Fludarabine Phosphate

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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C₁₀H₁₃FN₅O₇P     365.21

9H-Purin-6-amine, 2-uoro-9-(5-O-phosphono-β-d-arabinofuranosyl)-;

9-β-d-Arabinofuranosyl-2-uoroadenine 5′-(dihydrogen phosphate) CAS RN®: 75607-67-9; UNII: 1X9VK9O1SC.

1 DEFINITION

Fludarabine Phosphate contains NLT 98.0% and NMT 102.0% of fludarabine phosphate (C₁₀H₁₃FN₅O₇P), calculated on the anhydrous, solvent free basis.

[Caution—Fludarabine phosphate is potentially cytotoxic. Great care should be taken to prevent inhaling particles and exposing the skin to it.]

2 IDENTIFICATION

Change to read:

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

B. 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

Procedure

Solution A: 10 mM monobasic potassium phosphate

Mobile phase: Methanol and Solution A (6:94)

Standard solution: 0.02 mg/mL of USP Fludarabine Phosphate RS in Mobile phase

Sample solution: 0.02 mg/mL of Fludarabine Phosphate in Mobile phase

3.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 260 nm

Column: 4.6-mm × 15-cm; 5-µm packing L1

Flow rate: 1.0 mL/min

Injection volume: 10 µL

3.2 System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 1.0%

3.3 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of udarabine phosphate (C₁₀H₁₃FN₅O₇P) in the portion of Fludarabine Phosphate 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 Fludarabine Phosphate RS in the Standard solution (mg/mL)

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

Acceptance criteria: 98.0%–102.0% on the anhydrous, solvent-free basis

4 IMPURITIES

4.1 Chloride

Standard stock solution: 82.4 µg/mL of sodium chloride in water

Standard solution: Transfer 2.0 mL of Standard stock solution to a test tube, add 13.0 mL of water, and mix.

Sample solution: Transfer 50.0 mg of Fludarabine Phosphate to a test tube, add 15 mL of water to dissolve, and heat gently if necessary. Analysis: Add 1.0 mL of nitric acid to the Standard solution and Sample solution, and place each in separate, colorless test tubes containing 1.0 mL of silver nitrate TS.

Acceptance criteria: NMT 0.2%; the Sample solution shows less turbidity than the Standard solution.

4.2 Limit of Free Phosphate

Reagent solution: Mix 4 g of finely powdered ammonium molybdate and 0.1 g of finely powdered ammonium vanadate in a 150-mL beaker. Add 70 mL of water, and grind the particles using a glass rod. A clear solution is obtained within a few minutes. Add 20 mL of nitric acid, adjust to room temperature, and dilute with water to 100 mL.

Standard solution: 7.16 µg/mL of potassium dihydrogen phosphate in water. Transfer 2.0 mL of this solution to a test tube.

Sample solution: Transfer 10 mg of Fludarabine Phosphate in 2.0 mL of water to a test tube and heat gently.

Blank: 2.0 mL of water in a test tube

Analysis: To each of the test tubes containing the Standard solution, Sample solution, and Blank, add 2.0 mL of Reagent solution. Acceptance criteria: NMT 0.1%; the color of the Standard solution must be more intense than that of the Blank. Viewed downward in diffuse daylight against a white background, the yellow coloration of the Sample solution must not be more intense than that of the Standard solution.

4.3 Limit of Sodium

Standard stock solution: 2.54 mg/mL of sodium chloride in water. Sodium chloride is previously dried at 105° for 2 h.

Standard solution: 1 µg/mL of sodium in water, from Standard stock solution

Sample solution: 0.5 mg/mL of Fludarabine Phosphate in water

Instrumental conditions Mode: Flame photometry

Analytical wavelength: Sodium emission line at 589.0 nm

Blank: Water

Analysis

Samples: Standard solution and Sample solution

Acceptance criteria: NMT 0.2%; the emission response of the Sample solution is NMT that of the Standard solution.

4.4 Organic Impurities, Procedure 1: Early-Eluting Impurities

Mobile phase, Standard solution, and Chromatographic system: Proceed as directed in the Assay.

System suitability solution: 10 mg of Fludarabine Phosphate in 10 mL of 0.1 N hydrochloric acid. Heat the solution at 80° in a water bath for 15 min.

