A: Infrared Absorption 197M: previously dried at a pressure of not more than 5 mm of mercury at 60 for 3 hours.

B: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.

Test solution: 10 mg per mL, in water.

Phosphate buffer— Prepare a solution containing 0.01 M monobasic sodium phosphate and 0.01 M dibasic sodium phosphate in a suitable container. Adjust with 0.1 M sodium hydroxide or 0.1 M phosphoric acid to a pH of 7.0.

Solution A— Prepare a filtered and degassed mixture of Phosphate buffer and methanol (49:1). Make adjustments if necessary (see System Suitability under Chromatography 621). Prepare this solution fresh daily.

Solution B— Prepare a filtered and degassed mixture of Phosphate buffer and methanol (7:3). Make adjustments if necessary (see System Suitability under Chromatography 621). Prepare this solution fresh daily.

Mobile phase— Use variable mixtures of Solution A and Solution B as directed under Chromatographic system.

System suitability solution— Dissolve suitable quantities of uridine, USP Uracil Arabinoside RS, and USP Cytarabine RS in water to obtain a solution containing about 0.02, 0.02, and 5.0 mg per mL, respectively.

Standard solution— Dissolve an accurately weighed quantity of USP Cytarabine RS in water, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 4 µg per mL.

Test solution— Transfer about 25 mg of Cytarabine, accurately weighed, to a 5.0-mL volumetric flask, dissolve in and dilute with water to volume, and mix. [NOTE—Prepare this solution fresh daily.]

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1 mL per minute. The chromatograph is programmed to provide variable mixtures of Solution A and Solution B, the percentage of Solution B being 0% at the time of injection. This composition is held for 10 minutes. Solution B is then linearly increased to 100% over a period of 10 minutes. After maintaining this composition for 5 minutes, the percentage of Solution B is then linearly decreased to 0% over a period of 5 minutes. This composition is maintained for 20 minutes to equilibrate the system. Chromatograph the System suitability solution, and record the peak responses as directed under Procedure: the relative retention times are about 0.55 for uracil, 1.14 for uridine, 1.62 for uracil arabinoside, and 1.0 for cytarabine; and the resolution, R, between cytarabine and uridine is not less than 1.25. Chromatograph the Standard solution, and record the peak responses as directed under Procedure: the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage of uracil arabinoside in the portion of Cytarabine taken by the formula: in which C is the concentration, in mg per mL, of USP Cytarabine RS in the Standard solution; W is the weight, in mg, of the specimen, 1.34 is the relative response factor for uracil arabinoside; ri is the peak response of uracil arabinoside in the Test solution; and rS is the peak response of USP Cytarabine RS in the Standard solution: not more than 0.30% is found.

500(C / W)(ri / FrS),

(Official January 1, 2007)

Phosphate buffer— Dissolve 0.73 g of monobasic sodium phosphate and 1.4 g of dibasic sodium phosphate in 1 L of water, mix, and filter.

Mobile phase— Prepare a filtered and degassed mixture of Phosphate buffer and methanol (95:5). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Cytarabine RS in water, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 0.1 mg per mL.

Resolution solution— Dissolve an accurately weighed quantity of USP Uracil Arabinoside RS in Standard preparation, and dilute quantitatively, and stepwise if necessary, with Standard preparation to obtain a solution having a known concentration of about 0.1 mg per mL.

Assay preparation— Transfer about 10 mg of Cytarabine, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with water to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1.0 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the relative retention times are about 1.0 for cytarabine and 1.3 for uracil arabinoside; and the resolution, R, between cytarabine and uracil arabinoside is not less than 2.5. 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%. [NOTE—After chromatography has been completed, flush the column with a mixture of water and methanol (7:3).]

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 major peaks. Calculate the quantity, in mg, of C9H13N3O5 in the portion of Cytarabine taken by the formula: in which C is the concentration, in mg per mL, of USP Cytarabine RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.