A: 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.

B: It responds to the tests for Sulfate 191.

Test solution: 30 mg per mL, in water.

Dilute phosphoric acid, Mobile phase, Resolution solution, Assay preparation, and Chromatographic system— Proceed as directed in the Assay.

Test solution— Use the Assay preparation.

Reference solution— Transfer 1.0 mL of the Test solution to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Procedure— Separately inject equal volumes (about 20 µL) of the Test solution and the Reference solution into the chromatograph, and measure the area responses for all the peaks, except those due to the solvent. Calculate the percentage of each impurity in the portion of Netilmicin Sulfate taken by the formula: in which ri is the peak response of each impurity in the chromatogram obtained from the Test solution, and rS is the netilmicin peak response in the chromatogram obtained from the Reference solution: not more than 1% of any individual impurity is found, and not more than 5% of total impurities is found.

(Official January 1, 2007)

Dilute phosphoric acid— Dilute 5.0 mL of phosphoric acid to 1000 mL with water, and mix.

Mobile phase— Dissolve 20.22 g of sodium heptanesulfonate in Dilute phosphoric acid, dilute with Dilute phosphoric acid to 1000 mL, and mix. To 620 mL of this solution add 380 mL of acetonitrile, mix, and pass through a filter having a 0.45-µm porosity. Make adjustments if necessary (see System Suitability under Chromatography 621).

Resolution solution— Prepare a solution in Mobile phase containing about 1 mg of USP Netilmicin Sulfate RS and 1 mg of USP Sisomicin Sulfate RS per mL.

Standard preparation— Transfer about 50 mg of USP Netilmicin Sulfate RS to a low-actinic, previously accurately tared, 50-mL volumetric flask. Place the flask in a vacuum desiccator under a vacuum of less than 5 mm of mercury for 1 hour. Accurately weigh the flask, and determine the dry weight of the USP Netilmicin Sulfate RS taken. Dissolve in and dilute with Mobile phase to volume, and mix.

Assay preparation— Transfer about 50 mg of Netilmicin Sulfate to a low-actinic, previously accurately tared, 50-mL volumetric flask. Place the flask in a vacuum desiccator under a vacuum of less than 5 mm of mercury for 1 hour. Accurately weigh the flask, and determine the dry weight of the Netilmicin Sulfate taken. Dissolve in and dilute with Mobile phase to volume, and mix.

Chromatographic system— (see Chromatography 621)—The chromatograph is equipped with a 205-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The flow rate is about 1 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the resolution, R, between sisomicin and netilmicin is not less than 1. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency is not less than 3000 theoretical plates; the tailing factor is not more than 2; and the relative standard deviation for replicate injections is not more than 1%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, and measure the area responses for the main peaks. Calculate the quantity, in µg, of netilmicin (C21H41N5O7) per mg of Netilmicin Sulfate taken by the formula: in which WS is the dry weight, in mg, of USP Netilmicin Sulfate RS taken to prepare the Standard preparation; P is the designated potency, in µg of netilmicin (C21H41N5O7) per mg, of the USP Netilmicin Sulfate RS; WU is the dry weight, in mg, of the Netilmicin Sulfate taken to prepare the Assay preparation; and rU and rS are the netilmicin peak responses obtained from the Assay preparation and the Standard preparation, respectively.