Fluorouracil 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

Fluorouracil Injection is a sterile solution of Fluorouracil in Water for Injection, prepared with the aid of Sodium Hydroxide. It contains NLT 90.0% and NMT 110.0% of the labeled amount of fluorouracil (C₄H₃FN₂O₂).

[Note-If a precipitate is formed as a result of exposure to low temperatures, redissolve it by heating to 60° with vigorous shaking, and allow to cool to body temperature prior to use.]

2 IDENTIFICATION

A. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

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B. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197A or (USP 1-Aug-2024) 197M

Sample for 197M: Carefully acidify a portion of Injection, equivalent to 100 mg of fluorouracil, with glacial acetic acid. Stir, and slightly chill the solution to precipitate the fluorouracil. Collect the precipitate, wash with 1 mL of water, and then dry under vacuum over phosphorus pentoxide at 80° for 4 h.

Acceptance criteria: Meets the requirements

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C. The UV spectrum of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay. (USP 1-Aug-2024)

3 ASSAY

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3.1 Procedure

Buffer: 6.8 g/L of potassium phosphate, monobasic in water. Adjust with 5 M potassium hydroxide to a pH of 5.7 ± 0.1.

Mobile phase: Acetonitrile and Buffer (5:95)

Standard solution: 10 μg/mL of USP Fluorouracil RS in water

Sample solution: Nominally 10 μg/mL of fluorouracil in water, from Injection

3.2 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 254 nm. For Identification C, use a diode array detector in the range of 200–400 nm. (USP 1-Aug-2024)

Column: 4.6-mm × 25-cm; 5-μm packing L1

Flow rate: 1.0 mL/min

Injection volume: 20 μL

3.3 System suitability

Sample: Standard solution

3.4 Suitability requirements

Relative standard deviation: NMT 0.73%

Tailing factor: NMT 1.5

3.5 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of fluorouracil (C₄H₃FN₂O₂) in the portion of Injection taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of fluorouracil (USP 1-Aug-2024) from the Sample solution

r_S = peak response of fluorouracil (USP 1-Aug-2024) from the Standard solution

C_S = concentration of USP Fluorouracil RS in the Standard solution (μg/mL)

C_U = nominal concentration of fluorouracil in the Sample solution (μg/mL)

Acceptance criteria: 90.0%–110.0%

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4 IMPURITIES

4.1 Organic Impurities

Mobile phase: 0.77 g/L of ammonium acetate in water. Adjust with formic acid to a pH of 5.60–5.65.

System suitability solution: 0.2 mg/mL of USP Fluorouracil RS and 1.0 μg/mL each of USP Fluorouracil Related Compound A RS, USP Fluorouracil Related Compound B RS, USP Uracil RS, and USP Fluorouracil Related Compound E RS in water

Sensitivity solution: 0.1 μg/mL of USP Fluorouracil Related Compound E RS in water

Standard solution: 0.3 μg/mL each of USP Fluorouracil RS, USP Fluorouracil Related Compound A RS, USP Fluorouracil Related Compound B RS, USP Uracil RS, and USP Fluorouracil Related Compound E RS in water

Sample solution: Nominally 0.2 mg/mL of fluorouracil in water, from Injection

4.1.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; 4-μm packing L11

Flow rate: 1.0 mL/min

Injection volume: 10 μL

Run time: NLT 3 times the retention time of fluorouracil

4.1.2 System suitability

Samples: System suitability solution, Sensitivity solution, and Standard solution

4.1.3 Suitability requirements

Resolution: NLT 2.0 between fluorouracil related compound A and fluorouracil related compound B; NLT 2.0 between fluorouracil related compound B and uracil; NLT 2.0 between uracil and fluorouracil; NLT 2.0 between fluorouracil and fluorouracil related compound E,

4.1.4 System suitability solution

Relative standard deviation: NMT 5.0% for each component, Standard solution

Signal-to-noise ratio: NLT 10 for flurouracil related compound E, Sensitivity solution

4.1.5 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of fluorouracil related compound A, fluorouracil related compound B, uracil, and fluorouracil related compound E in the portion of Injection taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak area of each specified impurity from the Sample solution

r_S = peak area of each specified impurity from the Standard solution

C_S = concentration of corresponding Reference Standard in the Standard solution (mg/mL)

C_U = nominal concentration of fluorouracil in the Sample solution (mg/mL)

Calculate the percentage of any unspecified impurity in the portion of Injection taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak area of each unspecified impurity from the Sample solution

r_S = peak area of fluorouracil from the Standard solution

C_S = concentration of USP Fluorouracil RS in the Standard solution (mg/mL)

C_U = nominal concentration of fluorouracil in the Sample solution (mg/mL)

Acceptance criteria: See Table 1. The reporting threshold is 0.05%.

Table 1

^a Total impurities does not include urea, which is tested separately.

4.2 Limit of Urea

Mobile phase: 12.7 g/L of sodium nitrate with 0.1% (v/v) of triethylamine in water

Standard solution: 0.5 mg/mL of USP Urea RS in Mobile phase

Sample solution: Nominally 25 mg/mL of fluorouracil in Mobile phase, from Injection

4.2.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: Refractive index

Column: 7.8-mm × 30-cm; 6-μm packing L39

4.2.2 Temperatures

Detector: 45°

Column: 35°

Flow rate: 0.8 mL/min

Injection volume: 50 μL

Run time: NLT 1.5 times the retention time of urea

Attenuation: 250,000 nano Refractive Index Units

4.2.3 System suitability

Sample: Standard solution

4.2.4 Suitability requirements

Relative standard deviation: NMT 3.0%

Tailing factor: NMT 2.0

4.2.5 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of urea in the portion of Injection taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak area of urea from the Sample solution

r_S = peak area of urea from the Standard solution

C_S = concentration of USP Urea RS in the Standard solution (mg/mL)

C_U = nominal concentration of fluorouracil in the Sample solution (mg/mL)

Acceptance criteria: NMT 3.0% (USP 1-Aug-2024)

5 SPECIFIC TESTS

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Bacterial Endotoxins Test 〈85〉: Meets the requirements (USP 1-Aug-2024)

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Particulate Matter in Injections 〈788〉: Meets the requirements (USP 1-Aug-2024)

pH 〈791〉: 8.6–9.4

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Sterility Test 〈71〉: Meets the requirements (USP 1-Aug-2024)

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

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in single-dose containers, preferably of Type I glass, and store at controlled room temperature. Avoid freezing and exposure to light.

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Labeling: Label it to indicate the expiration date (USP 1-Aug-2024) .

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USP Reference Standards 〈11〉

USP Fluorouracil RS

USP Fluorouracil Related Compound A RS

Pyrimidine-2,4,6(1H,3H,5H)-trione.

C₄H₄N₂O₃ 128.09

USP Fluorouracil Related Compound B RS

Dihydropyrimidine-2,4,5(3H)-trione.

C₄H₄N₂O₃ 128.09

USP Fluorouracil Related Compound E RS

5-Chloropyrimidine-2,4(1H,3H)-dione.

C₄H₄ClN₂O₂ 146.53

USP Uracil RS

Uracil.

C₄H₄N₂O₂ 112.09

USP Urea RS

Urea.

CH₄N₂O 60.06 (USP 1-Aug-2024)