A: Infrared Absorption 197K.

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: 5 mg per mL, in a mixture of chloroform and methanol (9:1).

Buffer solution— Prepare as directed in the Assay.

Solution A— Prepare a filtered and degassed mixture of Buffer solution and acetonitrile (80:20).

Solution B— Prepare a filtered and degassed mixture of acetonitrile and Buffer solution (60:40).

Mobile phase— Use variable mixtures of Solution A and Solution B as directed under Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

Diluting solution— Prepare a filtered mixture of 0.02 M sodium acetate, previously adjusted with acetic acid to a pH of 4.0, and acetonitrile (70:30).

Standard solution— Dissolve an accurately weighed quantity of USP Etoposide RS in Diluting solution to obtain a Standard stock solution having a known concentration of about 2.0 mg per mL. Dilute this Standard stock solution quantitatively and stepwise with Diluting solution to obtain a solution having a known concentration of about 10 µg per mL.

System suitability solution— Transfer about 20 mg of n-propylparaben, accurately weighed, to a 100-mL volumetric flask, and dissolve in and dilute with Diluting solution to volume. Transfer 5 mL of this solution and 5 mL of the Standard stock solution to a 50-mL volumetric flask, and dilute with Diluting solution to volume. Transfer 5.0 mL of this solution to a 100-mL volumetric flask, dilute with Diluting solution to volume, and mix.

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

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm × 15-cm column that contains packing L11 having a diameter of less than 5 µm. The flow rate is about 1.5 mL per minute. Chromatograph the System suitability solution using Solution A, and record the peak responses as directed for Procedure: the relative retention times are about 0.20 for lignan P, 1.0 for etoposide, and 1.43 for picroetoposide; and the resolution, R, between propylparaben and etoposide is not less than 1.1. The chromatograph is programmed for Procedure as follows.

Procedure— Separately inject equal volumes (about 25 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms for at least 40 minutes, and measure the peak responses. Calculate the percentages of lignan P and picroetoposide in the portion of Etoposide taken by the formula: in which C is the concentration, in mg per mL, of USP Etoposide RS in the Standard solution; W is the weight, in mg, of Etoposide taken to prepare the Test solution; ri is the peak response for each related compound obtained from the Test solution; and rS is the peak response for etoposide obtained from the Standard solution: not more than 0.5% of lignan P and 1.0% of picroetoposide is found. Calculate the quantity of any other impurity observed in the chromatogram of the Test solution by the same formula: not more than 2.0% of all related compounds and other impurities is found.

(Official January 1, 2007)

Buffer solution— Dissolve 5.44 g of sodium acetate in 2000 mL of water, adjust with glacial acetic acid to a pH of 4.0, and filter.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solution and acetonitrile (74:26). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Etoposide RS in acetonitrile to obtain a Standard stock solution having a known concentration of about 2.0 mg per mL. Transfer 5.0 mL of this Standard stock solution to a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix.

System suitability solution— Dissolve an accurately weighed quantity of USP Etoposide Resolution Mixture RS in Mobile phase to obtain a solution having a known concentration of 0.3 mg per mL.

Assay preparation— Transfer about 100 mg of Etoposide, accurately weighed, to a 50-mL volumetric flask, dissolve in and dilute with acetonitrile to volume, and mix. Transfer 5.0 mL of this solution to a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm × 30-cm column that contains packing L11. The flow rate is about 1 mL per minute. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the resolution, R, between the etoposide and -etoposide peaks is not less than 1.35. 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 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, and allow the Assay preparation to elute for not less than 1.5 times the retention time of etoposide. Record the chromatograms, and measure the responses for all the peaks. Calculate the quantity, in mg, of C29H32O13 in the portion of Etoposide taken by the formula: in which C is the concentration, in mg per mL, of USP Etoposide RS in the Standard preparation; and rU and rS are the etoposide peak responses obtained from the Assay preparation and the Standard preparation, respectively.