Fluorouracil

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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C₄H₃FN₂O₂ 130.08

2,4(1H,3H)-Pyrimidinedione, 5-fluoro-;

5-Fluorouracil CAS RN®: 51-21-8; UNII: U3P01618RT.

1 DEFINITION

Fluorouracil contains NLT 98.0% and NMT 102.0% of fluorouracil (C₄H₃FN₂O₂), calculated on the dried basis.

[Caution-Great care should be taken to prevent inhaling particles of Fluorouracil and exposing the skin to it.]

2 IDENTIFICATION

Change to read:

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197M (CN 1-May-2020)

Change to read:

B. Spectroscopic Identification Tests 〈197〉, Ultraviolet-Visible Spectroscopy: 197U (CN 1-May-2020)

Medium: pH 4.7 acetate buffer prepared from 8.4 g of sodium acetate and 3.35 mL of glacial acetic acid mixed with water to make 1000 mL

Sample solution: 10 μg/mL in Medium

Acceptance criteria: Meets the requirements

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

3 ASSAY

3.1 Procedure

Buffer: 6.8 g/L of monobasic potassium phosphate 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: 10 μg/mL of Fluorouracil in water

3.2 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 254 nm

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%, Standard solution

Tailing factor: NMT 1.5, Standard solution

3.5 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of fluorouracil (C₄H₃FN₂O₂) in the portion of Fluorouracil taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution

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

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

Acceptance criteria: 98.0%–102.0% on the dried basis

4 IMPURITIES

Residue on Ignition 〈281〉: NMT 0.1%

4.1 Organic Impurities

Protect the Standard solution and Sample solution from light.

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

Standard solution: 0.1 μg/mL each of USP Fluorouracil RS, USP Fluorouracil Related Compound A RS, USP Fluorouracil Related Compound B RS, and USP Uracil RS, and 0.2 μg/mL of USP Fluorouracil Related Compound E RS in Mobile phase

Sample solution: 0.1 mg/mL of Fluorouracil in Mobile phase

4.1.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 266 nm

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

Flow rate: 1.0 mL/min

Injection volume: 20 μL

Run time: NLT 3 times the retention time of the fluorouracil peak

4.1.2 System suitability

Sample: Standard solution

4.1.3 Suitability requirements

Resolution: NLT 2 between the uracil and fluorouracil peaks

4.1.4 Analysis

Samples: Standard solution and Sample solution

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

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of relevant fluorouracil related compound from the Sample solution

r_S = peak response of relevant fluorouracil related compound from the Standard solution

C_S = concentration of relevant fluorouracil related compound in the Standard solution (mg/mL)

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

Calculate the percentage of 5-methoxyuracil, fluorouracil related compound E, and any unspecified impurity in the portion of Fluorouracil taken:

Result = (r_U/r_S) × (C_S/C_U) × (1/F) × 100

r_U = peak response of each impurity from the Sample solution

r_S = peak response of fluorouracil from the Standard solution

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

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

F = relative response factor for each individual impurity (see Table 1)

Acceptance criteria: See Table 1. Disregard any impurity peaks less than 0.05%.

Table 1

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

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

^c 5-Methoxypyrimidine-2,4(1H,3H)-dione.

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

4.2 Limits of Fluorouracil Related Compound F and Urea

Diluent: Methanol and water (1:1)

Standard solution A: 0.025 mg/mL of USP Fluorouracil Related Compound F RS in Diluent

Standard solution B: 0.02 mg/mL of USP Urea RS in methanol

Sample solution: 10 mg/mL of Fluorouracil in Diluent

4.2.1 Chromatographic system

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

Mode: TLC

Adsorbent: Chromatographic silica gel mixture

Application volume: 10 μL

Developing solvent system: Ethyl acetate, methanol, and water (70:15:15)

Reagent: Prepare a 10-mg/mL solution of p-dimethylaminobenzaldehyde in anhydrous alcohol. Prepare a mixture of this solution and hydrochloric acid (10:1).

4.2.2 Analysis

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

Procedure: Develop with Developing solvent system, followed by air drying. Examine the plate under UV light at 254 nm for fluorouracil related compound F. Spray the plate at least twice with Reagent, and dry the plate in an oven at 80° for 3–4 min. Examine the plate under daylight for urea. [Note—The urea produces a yellow spot, and fluorouracil is not detected by the spray.]

Acceptance criteria: The spot of fluorouracil related compound F in the Sample solution is not more intense than the spot of fluorouracil related compound F from Standard solution A (NMT 0.25%). The spot of urea in the Sample solution is not more intense than the spot of urea from Standard solution B (NMT 0.2%).

5 SPECIFIC TESTS

Loss on Drying 〈731〉

Analysis: Dry under vacuum over phosphorus pentoxide at 80° for 4 h.

Acceptance criteria: NMT 0.5%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight, light-resistant containers.

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 Fluorouracil Related Compound F RS

2-Ethoxy-5-fluoropyrimidin-4(1H)-one.

C₆H₇FN₂O₂ 158.13

USP Uracil RS

Uracil.

C₄H₄N₂O₂ 112.09

USP Urea RS

Urea.

CH₄N₂O 60.06