(Official January 1, 2007)

Mobile phase— Prepare a mixture of water, methanol, and glacial acetic acid (68:30:2). Make adjustments if necessary (see System Suitability under Chromatography 621).

Extractant— Prepare a mixture of 0.15 N hydrochloric acid and methanol (3:1).

Standard preparation— Prepare a solution of USP Sulfamethazine RS in Extractant having a known concentration of about 0.01 mg per mL.

Assay preparation— Transfer about 5 g of Sulfamethazine Granulated, accurately weighed, to a suitable container, add 250.0 mL of Extractant, and shake by mechanical means for 2 hours. Allow the mixture to settle, storing the mixture in a refrigerator if settling is allowed to continue overnight. Filter a portion of the supernatant, and transfer 10.0 mL of the clear filtrate to a 100-mL volumetric flask. Dilute with Extractant to volume, and mix. Transfer 5.0 mL of this solution to a 200-mL volumetric flask, dilute with Extractant to volume, and mix. Filter a portion of this solution through a filter having a porosity of 0.5 µm or finer, and use the filtrate as the Assay preparation. This solution contains about 0.01 mg of sulfamethazine per mL.

Derivatizing reagent— Dissolve 6.0 g of dimethylaminobenzaldehyde in 200 mL of glacial acetic acid, add 120 mL of methanol and 80 mL of water, mix, and degas. Prepare this reagent daily.

Chromatographic system— The liquid chromatograph is equipped with a guard column that contains packing L1, a 4.6-mm × 25-cm analytical column that contains packing L1, a separate pump to deliver the Derivatizing reagent via a T-junction installed immediately post-column, a post-column derivatization coil consisting of 3-m × 0.5-mm inside-diameter polytef tubing, a flow cell, and a 450-nm detector. The Mobile phase flow rate is about 2 mL per minute, and the Derivatizing reagent flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the capacity factor, k¢, for the sulfamethazine peak is not less than 2.0, and the relative standard deviation for replicate injections is not more than 3.5%.

Procedure— Separately inject equal volumes (about 100 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the sulfamethazine peaks. Calculate the quantity, in mg, of sulfamethazine (C12H14N4O2S) in each g of the Sulfamethazine Granulated taken by the formula: in which C is the concentration, in mg per mL, of USP Sulfamethazine RS in the Standard preparation, W is the quantity, in g, of Sulfamethazine Granulated taken to prepare the Assay preparation, and rU and rS are the sulfamethazine peak responses obtained from the Assay preparation and the Standard preparation, respectively.