potassium iodobismuthate solution TS2.

The principal spots obtained with solution (A) correspond in position, appearance and intensity to those obtained with solution (B).

B. See the test described under “Assay”, Method A. The retention times of the two principal peaks in the chromatogram obtained with solution (1) correspond to those in the chromatogram obtained with solution (2).

pH value (1.13). pH of the oral suspension, 5.0–6.5.

Related substances

Trimethoprim related substance

Carry out the test as described under 1.14.1. Thin-layer chromatography using silica gel R6 as the coating substance and a mixture of 80 volumes of chloroform R, 20 volumes of methanol R and 3 volumes of ammonia (~260 g/l) TS as the mobile phase. Prepare a solvent mixture as follows: mix 8 volumes of chloroform R and 2 volumes of methanol R. Apply separately to the plate 5 µl of each of the following two solutions. For solution (A) transfer an accurately measured volume of the oral suspension containing about 40 mg of Trimethoprim to a separatory funnel. Extract with three portions of 25 mL of the solvent mixture collecting the extracts in a 125 mL conical flask. Evaporate the combined extracts to dryness in a current of air on a steam bath. Dissolve the residue in 2.0 mL of the solvent mixture then centrifuge. For solution (B) use a solution of 0.1 mg of trimethoprim RS per mL solvent mixture. After removing the plate from the chromatographic chamber allow it to dry in air and examine the chromatogram in ultraviolet light (254 nm).

Trimethoprim and related substance have the following R_f values: trimethoprim about 0.7; trimethoprim degradation product about 0.3–0.5. In the chromatogram obtained with solution (A) any spot corresponding to the trimethoprim degradation product is not greater in size and intensity than the spot obtained with solution (C) (0.5%).

Sulfamethoxazole related substances.

Carry out the test as described under 1.14.1. Thin-layer chromatography using silica gel R5 as the coating substance.. As the mobile phase use a mixture of 25 volumes of ethanol/methanol (95/5) TS, 25 volumes of heptane R, 25 volumes of dichloromethane R and 7 volumes of glacial acetic acid R. Apply separately to the plate 50 µl of each of the following five solutions. For solution (A) transfer an accurately measured volume of the oral suspension, containing about 200 mg of Sulfamethoxazole, to a 100 mL volumetric flask containing 10 mL of ammonia (~260 g/l) TS. Add 50 mL of methanol R, shake, dilute to volume with methanol R and centrifuge a portion of this solution. For solution (B) transfer 10 mg of sulfanilamide R into a 50 mL volumetric flask, dissolve in 5.0 mL of ammonia (~260 g/l) TS and dilute to volume with methanol R. Transfer 5.0 mL of this solution into a 100 mL volumetric flask, add 10 mL of ammonia (~260 g/l) TS and dilute to volume with methanol R. For solution (C) transfer 10 mg of sulfanilic acid R into a 50 mL volumetric flask, dissolve in 5 mL of ammonia (~260 g/l) and dilute to volume with methanol R. Transfer 3.0 mL of this solution into a 100 mL volumetric flask, add 10 mL of ammonia (~260 g/l) TS and dilute to volume with methanol R. For solution (D) transfer 3 mg of sulfamethoxazole N₄-glucoside R into a 50 mL volumetric flask, dissolve in 5 mL of ammonia (~260 g/l) TS and dilute to volume with methanol R. For solution (E) transfer 10 mg of sulfanilamide R, 6 mg of sulfanilic acid R and 60 mg of sulfamethoxazole N₄-glucoside R into a 100 mL volumetric flask, dissolve in 10 mL of ammonia (~260 g/l) TS and dilute to volume with methanol R. Transfer 1.0 mL of this solution into a 10 mL volumetric flask containing 20 mg of sulfamethoxazole RS, add 1 mL of ammonia (~260 g/l) TS and dilute to volume with methanol R to volume.

After removing the plate from the developing chamber allow it to dry in air, spray with 4-dimethylaminobenzaldhyde TS7 and allow the plate to stand for 15 minutes.

In the chromatogram obtained with solution (A) any spot corresponding to sulfanilamide is not greater in size and intensity than the spot obtained with solution (B) (0.5%), any spot corresponding to sulfanilic acid is not greater in size and intensity than the spot obtained with solution (C) (0.3%) and any spot corresponding to sulfamethoxazole N₄-glucoside is not greater in size and intensity than the spot obtained with solution (D) (3.0%). The test is not valid unless the chromatogram obtained with solution (E) shows four clearly separated principal spots.

Assay

• Either method A or methods B and C may be applied.

