Sulfamethoxazole and Trimethoprim Injection

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Sulfamethoxazole and Trimethoprim Injection is a sterile solution of Sulfamethoxazole and Trimethoprim in Water for Injection, which, when diluted with Dextrose Injection, is suitable for intravenous infusion. It contains NLT 90.0% and NMT 110.0% of the labeled quantities of sulfamethoxazole (C₁₀H₁₁N₃O₃S) and trimethoprim (C₁₄H₁₈N₄O₃).

2 IDENTIFICATION

A. The R values of the principal spots of the Sample solution correspond to those of the Standard solutions of USP Trimethoprim RS (R about 0.5) and USP Sulfamethoxazole RS (R about 0.7), as obtained in the Impurities tests.

3 ASSAY

Procedure

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 pass through a filter of 0.45-μm pore size.

Standard stock solution: 0.32 mg/mL of USP Trimethoprim RS and 0.32J mg/mL of USP Sulfamethoxazole RS in methanol, where J is the ratio of the labeled amount, in mg, of sulfamethoxazole to the labeled amount, in mg, of trimethoprim in the dosage form

Standard solution: 0.032 mg/mL of USP Trimethoprim RS per mL and 0.032J mg/mL of USP Sulfamethoxazole RS per mL in Mobile phase from Standard stock solution

Sample stock solution: Transfer a volume of Injection, equivalent to 80 mg of sulfamethoxazole, to a 50-mL volumetric flask, and dilute with methanol to volume.

Sample solution: Nominally 0.16 mg/mL of sulfamethoxazole in Mobile phase from Sample stock solution

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 3.9-mm × 30-cm; packing L1

Flow rate: 2 mL/min

Injection volume: 20 μL

System suitability

Sample: Standard solution

[Note—The relative retention times for trimethoprim and sulfamethoxazole are 1.0 and 1.8, respectively.]

Suitability requirements

Resolution: NLT 5.0 between sulfamethoxazole and trimethoprim

Tailing factor: NMT 2.0 for trimethoprim and sulfamethoxazole

Relative standard deviation: NMT 2.0% for replicate injections

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of trimethoprim (C₁₄H₁₈N₄O₃) and sulfamethoxazole (C₁₀H₁₁N₃O₃S) in the portion of

Injection taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of trimethoprim or sulfamethoxazole from the Sample solution

r_S = peak response of trimethoprim or sulfamethoxazole from the Standard solution

C_S = concentration of the USP Trimethoprim RS or USP Sulfamethoxazole RS in the Standard solution (mg/mL)

C_U = nominal concentration of trimethoprim or sulfamethoxazole in the Sample solution (mg/mL)

Acceptance criteria: 90.0%–110.0% each of sulfamethoxazole and trimethoprim

4 IMPURITIES

Limit of Trimethoprim Degradation Product

Solution A: 0.06 N hydrochloric acid solution

Standard solution A: 48 mg/mL of USP Trimethoprim RS in chloroform and methanol (1:1)

Standard solution B: 240 μg/mL of USP Trimethoprim RS in a mixture of chloroform and methanol (1:1) from Standard solution A

Sample solution: Transfer a volume of Injection, equivalent to 48 mg of trimethoprim and 240 mg of sulfamethoxazole, to a glass-stoppered, 50-mL centrifuge tube. Add 15 mL of Solution A, and mix. Add 15 mL of chloroform, shake for 30 s, and centrifuge at high speed for 3 min.

Transfer the supernatant layer to a 125-mL separator. Extract the chloroform layer in the centrifuge tube with 15 mL of Solution A, centrifuge at high speed, and add the extract to the separator. Add 2 mL of sodium hydroxide solution (100 mg/mL) to the solution in the separator, and extract with three 20-mL portions of chloroform, collecting the organic layer in a 125-mL conical flask. Evaporate the chloroform under a stream of nitrogen to dryness. Dissolve the residue in 1 mL of a mixture of chloroform and methanol (1:1).

