Fludarabine Phosphate 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

Fludarabine Phosphate Injection is a sterile solution of Fludarabine Phosphate in Water for Injection. It contains NLT 95.0% and NMT 105.0% of the labeled amount of fludarabine phosphate (C₁₀H₁₃FN₅O₇P).

[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〉, Ultraviolet-Visible Spectroscopy: 197U (CN 1-May-2020)

Solution: 27 µg/mL in 0.1 M hydrochloric acid

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: 6.9 g/L of monobasic sodium phosphate monohydrate in water (50 mM). Adjust with 1.0 N sodium hydroxide to a pH of 4.5 ± 0.2. Mobile phase: Methanol and Solution A (3:47)

Standard solution: 0.1 mg/mL of USP Fludarabine Phosphate RS in Solution A

Sample solution: Equivalent to 0.1 mg/mL of fludarabine phosphate from Injection diluted with Solution A

3.1 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 mL/min

Injection size: 20 µL

3.2 System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 1.8

Relative standard deviation: NMT 1%

3.3 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of Fludarabine Phosphate (C₁₀H₁₃FN₅O₇P) in the portion of Injection 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 = nominal concentration of fludarabine phosphate in the Sample solution (mg/mL)

Acceptance criteria: 95.0%–105.0%

4 IMPURITIES

Organic Impurities

4.1 Procedure 1: Early-Eluting Impurities (Impurities Eluting Before Fludarabine)

Solution A: 10 mM monobasic potassium phosphate in water

Mobile phase: Solution A and methanol (47:3)

System suitability solution: 1 mg/mL of fludarabine phosphate in 0.1 N hydrochloric acid. Heat the solution at 80° in a water bath for 15 min.

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

Quantitative limit solution: 0.5 µg/mL of USP Fludarabine Phosphate RS in Mobile phase from the Standard solution Sample solution: Equivalent to 1 mg/mL of fludarabine phosphate from Injection diluted with Mobile phase

4.1.1 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 mL/min

Injection size: 10 µL

4.1.2 System suitability

Samples: Standard solution, System suitability solution, and Quantitative limit 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, Quantitative limit solution

4.1.3 Analysis

Samples: Standard solution and Sample solution

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

Result = (r_U /r_S) × (1/F) × 100

r_U = peak response of each impurity from the Sample solution

r_S = peak response of fludarabine phosphate from the Sample solution

F = relative response factor (see Impurity Table 1) Acceptance criteria See Impurity Table 1.

Impurity 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). It is a process impurity and controlled in the drug substance monograph.

4.2 Procedure 2: Late-Eluting Impurities (Impurities Eluting After Fludarabine)

Solution A, Standard solution, Quantitative limit solution, Sample solution, and Chromatographic system: Proceed as directed in Procedure 1:Early-Eluting Impurities

Mobile phase: Solution A and methanol (4:1)

4.2.1 System suitability

Samples: Standard solution and Quantitative limit solution

Suitability requirements

Tailing factor: NMT 2.0, Standard solution

Relative standard deviation: NMT 2.0%, Standard solution

Signal-to-noise ratio: NLT 10, Quantitative limit solution

4.2.2 Analysis

Samples: Standard solution and Sample solution

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

Result = (r_U /r_S) × (1/F) × 100

r_U = peak response of each impurity from the Sample solution

r_S = peak response of fludarabine phosphate from the Sample solution

F = relative response factor (see Impurity Table 2)

Acceptance criteria

Individual impurities: See Impurity Table 2.

Total impurities: The sum of all fludarabine phosphate degradation products found in Procedure 1 and Procedure 2 is NMT 2.0%.

Impurity 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). It is a process impurity and controlled in the drug substance monograph.

5 SPECIFIC TESTS

Bacterial Endotoxins Test 〈85〉: NMT 7.7 USP Endotoxin Units/mg of fludarabine phosphate

Sterility Tests 〈71〉: Meets the requirements when tested as directed under Test for Sterility of the Product to be Examined, Membrane Filtration

pH 〈791〉: 6.0–7.1

Particulate Matter in Injections 〈788〉: Meets the requirements Injections and Implanted Drug Products 〈1〉: Meets the requirements

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in well-closed containers, preferably of Type I glass, protected from light. Store in a refrigerator.

USP Reference Standards 〈11〉

USP Fludarabine Phosphate RS.