Fludarabine Phosphate Injection
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.
]
IDENTIFICATION
• A. Ultraviolet Absorption 
197U
197U
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.
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
Chromatographic system
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
System suitability
Sample:
Standard solution
Suitability requirements
Tailing factor:
NMT 1.8
Relative standard deviation:
NMT 1%
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 = (rU/rS) × (CS/CU) × 100
| rU | = | = peak response from the Sample solution |
| rS | = | = peak response from the Standard solution |
| CS | = | = concentration of USP Fludarabine Phosphate RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of fludarabine phosphate in the Sample solution (mg/mL) |
Acceptance criteria:
95.0%–105.0%
IMPURITIES
Organic Impurities
• 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.
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
Chromatographic system
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
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
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentage of each early-eluting impurity in the portion of Injection taken:
Result = (rU/rS) × (1/F) × 100
| rU | = | = peak response of each impurity from the Sample solution |
| rS | = | = 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
| Name | Relative Retention Time |
Relative Response Factor |
Acceptance Criteria, NMT (%) |
|---|---|---|---|
| Iso-ara-guanine-monophosphatea | 0.26 | 0.25 | 1.0 |
| Isoguanineb | 0.34 | 0.40 | 0.2 |
| 3',5'-Diphosphate analogc | 0.42 | — | — |
| Fludarabine | 1.0 | — | — |
| Any individual degradation product | <1.0 | 1.0 | 0.2 |
|
a
6-Amino-9-
 -d-arabinofuranosyl-2-oxo-1H-purine 5¢-(dihydrogen phosphate).
b
6-Amino-1H-purin-2(9H)-one.
c
9-
-d-Arabinofuranosyl-2-fluoroadenine 3¢,5¢-bis(dihydrogen phosphate). It is a process impurity and controlled in the drug substance monograph.
|
|||
• 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)
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
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentage of each late-eluting impurity in the portion of Injection taken:
Result = (rU/rS) × (1/F) × 100
| rU | = | = peak response of each impurity from the Sample solution |
| rS | = | = 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
| Name | Relative Retention Time |
Relative Response Factor |
Acceptance Criteria, NMT (%) |
|---|---|---|---|
| Fludarabine | 1.0 | — | — |
| 2-Fluoroadeninea | 1.5 | 2.0 | 0.2 |
| 2-Fluoro-ara-adenineb | 1.9 | 1.7 | 0.2 |
| 2-Ethoxyphosphate analogc | 2.5 | — | — |
| Any individual degradation product | >1.0 | 1.0 | 0.2 |
|
a
2-Fluoro-9H-purin-6-amine.
b
9-
-d-Arabinofuranosyl-2-fluoroadenine.
c
2-Ethoxy-9-
-d-arabinofuranosyladenine 5¢-(dihydrogen phosphate). It is a process impurity and controlled in the drug substance monograph.
|
|||
SPECIFIC TESTS
• Bacterial Endotoxins Test 85:
NMT 7.7 USP Endotoxin Units/mg of fludarabine phosphate
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
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
791:
6.0–7.1
• Particulate Matter in Injections 788:
Meets the requirements
788:
Meets the requirements
• Injections 1:
Meets the requirements
1:
Meets the requirements
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in well-closed containers, preferably of Type I glass, protected from light. Store in a refrigerator.
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Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Feiwen Mao, M.S.
Senior Scientific Liaison 1-301-816-8320 |
(SM32010) Monographs - Small Molecules 3 |
| 85 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| 71 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 3213
Pharmacopeial Forum: Volume No. 36(2) Page 402