Fludarabine Phosphate for Injection
»Fludarabine Phosphate for Injection contains not less than 95.0percent and not more than 105.0percent of the labeled amount of C10H13FN5O7P.
Caution—Fludarabine Phosphate is potentially cytotoxic.Great care should be taken to prevent inhaling particles and exposing the skin to it.
Packaging and storage— Preserve in Containers for Sterile Solids,as described under Injections á1ñ,at 2to 30,or at controlled room temperature.
Constitued solution— At the time of use,it meets the requirements for Constituted Solutionsunder Injections á1ñ.
Identification—
A: Ultraviolet Absorption á197Uñ—
Solution: 27µg per mL.
Medium: 0.1Mhydrochloric acid.
B: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparation,as obtained in the Assay.
Water,Method Iá921ñ: not more than 5.0%.
pHá791ñ: between 7.2and 8.2.
Bacterial endotoxins á85ñ— It contains not more than 7.7USP Endotoxin Units per mg of fludarabine phosphate.
Sterility á71ñ— It meets the requirements when tested as directed for Membrane Filtrationunder Test for Sterility of the Product to be Examined.
Related compounds—
TEST A(EARLY-ELUTING IMPURITIES)—
Mobile phase— Prepare as directed in the Assay.
Standard solution— Prepare as directed for Standard preparationin the Assayunder Fludarabine Phosphate.
System suitability solution— Prepare as directed forSystem suitability solutionin Chromatographic purity Test Aunder Fludarabine Phosphate.
Sensitivity check solution— Dilute the Standard solutionwith Mobile phaseto obtain a solution having a concentration of 0.0005mg per mL.
Test solution— Inject 2.0mLof water into each of 5vials of Fludarabine Phosphate for Injection.Quantitatively transfer the contents of the vials into a single 250-mLvolumetric flask,using water rinses.Dilute the volumetric flask with Mobile phaseto volume,and mix to obtain a solution having a concentration of about 1mg of fludarabine phosphate per mL.
Chromatographic system— Proceed as directed for Chromatographic systemin Chromatographic purity Test Aunder Fludarabine Phosphate.
Procedure— Separately inject equal volumes (about 10µL)of the Standard solutionand the Test solution,record the chromatograms,and measure all of the peak responses up to and including the fludarabine phosphate peak.Calculate the percentage of each early-eluting impurity present in the portion of Fludarabine Phosphate taken by the formula:
100F1(ru/rs),
in which F1is a relative response factor equal to the values given in Table 1,and equal to 1.0for any other individual early-eluting degradation product not appearing in Table 1;rUis the response for each individual impurity in the Test solution;and rSis the response for the fludarabine phosphate peak in the Test solution.In addition to meeting the limits for the individual degradation products given in Table 1,not more than 0.2%of any other early-eluting fludarabine phosphate degradation peak is found.
Table 1.
Relative
Retention Time		 Relative Response
Factor (F1)		 Relative Response
Factor (F2)		 Impurity Name Limit
(w/w%)
0.26 4.0 Iso-ara-guanine-monophosphate 1.0
0.34 2.5 Isoguanine 0.2
1.5 0.5 2-fluoroadenine 0.2
1.9 0.6 2-fluoro-ara-adenine 0.2
TEST B(LATE-ELUTING-IMPURITIES)—
Mobile phase— Prepare a mixture of filtered,degassed 10mMmonobasic potassium phosphate and methanol (4:1).
Standard solution,System suitability solution,and Sensitivity check solution— Prepare as directed for Chromatographic purity Test Aunder Fludarabine Phosphate.
Test solution— Prepare as directed for Related compounds Test A.
Chromatographic system— Proceed as directed for Chromatographic purity Test Bunder Fludarabine Phosphate.
Procedure— Separately inject equal volumes (about 10mL)of the Standard solution and the Test solution,record the chromatograms at 260nm,and measure all of the peak responses starting with the fludarabine phosphate peak.Calculate the percentage of each late-eluting impurity present in the portion of Fludarabine Phosphate taken by the formula:
100F2(rU/rS),
in which F2is a relative response factor equal to the values given in Table 1,and 1.0for any other individual late-eluting degradation peak not appearing in Table 1;rUis the response for each individual impurity in the Test solution;and rSis the response for the fludarabine phosphate peak in the Test solution.In addition to meeting the limits for the individual degradation products given in Table 1,not more than 0.2%of any other late-eluting fludarabine phosphate degradation product is found;and the sum of all fludarabine phosphate degradation products found inTest AandTest Bis not more than 2.0%.
Uniformity of dosage units á905ñ: meets the requirements.
Assay—
Mobile phase— Prepare a mixture of filtered,degassed 10mMmonobasic potassium phosphate and methanol (47:3).
Standard preparation— Dissolve an accurately weighed quantity of USP Fludarabine Phosphate RSin Mobile phase,and dilute quantitatively,and stepwise if necessary,in Mobile phaseto obtain a solution having a known concentration of 0.02mg per mL.
Assay preparation— Inject 2.0mLof Mobile phaseinto each of 5vials of Fludarabine Phosphate for Injection.Quantitatively transfer the contents of the vials into a 250-mLvolumetric flask,using Mobile phaserinses.Dilute with Mobile phaseto volume,and mix.Transfer 5.0mLof the solution to a 250-mLvolumetric flask,dilute with Mobile phaseto volume,and mix to obtain a solution having a concentration of about 0.02mg of fludarabine phosphate per mL.
Chromatographic system— The liquid chromatograph is equipped with a 260-nm detector and a 4.6-mm ×15-cm column containing 5-µm packing L1.The flow rate is 1.0mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 2.0%.
Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand Assay preparationinto the chromatograph,record the chromatograms,and measure the response for the fludarabine phosphate peak.Calculate the quantity,in mg,of C10H13FN5O7Pin the portion of Fludarabine Phosphate for Injection taken by the formula:
2500C(rU/rS),
in which Cis the concentration,in mg per mL,of USP Fludarabine Phosphate RSin the Standard preparation;and rUand rSare the peak responses for fludarabine in the Assay preparationand the Standard preparation,respectively.
Auxiliary Information— Staff Liaison:Lawrence Evans,III,Ph.D.,Scientist
Expert Committee:(PA6)Pharmaceutical Analysis 6
USP28–NF23Page 832
Pharmacopeial Forum:Volume No.28(6)Page 1799
Phone Number:1-301-816-8389