Fludarabine Phosphate, chemical structure, molecular formula, Reference Standards

Fludarabine Phosphate

C10H13FN5O7P 365.21
9H-Purin-6-amine,2-fluoro-9-(5-O-phosphono-b-D-arabinofuranosyl)-.
9-b-D-Arabinofuranosyl-2-fluoroadenine 5¢-(dihydrogen phosphate) [75607-67-9].

»Fludarabine Phosphate contains not less than 98.0percent and not more than 102.0percent of 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.

Packaging and storage— Preserve in well-closed,light-resistant containers,and store in a refrigerator.

USP Reference standards á11ñ— USP Fludarabine Phosphate RS.

Identification, Infrared Absorption á197Kñ.

Specific rotation á781Sñ: between +10

and +14

Test solution: 5mg per mL,in water.

Microbial limits á61ñ— The total aerobic microbial count does not exceed 1000cfu per g.

Water,Method Iá921ñ: not more than 3.0%.

Chloride á221ñ: not more than 0.2%.

Limit of ethanol—

Standard solution— Prepare a solution of alcohol in dimethylformamide to obtain a solution having a known concentration of about 0.50mg of alcohol (C2H5OH)per mL.

Test solution— Dissolve an accurately weighed portion of Fludarabine Phosphate in dimethylformamide to obtain a solution having a concentration of about 50mg per mL.

Blank solution— Use dimethylformamide.

Chromatographic system (see Chromatography á621ñ)— The gas chromatograph is equipped with a headspace injector,a flame-ionization detector,and a 0.25-mm ×30-m capillary column,the internal wall of which is coated with a 1.4-µm film of liquid phase G43.The column temperature is programmed as follows.Initially the temperature of the column is equilibrated at 40

for 10minutes,then increased at a rate of 5

per minute to 70

,and then increased at a rate of 30

per minute to 220

.The injection port temperature is maintained at 160

,and the detector temperature is maintained at 250

.The carrier gas is helium,flowing at a rate of about 27cm per second.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the retention time for alcohol is about 3minutes;and the relative standard deviation for three injections of the Standard solutionis not more than 4.0%.Chromatograph the Blank solution,and record the peak responses as directed for Procedure:the chromatogram shows no peak at the retention time for alcohol.

Procedure— Transfer 2.0mLeach of the Test solution,the Standard solution,and the Blank solutionto separate headspace vials,then seal the vials using a flanged cap so that the cap can no longer be turned.The vials are maintained at 80

for 60minutes prior to headspace injection.Record the chromatograms,and measure the peak area for alcohol.Calculate the percentage of alcohol in the portion of fludarabine phosphate taken by the formula:

100(CS/CU)(rU/rS),

in which CSis the concentration,in mg per mL,of alcohol (C2H5OH)in the Standard solution;CUis the concentration,in mg per mL,of fludarabine phosphate in theTest solution;and rUand rSare the alcohol peak areas in the chromatograms obtained from the Test solutionand the Standard solution,respectively:not more than 1.0%of alcohol (C2H5OH)is found.Use the percentage obtained to calculate the Assayresult on the solvent-free basis.

Limit of free phosphate—

Standard stock solution— Transfer an accurately weighed quantity of potassium dihydrogen phosphate into a 1000-mLvolumetric flask,dissolve in and dilute with water to volume,and mix to obtain a solution having a known concentration of about 0.716mg of potassium dihydrogen phosphate per mL.

Standard working solution— Transfer 1.0mLof the Standard stock solutioninto a 100-mLvolumetric flask,and dilute with water to volume.Transfer 2.0mLof this solution to a test tube.

Blank solution— Place 2.0mLof water in a test tube.

Test solution— Transfer 10mg of Fludarabine Phosphate,accurately weighed,to a test tube,and dissolve in 2.0mLwater,heating gently.

Molybdovanadic reagent— In a 150-mLbeaker,mix 4g of finely powdered ammonium molybdate and 0.1g of finely powdered ammonium vanadate.Add 70mLof water,and grind the particles using a glass rod.Aclear solution is obtained within a few minutes.Add 20mLof nitric acid,adjust to room temperature,and dilute with water to 100mL.

Procedure— To each of the test tubes containing the Standard working solution,Test solution,and Blank solution,add 2.0mLMolybdovanadic reagent:the color of the Standard working solutionmust be more intense than that of the Blank solution.Viewed downward in diffuse daylight against a white background,the yellow coloration of the Test solutionmust not be more intense than that of the Standard working solution (0.1%).

