Fluorouracil
C4H3FN2O2 130.08
2,4(1H,3H)-Pyrimidinedione, 5-fluoro-;
5-Fluorouracil [51-21-8]; UNII: U3P01618RT.
DEFINITION
Fluorouracil contains NLT 98.0% and NMT 102.0% of uorouracil (C4H3FN2O2), calculated on the dried basis.
[ CAUTION- Great care should be taken to prevent inhaling particles of Fluorouracil and exposing the skin to it.]
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.
ASSAY
• 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
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
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 0.73%, Standard solution
Tailing factor: NMT 1.5, Standard solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of uorouracil (C4H3FN2O2) in the portion of Fluorouracil taken:
Result = (ru /rs ) × (Cs /Cu ) × 100
ru = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of USP Fluorouracil RS in the Standard solution (µg/mL)
Cu = concentration of Fluorouracil in the Sample solution (µg/mL)
Acceptance criteria: 98.0%-102.0% on the dried basis
IMPURITIES
• RESIDUE ON IGNITION <281>: NMT 0.1%
• 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
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 uorouracil peak
System suitability
Sample: Standard solution
Suitability requirements
Resolution: NLT 2 between the uracil and uorouracil peaks
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of uorouracil related compound A, uorouracil related compound B, and uracil in the portion of
Fluorouracil taken:
Result = (ru /rs ) × (Cs /Cu ) × 100
ru = peak response of relevant uorouracil related compound from the Sample solution
rs = peak response of relevant uorouracil related compound from the Standard solution
Cs = concentration of relevant uorouracil related compound in the Standard solution (mg/mL)
Cu = concentration of Fluorouracil in the Sample solution (mg/mL)
Calculate the percentage of 5-methoxyuracil, uorouracil related compound E, and any unspecied impurity in the portion of
Fluorouracil taken:
Result = (ru /rs ) × (Cs /Cu ) × (1/F) × 100
ru = peak response of each impurity from the Sample solution
rs = peak response of uorouracil from the Standard solution
Cs = concentration of USP Fluorouracil RS in the Standard solution (mg/mL)
Cu = 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
Name Relative Retention Time Relative Response Factor Acceptance Criteria, NMT (%)
Fluorouracil related compound A a 0.5 – 0.15
Fluorouracil related compound B b 0.7 – 0.15
Uracil 0.9 – 0.15
Name Relative Retention Time Relative Response Factor Acceptance Criteria, NMT (%)
Fluorouracil 1.0 – -
5-Methoxyuracil c 1.6 0.67 0.15
Fluorouracil related compound E d 1.9 0.77 0.15
Any individual unspecied impurity – 1.0 0.10
Total impurities – - 0.5
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.
• 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
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).
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
uorouracil 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 uorouracil is not detected by the spray.]
Acceptance criteria: The spot of uorouracil related compound F in the Sample solution is not more intense than the spot of
uorouracil 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%).
SPECIFIC TESTS
• LOSS ON DRYING <731>
Analysis: Dry under vacuum over phosphorus pentoxide at 80° for 4 h.
Acceptance criteria: NMT 0.5%
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.
C4H3ClN2O2 146.53
USP Fluorouracil Related Compound F RS
2-Ethoxy-5-uoropyrimidin-4(1H)-one.
C6H7FN2O2 158.13
USP Uracil RS
Uracil.
C4H4N2O2 112.09
Post time: Mar-30-2024