Chromatographic system

(See Chromatography 〈621〉, Thin-Layer Chromatography.)

Mode: TLC

Adsorbent: 0.25-mm layer of chromatographic silica gel

Application volume: 10 μL

Developing solvent system: Chloroform, methanol, and ammonium hydroxide (97:7.5:1)

Spray reagent: Freshly prepared mixture of ferric chloride solution (100 mg/mL) and potassium ferricyanide solution (50 mg/mL) (1:1)

Analysis

Samples: Standard solution A, Standard solution B, and Sample solution

Apply the Samples to separate points on the plate. Develop the chromatogram using Developing solvent system until the solvent front has moved at least 12 cm. Remove the plate from the developing chamber, air-dry, and spray with Spray reagent. Locate the bands by viewing under short-wavelength UV light. Trimethoprim produces a spot at about R 0.5, and the trimethoprim degradation product produces a spot at about R 0.6–0.7. [Note—There may be spots due to concentrate excipients at about R 0.1.]

Acceptance criteria: Any spot from the Sample solution at about R 0.6–0.7 is not greater in size and intensity than the spot produced by Standard solution B at about R 0.5, corresponding to NMT 0.5%.

Limit of Sulfanilamide and Sulfanilic Acid

Solution A: Methanol and dehydrated alcohol (5:95)

Solution B: Dilute 1 mL of ammonium hydroxide with Solution A to 100 mL

Standard solution A: 10 mg/mL of USP Sulfamethoxazole RS in Solution B

Standard solution B: 0.05 mg/mL of USP Sulfanilamide RS in Solution B

Standard solution C: 0.03 mg/mL of USP Sulfanilic Acid RS in Solution B

Sample solution: Transfer a volume of Injection, equivalent to 32 mg of trimethoprim and 160 mg of sulfamethoxazole, into a 25-mL graduated cylinder. Dilute with Solution B to 16 mL.

Chromatographic system

(See Chromatography 〈621〉, Thin-Layer Chromatography.)

Mode: TLC

Adsorbent: 0.25-mm layer of chromatographic silica gel

Application volume: 10 μL

Developing solvent system: Solution A, heptane, chloroform, and glacial acetic acid (30:30:30:10)

Spray reagent (Modified Ehrlich's reagent): 100 mg of p-dimethylaminobenzaldehyde in 1 mL of hydrochloric acid. Dilute with alcohol to 100 mL.

Analysis

Samples: Standard solution A, Standard solution B, Standard solution C, and Sample solution

Apply the Samples to separate points on the plate. Develop the chromatogram in the Developing solvent system until the solvent front has moved NLT 12 cm. Remove the plate from the developing chamber, air-dry, spray with Spray reagent, and allow the plate to stand for 15min. Sulfamethoxazole produces a spot at about R 0.7.

Acceptance criteria: Any spots from the Sample solution at about R 0.5 or 0.1 are not greater in size or intensity than spots produced by Standard solution B and Standard solution C, respectively, corresponding to NMT 0.5% of sulfanilamide and 0.3% of sulfanilic acid.

5 SPECIFIC TESTS

pH 〈791〉: 9.5–10.5

Pyrogen Test 〈151〉: Meets the requirements, the test dose being 0.5 mL/kg

Particulate Matter in Injections 〈788〉: Meets the requirements for small-volume injections

Other Requirements: It meets the requirements in Injections and Implanted Drug Products 〈1〉.

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in single-dose, light-resistant containers, preferably of Type I glass. It may be packaged in 50-mL multiple-dose containers.

Labeling: Label it to indicate that it is to be diluted with 5% Dextrose Injection before administration.

USP Reference Standards 〈11〉

USP Sulfamethoxazole RS

USP Sulfanilamide RS

p-Aminobenzenesulfonamide.

C₆H₈N₂O₂S 172.20

USP Sulfanilic Acid RS

Benzenesulfonic acid, 4-amino-.

C₆H₇NO₃S 173.19

USP Trimethoprim RS