A. Carry out the test as described under 1.14.1 Chromatography, High-performance liquid chromatography using a stainless steel column (25 cm x 4.6 mm) packed with particles of base-deactivated silica gel, the surface of which has been modified with chemically-bonded octadecylsilyl groups (5 µm). As the mobile phase use a solution prepared as follows: mix 1400 mL of water R, 400 mL of acetonitrile R and 2.0 mL of triethylamine R in a 2000 mL volumetric flask. Allow to equilibrate to room temperature and adjust with acetic acid (~10 g/l) TS to pH 5.9. Dilute to volume with water R and filter.

Prepare the following solutions. For solution (1) transfer an accurately weighed quantity of the oral suspension, containing about 80 mg of Sulfamethoxazole, to a 50 mL volumetric flask using about 30 mL of methanol R. Sonicate the mixture for about 10 minutes with occasional shaking. Allow to cool to room temperature, make up to volume with methanol R, mix and filter. Transfer 5.0 mL of the filtrate into a 50 mL volumetric flask, make up to volume with the mobile phase and mix. For solution (2) prepare a solution of 0.32 mg of trimethoprim RS and 1.60 mg of sulfamethoxazole RS per mL of methanol R. Transfer 5.0 mL of this solution into a 50 mL volumetric flask, make up to volume with the mobile phase.

Operate with a flow rate of 1.0 mL per minute. As a detector use an ultraviolet spectrophotometer set at a wavelength of 254 nm. Inject separately 20 µl of solutions (1) and (2) and record the chromatogram for 1.5 times the retention time of sulfamethoxazole. In the chromatogram obtained with solution (2) the peak due to trimethoprim is eluted at a relative retention of 0.53 with reference to sulfamethoxazole (retention time about 11 minutes). The test is not valid unless the resolution factor between the peaks due to sulfamethoxazole and to trimethoprim is at least 5.0.

Measure the areas of the peak responses obtained in the chromatograms for solutions (1) and (2). Determine the weight per mL (1.3.1) of the oral suspension and calculate the content of Sulfamethoxazole (C₁₀H₁₁N₃O₃S) and Trimethoprim (C₁₄H₁₈N₄O₃) weight in volume in the oral suspension using the declared content of C₁₀H₁₁N₃O₃S and C₁₄H₁₈N₄O₃ in sulfamethoxazole RS and trimethoprim RS.

B. Prepare the following solutions. For solution (A) transfer an accurately weighed quantity of the oral suspension, containing about 0.25 g of Sulfamethoxazole, to a separatory funnel, add 30 mL of sodium hydroxide (~4 g/l) TS, shake and extract with four quantities, each of 50 mL, of dichloromethane R, washing each extract with the same two quantities, each of 10 mL, of sodium hydroxide (~4 g/l) TS. Reserve the combined dichloromethane extracts for “Assay”, Method C. Dilute the combined aqueous solution and washings to 250.0 mL with water R, filter and dilute 5.0 mL of the filtrate to 200.0 mL with water R. For solution (B) dissolve 0.25 g of sulfamethoxazole RS in 50 mL of sodium hydroxide (~4 g/l) TS and dilute to 250.0 mL with water R. Dilute 5.0 mL of this solution to 200.0 mL with water R. For solution (C) use water R.

Carry out the following procedure protected from light. To 2.0 mL of each of solution (A), (B) and (C) add 0.5 mL of hydrochloric acid (~146 g/l) TS and 1 mL of sodium nitrite (~1 g/l) TS and allow to stand for 2 minutes. Add 1 mL of ammonium sulfamate (~5 g/l) TS and allow to stand for 3 minutes. Add 1 mL of N-(1-napthyl)ethylenediamine hydrochloride (1 g/l) TS and allow to stand for 10 minutes. Dilute the resulting solutions to 25.0 mL with water R and measure the absorbances at 538 nm, with solution (C) in the reference cell.

Calculate the content of Sulfamethoxazole (C₁₀H₁₁N₃O₃S) from the values of the obtained absorbances. Determine the weight per mL (1.3.1) of the oral suspension and calculate the content of Sulfamethoxazole (C₁₀H₁₁N₃O₃S) weight in volume of the oral suspension.

C. Extract the dichloromethane solution reserved in “Assay”, Method ) with four quantities, each of 50 mL, of acetic acid (~60 g/l) TS. Wash the combined extracts with 5 mL of dichloromethane R and dilute the aqueous extracts to 250.0 mL with acetic acid (~60 g/l) TS. To 10.0 mL of this solution add 10 mL of acetic acid (~60 g/l) TS and sufficient water R to produce 100.0 mL. Measure the absorbance of the resulting solution at 271 nm.

Determine the weight per mL (1.3.1) of the oral suspension and calculate the content of Trimethoprim (C₁₄H₁₈N₄O₃) weight in volume of the oral suspension, using the absorptivity value of 20.4 ( = 204).