Sensitivity solution: 0.5 µg/mL of USP Fludarabine Phosphate RS in Mobile phase, from the Standard solution Sample solution: 1 mg/mL of Fludarabine Phosphate in Mobile phase

4.4.1 System suitability

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

Suitability requirements

Resolution: NLT 2.0 between the iso-ara-guanine monophosphate and isoguanine peaks, System suitability solution Relative standard deviation: NMT 2.0%, Standard solution

Signal-to-noise ratio: NLT 10, Sensitivity solution

4.4.2 Analysis

Sample: Sample solution

Calculate the percentage of each early-eluting impurity in the portion of Fludarabine Phosphate taken:

Result = (r_U /r_S ) × (1/F₁) × 100

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

r_S = peak response of udarabine phosphate from the Sample solution

F₁ = relative response factor for each individual impurity (see Table 1)

Acceptance criteria: See Table 1.

Table 1

^a 6-Amino-9-β-D-arabinofuranosyl-2-oxo-1H-purine 5’-(dihydrogen phosphate).

^b 6-Amino-1H-purin-2(9H)-one.

^c 9-β-D-Arabinofuranosyl-2-uoroadenine 3’,5’-bis(dihydrogen phosphate).

4.5 Organic Impurities, Procedure 2: Late-Eluting Impurities

Solvent A: 10 mM monobasic potassium phosphate

Mobile phase: Methanol and Solvent A (1:4)

Standard solution and Chromatographic system: Proceed as directed in the Assay.

Sensitivity solution and Sample solution: Prepare as directed in Organic Impurities, Procedure 1: Early-Eluting Impurities.

4.5.1 System suitability

Samples: Standard solution and Sensitivity solution

Suitability requirements

Tailing factor: NMT 2.0, Standard solution

Relative standard deviation: NMT 2.0%, Standard solution

Signal-to-noise ratio: NLT 10, Sensitivity solution

4.5.2 Analysis

Sample: Sample solution

Calculate the percentage of each late-eluting impurity in the portion of Fludarabine Phosphate taken:

Result = (r_U /r_S) × (1/F₂) × 100

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

r_S = peak response of udarabine phosphate from the Sample solution

F₂ = relative response factor for each individual impurity (see Table 2)

Acceptance criteria: See Table 2.

Table 2

^a 2-Fluoro-9H-purin-6-amine.

^b 9-β-D-Arabinofuranosyl-2-fluoroadenine.

^c 2-Ethoxy-9-β-D-arabinofuranosyladenine 5’-(dihydrogen phosphate).

^d The sum of all unspecified impurities found in Organic Impurities, Procedure 1: Early-Eluting Impurities and Organic Impurities, Procedure 2: Late-Eluting Impurities.

^e The sum of all impurities found in Organic Impurities, Procedure 1: Early-Eluting Impurities and Organic Impurities, Procedure 2: Late-Eluting Impurities.

4.6 Limit of Alcohol

Standard solution: 0.50 mg/mL of alcohol in dimethylformamide

Sample solution: 50 mg/mL of Fludarabine Phosphate in dimethylformamide

Blank: Dimethylformamide

4.6.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.) Mode: GC equipped with a headspace injector Detector: Flame ionization

Column: 0.25-mm × 30-m; 1.4-µm coating of phase G43

Temperatures

Injection port: 160°

Detector: 250°

Column: See Table 3.

Table 3

Carrier gas: Helium

Flow rate: 27 cm/s

Sample

Volume: 2 mL/vial. [Note—Seal the vials using a flanged cap so that the cap can no longer be turned.]

Conditioning temperature: 80°

Conditioning time: 60 min

Injection volume: 1.0 mL

4.6.2 System suitability

Samples: Standard solution and Blank

[Note—The retention time for alcohol is about 3 min.]

Suitability requirements

Relative standard deviation: NMT 4.0% for three injections, Standard solution

Peak interference: No peak at the retention time for alcohol, Blank

4.6.3 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of alcohol in the portion of Fludarabine Phosphate taken:

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

r_U = peak area of alcohol from the Sample solution

r_S = peak area of alcohol from the Standard solution

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

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

[Note—Use the percentage obtained to calculate the Assay result on the solvent-free basis.]

Acceptance criteria: NMT 1.0%

5 SPECIFIC TESTS

Microbial Enumeration Tests 〈61〉 and Tests for Specified Microorganisms 〈62〉: The total aerobic microbial count is NMT 103 cfu/g.

Optical Rotation 〈781S〉, Procedures, Specific Rotation

Sample solution: 5 mg/mL in water

Acceptance criteria: +10° to +14°

Water Determination 〈921〉, Method I: NMT 3.0%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in well-closed, light-resistant containers, and store in a refrigerator.

USP Reference Standards 〈11〉

USP Fludarabine Phosphate RS.