Flucytosine

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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C₄H₄FN₃O            129.09

Cytosine, 5-fluoro-;

5-Fluorocytosine CAS RN®: 2022-85-7; UNII: D83282DT06.

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DEFINITION

Flucytosine contains NLT 98.0% and NMT 102.0% (USP 1-May-2022) of flucytosine (C₄H₄FN₃O), calculated on the dried basis.

1 IDENTIFICATION

A. Spectroscopic Identification Tests 〈197〉, Ultraviolet-Visible Spectroscopy: 197U

Analytical wavelength: 285 nm

Medium: Dilute hydrochloric acid in water (1 in 100).

Sample solution: 8 µg/mL of flucytosine in Medium

Acceptance criteria: Absorptivities, calculated on the dried basis, do not differ by NMT 2.0%.

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B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay. (USP 1-May-2022)

2 ASSAY

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Procedure

Buffer: 13.6 g/L of monobasic potassium phosphate in water. Adjust with phosphoric acid to a pH of 2.0.

Solution A: Buffer and methanol (98:2)

Solution B: Buffer and methanol (85:15)

Mobile phase: See Table 1.

Table 1

Diluent: Dissolve 13.6 g of monobasic potassium phosphate in 980 mL of water. Add 20 mL of methanol and mix. Standard solution: 0.05 mg/mL of USP Flucytosine RS in Diluent

Sample solution: 0.05 mg/mL of Flucytosine in Diluent

2.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 284 nm

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

Flow rate: 1.1 mL/min

Injection volume: 20 µL

2.2 System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 2.0

Relative standard deviation: NMT 0.73%

2.3 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of flucytosine (C₄H₄FN₃O) in the portion of Flucytosine taken: 

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

r_U = peak response of flucytosine from the Sample solution

r_S = peak response of flucytosine from the Standard solution

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

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

Acceptance criteria: 98.0%–102.0% on the dried basis (USP 1-May-2022)

3 OTHER COMPONENTS

Delete the following:

Fluorouracil

Diluent: Glacial acetic acid and water (4:1)

Standard solution: 0.025 mg/mL solution of USP Fluorouracil RS in Diluent

Sample solution: 25 mg/mL flucytosine in Diluent

Application volume: 20 µL

3.1 Chromatographic system

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

Mode: TLC

Adsorbent: 0.5-mm layer of chromatographic silica gel mixture

Developing solvent system: Chloroform and glacial acetic acid (13:7)

3.2 Analysis

Samples: Standard solution and Sample solution

Locate the spots on the plate by observing under short-wavelength UV radiation.

Acceptance criteria: Any spot from the Sample solution is not greater in size and intensity than the spot at the respective R produced by the Standard solution, corresponding to NMT 0.1% of uorouracil. _{(USP 1-May-2022)}

4 IMPURITIES

Add the following:

4.1 Organic Impurities

Buffer, Solution A, Solution B, Mobile phase, and Diluent: Prepare as directed in the Assay.

Sensitivity solution: 0.1 µg/mL of USP Flucytosine RS in Diluent

Standard solution: 0.00030 mg/mL of USP Fluorouracil RS, 0.00045 mg/mL of USP Fluorouracil Related Compound F RS, and 0.00015 mg/mL of USP Flucytosine RS in Diluent

Sample solution: 0.3 mg/mL of Flucytosine in Diluent

4.1.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 260 nm

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

Flow rate: 1.1 mL/min

Injection volume: 20 µL

4.1.2 System suitability

Samples: Sensitivity solution and Standard solution

Suitability requirements

Relative standard deviation: NMT 5% each for flucytosine,fluorouracil, and fluorouracil related compound F, Standard solution Signal-to-noise ratio: NLT 10, Sensitivity solution

4.1.3 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of fluorouracil in the portion of Flucytosine taken:

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

r_U = peak response of fluorouracil 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 Flucytosine in the Sample solution (mg/mL)

Calculate the percentage of fluorouracil related compound F in the portion of Flucytosine taken:

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

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

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

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

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

Calculate the percentage of any individual impurity in the portion of Flucytosine taken:

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

r_U = peak response of any individual impurity from the Sample solution

r_S = peak response of flucytosine from the Standard solution

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

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

Acceptance criteria: See Table 2. The reporting threshold is 0.03%.

Table 2

Residue on Ignition 〈281〉: NMT 0.1%

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4.2 Limit of Fluoride Ion

(USP 1-May-2022)

[Note—All glassware and/or plasticware used in this test should be scrupulously clean and even free from trace amounts fluoride. The use of plasticware to contain the solutions while the potential is measured is recommended.]

Buffer: Transfer 110 g of sodium chloride into a 2-L volumetric ask, add 1 g of sodium citrate and 700 mL of water, and dissolve with shaking. Carefully add 150 g of sodium hydroxide, and dissolve with shaking. Cool to room temperature, and while stirring, cautiously add 450 mL of glacial acetic acid. Cool to room temperature, add 600 mL of isopropyl alcohol, dilute with water to volume, and mix. The pH of this solution is between 5.0 and 5.5.

Standard stock solution: 1 mg/mL of fluoride ion solution in water, prepared as follows. Transfer 2.211 g of sodium fluoride, previously dried at 150° for 4 h, into a 1-L volumetric ask, and dissolve in 200 mL of water. Add 1.0 mL of sodium hydroxide solution in water (1 in 250), dilute with water to volume, and mix. Store the solution in a closed plastic container.

Standard solutions: 1, 3, 5, and 10 µg/mL of fluoride from Standard stock solution in Buffer

Sample solution: 10 mg/mL of Flucytosine in Buffer

Analysis

Samples: Standard solutions and Sample solution

Concomitantly measure the potential (see Titrimetry 〈541〉), in mV, of the Standard solutions and the Sample solution, with a suitable pH meter equipped with a fluoride-specific ion electrode and a glass-sleeved calomel reference electrode that has been modified in the following manner. Mix 70 mL of freshly prepared saturated potassium chloride solution with 30 mL of isopropyl alcohol, ll the electrode with the clear supernatant, and allow the electrode to remain in the mixture for NLT 2 h before use, or preferably overnight. When taking the measurements, transfer the solution to a 150-mL beaker, and immerse the electrodes. Insert a polytef-coated stirring bar into the beaker, place the beaker on a magnetic stirrer having an insulated top, and allow to stir until equilibrium is attained (1–2 min). Rinse and dry the electrodes between measurements, taking care not to scratch the crystal in the specific ion electrode. Measure the potential of each Standard solution, and plot the fluoride concentration, in mg/100 mL, versus the potential, in mV, on semilogarithmic paper. Measure the potential of the Sample solution, and determine from the standard curve the fluoride concentration, in mg/100 mL.

Calculate the percentage of fluoride in the portion of Flucytosine taken:

Result = C/10

C = concentration of fluoride from the standard curve (mg/100 mL)

Acceptance criteria: NMT 0.05% of fluoride

5 SPECIFIC TESTS

Loss on Drying 〈731〉

Analysis: Dry at 105° for 4 h.

Acceptance criteria: NMT 1.5%

6 ADDITIONAL REQUIREMENTS

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

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USP Reference Standards 〈11〉

USP Flucytosine RS

USP Fluorouracil RS

USP Fluorouracil Related Compound F RS

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

C₆H₇FN₂O₂      158.13 _{(USP 1-May-2022)}