Related compounds—
TEST A (EARLY-ELUTING IMPURITIES)—
Mobile phase—
Prepare as directed in the Assay.
Standard solution—
Prepare as directed for
Standard preparation in the
Assay under
Fludarabine Phosphate.
System suitability solution—
Prepare as directed for
System suitability solution in
Chromatographic purity Test A under
Fludarabine Phosphate.
Sensitivity check solution—
Dilute the Standard solution with Mobile phase to obtain a solution having a concentration of 0.0005 mg per mL.
Test solution—
Inject 2.0 mL of water into each of 5 vials of Fludarabine Phosphate for Injection. Quantitatively transfer the contents of the vials into a single 250-mL volumetric flask, using water rinses. Dilute the volumetric flask with Mobile phase to volume, and mix to obtain a solution having a concentration of about 1 mg of fludarabine phosphate per mL.
Chromatographic system—
Proceed as directed for
Chromatographic system in
Chromatographic purity Test A under
Fludarabine Phosphate.
Procedure—
Separately inject equal volumes (about 10 µL) of the
Standard solution and 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
F1 is a relative response factor equal to the values given in
Table 1, and equal to 1.0 for any other individual early-eluting degradation product not appearing in
Table 1; 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 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 10 mM monobasic potassium phosphate and methanol (4:1).
Standard solution, System suitability solution, and Sensitivity check solution—
Prepare as directed for
Chromatographic purity Test A under
Fludarabine Phosphate.
Test solution—
Prepare as directed for Related compounds Test A.
Chromatographic system—
Proceed as directed for
Chromatographic purity Test B under
Fludarabine Phosphate.
Procedure—
Separately inject equal volumes (about 10 mL) of the
Standard solution and the
Test solution, record the chromatograms at 260 nm, 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
F2 is a relative response factor equal to the values given in
Table 1, and 1.0 for any other individual late-eluting degradation peak not appearing in
Table 1; 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 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 in
Test A and
Test B is not more than 2.0%.
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—
Inject 2.0 mL of Mobile phase into each of 5 vials of Fludarabine Phosphate for Injection. Quantitatively transfer the contents of the vials into a 250-mL volumetric flask, using Mobile phase rinses. 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 to obtain a solution having a concentration of about 0.02 mg 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 1.0 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 2.0%.
Procedure—
Separately inject equal volumes (about 10 µL) of the
Standard preparation and
Assay preparation into the chromatograph, record the chromatograms, and measure the response for the fludarabine phosphate peak. Calculate the quantity, in mg, of C
10H
13FN
5O
7P in the portion of Fludarabine Phosphate for Injection 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.
1
, at 2
to 30