Fludarabine Phosphate

(floo dar' a been fos' fate).

C10H13FN5O7P

365.21
9H-Purin-6-amine, 2-fluoro-9-(5-O-phosphono-

-d-arabinofuranosyl)-.
9-

-d-Arabinofuranosyl-2-fluoroadenine 5¢-(dihydrogen phosphate)

[75607-67-9].

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

—

USP Fludarabine Phosphate RS

Identification, Infrared Absorption

197K

Specific rotation

781S

: between +10

and +14

Test solution: 5 mg per mL, in water.

Microbial enumeration tests

and Tests for specified microorganisms

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

Water, Method I

921

: not more than 3.0%.

Chloride—

Standard chloride solution— Transfer 82.4 mg of sodium chloride to a 100-mL volumetric flask, and dissolve in and dilute with water to volume. Transfer 1.0 mL of this solution to a 10-mL volumetric flask, and dilute with water to volume. Transfer 2.0 mL of this solution to a test tube, add 13.0 mL of water, and mix.

Test solution— Transfer about 50.0 mg of Fludarabine Phosphate, accurately weighed, to a test tube, dissolve in 15 mL of water, and heat gently if necessary.

Procedure— Add 1.0 mL of nitric acid to the Standard chloride solution and the Test solution, and place each in separate colorless test tubes, containing 1.0 mL of silver nitrate TS. The Test solution shows less turbidity than the Standard chloride solution (0.2%).

Limit of alcohol—

Standard solution— Prepare a solution of alcohol in dimethylformamide to obtain a solution having a concentration of about 0.50 mg 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 50 mg per mL.

Blank solution— Use dimethylformamide.

Chromatographic system (see Chromatography

621

)— 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 10 minutes, 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 27 cm per second. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the retention time for alcohol is about 3 minutes; and the relative standard deviation for three injections of the Standard solution is 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 2.0 mL each of the Test solution, the Standard solution, and the Blank solution to 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 60 minutes 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 CS is the concentration, in mg per mL, of alcohol (C2H5OH) in the Standard solution; CU is the concentration, in mg per mL, of Fludarabine Phosphate in the Test solution; and rU and rS are the alcohol peak areas in the chromatograms obtained from the Test solution and the Standard solution, respectively: not more than 1.0% of alcohol (C2H5OH) is found. Use the percentage obtained to calculate the Assay result on the solvent-free basis.

Limit of free phosphate—

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

Standard working solution— Transfer 1.0 mL of the Standard stock solution into a 100-mL volumetric flask, and dilute with water to volume. Transfer 2.0 mL of this solution to a test tube.

Blank solution— Place 2.0 mL of water in a test tube.

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

Molybdovanadic reagent— In a 150-mL beaker, mix 4 g of finely powdered ammonium molybdate and 0.1 g of finely powdered ammonium vanadate. 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.

Procedure— To each of the test tubes containing the Standard working solution, Test solution, and Blank solution, add 2.0 mL Molybdovanadic reagent: the color of the Standard working solution must be more intense than that of the Blank solution. Viewed downward in diffuse daylight against a white background, the yellow coloration of the Test solution must 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 2 hours, to a 250-mL volumetric flask, dilute with water to volume, and mix to obtain a solution having a known concentration of about 2.54 mg 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 1.0 µg of sodium per mL.

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

Blank solution— Use water.

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

Heavy metals, Method I

231

: 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 the Standard preparation in the Assay.

System suitability solution— Dissolve 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 minutes.

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

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

Chromatographic system— The liquid chromatograph is equipped with a 260-nm detector and a 4.6-mm × 15-cm column that contains 5-µm packing L1. The flow rate is 1.0 mL per minute. Chromatograph the Sensitivity check solution at 260 nm, 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, the 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 10 µL) of the Standard solution and the Test solution, record the chromatograms at 260 nm, 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 F1 is a relative response factor equal to the values given in Table 1 for the tabulated impurities, and equal to 1.0 for any other impurities; rU is the response for each individual impurity in the Test solution; and rS is 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 10 mM monobasic potassium phosphate and methanol (4:1).

Standard solution— Prepare as directed for the Standard preparation in the Assay.

Sensitivity check solution and Test solution— Prepare as directed in 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 1.0 mL per minute. Chromatograph the Sensitivity check solution at 260 nm, and record the peak heights as directed for Procedure: the peak height of the fludarabine phosphate peak must be greater than 10 times the noise height, the 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 10 µL) of the Standard solution and 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 F2 is a relative response factor equal to the values given in Table 1, and 1.0 for all other individual peaks; rU is the response for each individual impurity in the Test solution; and rS is 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 in Test A and Test B, excluding those given in Table 1, is not more than 0.5%; and the sum of all impurities (Table 1 and others) found in Test A and Test B is not more than 1.5%.

Assay—

Mobile phase— Prepare a mixture of filtered, degassed 10 mM monobasic potassium phosphate and methanol (47:3).

Standard preparation— Dissolve an accurately weighed quantity of USP Fludarabine Phosphate RS in Mobile phase, and dilute quantitatively, and stepwise if necessary, in Mobile phase to obtain a solution having a known concentration of 0.02 mg per mL.

Assay preparation— Transfer 50 mg of Fludarabine Phosphate, accurately weighed, to a 50-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix. Transfer 5.0 mL of the solution to a 250-mL volumetric flask, dilute with Mobile phase to 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 1.0 mL per 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 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the fludarabine phosphate peak. Calculate the quantity, in mg, of C10H13FN5O7P in the portion of Fludarabine Phosphate taken by the formula:

2500C(rU / rS)

in which C is the concentration, in mg per mL, of USP Fludarabine Phosphate RS in the Standard preparation; and rU and rS are the peak responses for fludarabine in the Assay preparation and the Standard preparation, respectively.

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
Reference Standards	RS Technical Services 1-301-816-8129 rstech@usp.org
61	Radhakrishna S Tirumalai, Ph.D. Principal Scientific Liaison 1-301-816-8339	(GCM2010) General Chapters - Microbiology
62	Radhakrishna S Tirumalai, Ph.D. Principal Scientific Liaison 1-301-816-8339	(GCM2010) General Chapters - Microbiology

USP35–NF30 Page 3211

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