Limit of sodium—

Standard solution— Transfer an accurately weighed quantity of sodium chloride,previously dried at 105

for 2hours,to a 250-mLvolumetric flask,dilute with water to volume,and mix to obtain a solution having a known concentration of about 2.54mg of sodium chloride per mL.Dilute an accurately measured volume of this solution quantitatively,and stepwise if necessary,with water to obtain a solution containing 1.0µg of sodium per mL.

Test solution— Transfer about 50mg of Fludarabine Phosphate,accurately weighed,to a 100-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.

Blank solution— Use water.

Procedure— Using the Blank solutionto zero the instrument,concomitantly determine the atomic emission of the Standard solutionand the Test solutionat the sodium emission line at 589.0nm with a suitable flame photometer:the emission response obtained for the Test solutionis not greater than that obtained for the Standard solution(0.2%).

Heavy metals,Method Iá231ñ: not more than 0.002%.

Chromatographic purity—

TEST A(EARLY-ELUTING IMPURITIES)—

Mobile phase— Prepare as directed in the Assay.

Standard solution— Prepare as directed for Standard preparationin the Assay.

System suitability solution— Dissolve 10mg of Fludarabine Phosphate in 10mLof 0.1Nhydrochloric acid.Heat the solution at 80

in a water bath for 15minutes.

Test solution— Transfer 50mg of Fludarabine Phosphate,accurately weighed,to a 50-mLvolumetric flask,dissolve in and dilute with Mobile phaseto volume,and mix.

Sensitivity check solution— Dilute the Standard solutionwith Mobile Phase to obtain a solution having a concentration of 0.0005mg per mL.

Chromatgraphic system— The liquid chromatograph is equipped with a 260nm detector and a 4.6-mm ×15-cm column that contains 5-µm packing L1.The flow rate is 1.0mLper minute.Chromatograph the Sensitivity check solutionat 260nm,and record the peak heights as directed for Procedure:the ratio of the fludarabine phosphate peak height to the noise height is not less than 10,noise height being determined by a suitable procedure.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the resolution,R,between the iso-ara-guanine monophosphate peak (relative retention time about 0.26)and the isoguanine peak (relative retention time about 0.34)is not less than 2.0.Chromatograph the Standard solution,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 solutionand the Test solution,record the chromatograms at 260nm,and measure all of the areas for the major peaks 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 1for the tabulated impurities,and equal to 1.0for any other impurities;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 impurities given in Table 1,not more than 0.1%of any other impurity that elutes prior to fludarabine phosphate is found.

Table 1.

Relative Retention Time	Relative Response Factor (F1)	Relative Response Factor (F2)	Impurity	Limit (w/w%)
0.26	4.0		Iso-ara-guanine-monophosphate	0.8
0.34	2.5		Isoguanine	0.2
0.42	1.9		3¢,5¢-Diphosphate analog	0.4
1.5		0.5	2-Fluoroadenine	0.1
1.9		0.6	2-Fluoro-ara-adenine	0.2
2.5		1.8	2-Ethoxyphosphate analog	0.2

TEST B(LATE-ELUTING IMPURITIES)—

Mobile phase— Prepare a mixture of filtered,degassed 10mMmonobasic potassium phosphate and methanol (4:1).

Standard solution— Prepare as directed for Standard preparationin the Assay.

Sensitivity check solution andTest solution— Prepare as directed for Chromatographic purity Test A.

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 Sensitivity check solutionat 260nm,and record the peak heights as directed for Procedure:the peak height of the fludarabine phosphate peak must be greater than 10times the noise height,noise height being determined by a suitable procedure.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the tailing factor is not more than 2.0,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard solutionand the Test solution,record the chromatograms,and measure all of the areas for the major peaks 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 all other individual peaks;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 impurities given in Table 1,not more than 0.1%of any other impurity that elutes after fludarabine phosphate is found.The sum of all other impurities inTest AandTest B,excluding those given in Table 1,is not more than 0.5%;and the sum of all impurities (Table 1and others)found inTest AandTest Bis not more than 1.5%.

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— Transfer 50mg of Fludarabine Phosphate,accurately weighed,to a 50-mLvolumetric flask,dissolve in and dilute with Mobile phaseto volume,and mix.Transfer 5.0mLof the solution to a 250-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

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 1.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the fludarabine phosphate peak.Calculate the quantity,in mg,of C10H13FN5O7Pin the portion of Fludarabine Phosphate taken by the formula:

500C(rU/rS),

in which Cis the concentration,in mg per mL,of USP Fludarabine Phosphate in 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 830

Pharmacopeial Forum:Volume No.30(5)Page 1621

Phone Number:1-301-816-8389