A: Infrared Absorption 197M.

B: Ultraviolet Absorption 197U—

C: 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.

Internal standard solution— Prepare a solution of n-propyl alcohol in dimethyl sulfoxide having a concentration of about 0.05 µL per mL.

Standard solution— Prepare a solution in Internal standard solution having a concentration of 2.5 µg of chloroform, 5.0 µg of dehydrated alcohol, 5.0 µg of acetone, 5.0 µg of butyl alcohol, 5.0 µg of dioxane, 10.0 µg of methylene chloride, 15.0 µg of diisopropyl ether, 20.5 µg of acetonitrile, 50 µg of pentane, and 100 µg of methanol in each mL, and sonicate.

Test solution— Dissolve about 200 mg of Valrubicin, accurately weighed, in 4.0 mL of Internal standard solution, and sonicate.

Chromatographic system (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector and a 0.32-mm × 30-m fused-silica capillary column coated with a 5-µm film of G2 stationary phase. The carrier gas is helium, flowing at a rate of 30 mL per minute. The column temperature is maintained at 220. The injection port temperature and the detector block temperature are maintained at 250. Chromatograph the Standard solution, and record the responses as directed for Procedure: the relative retention times are about 0.48 for methanol, 0.66 for dehydrated alcohol, 0.71 for acetonitrile, 0.76 for acetone, 0.86 for pentane, 0.92 for methylene chloride, 1.0 for n-propyl alcohol, 1.19 for diisopropyl ether, 1.22 for chloroform, 1.35 for butyl alcohol, and 1.52 for dioxane; the component solvent peaks are resolved; and the relative standard deviation of the ratios of the peak area of each solvent to the peak area of n-propyl alcohol is not more than 10%.

Procedure— Separately inject equal volumes (about 1 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the concentration, in µg per g, of each residual solvent in the portion of Valrubicin taken by the formula: in which C is the concentration, in µg per mL, of the respective individual solvent in the Standard solution; W is the quantity, in mg, of Valrubicin taken to prepare the Test solution; and Ri and RS are the peak area ratios of the respective individual solvent to n-propyl alcohol obtained from the Test solution and the Standard solution, respectively: not more than 50 µg per g of chloroform, 100 µg per g of dehydrated alcohol, 100 µg per g of acetone, 100 µg per g of butyl alcohol, 100 µg per g of dioxane, 300 µg per g of methylene chloride, 410 µg per g of acetonitrile, 500 µg per g of diisopropyl ether, 1000 µg per g of pentane, and 2000 µg per g of methanol are found.

Mobile phase— Prepare as directed in the Assay.

Resolution solution— Prepare a solution of USP Valrubicin Related Compound A RS and USP Valrubicin RS in acetonitrile having known concentrations of about 0.25 mg per mL and 1 mg per mL, respectively.

Test solution— Use the Assay preparation.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector, a guard column, and a 5-mm × 10-cm analytical column that contains a 4-µm packing L1. The flow rate is about 3.5 mL per minute. Chromatograph the Resolution solution, and record the responses as directed for Procedure: the relative retention times are about 0.8 for valrubicin related compound A and 1.0 for valrubicin; and the resolution, R, between valrubicin related compound A and valrubicin is not less than 2.

Procedure— Inject a volume (about 10 µL) of the Test solution into the chromatograph, record the chromatogram, and measure the areas for the major peaks. Calculate the percentage of each impurity in the portion of Valrubicin taken by the formula: in which ri is the peak area for each impurity; and rs is the sum of the areas of all the peaks. Do not consider any peaks due to solvent or excipients. Not more than 0.3% of any individual impurity with a relative retention time of 0.06, 0.17, 0.27, or 0.52 is found; not more than 0.6% of any impurity with a relative retention time of about 0.14 is found; not more than 0.2% of any other individual impurity is found; not more than 1.0% of total other impurities that are not specified by relative retention time is found; and not more than 2.5% of total impurities that are not less than 0.1% is found.

(Official January 1, 2007)

Mobile phase— Prepare a filtered and degassed mixture of 0.015 M phosphoric acid and acetonitrile (57:43). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Valrubicin RS in acetonitrile, and dilute quantitatively with acetonitrile to obtain a solution having a known concentration of about 1 mg per mL.

Assay preparation— Transfer about 25 mg of Valrubicin, accurately weighed, to a 25-mL volumetric flask, dissolve in and dilute with acetonitrile to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector, a guard column, and a 5-mm × 10-cm analytical column that contains a 4-µm packing L1. The flow rate is about 3.5 mL per minute. Chromatograph the Standard preparation, and record the responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2%.

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 areas for the major peaks. Calculate the quantity, in mg, of C34H36F3NO13 in the portion of Valrubicin taken by the formula: in which C is the concentration, in mg per mL, of USP Valrubicin RS in the Standard preparation; P is the specified percentage of valrubicin in USP Valrubicin RS; and rU and rS are the valrubicin peak areas obtained from the Assay preparation and the Standard preparation, respectively.