A: Infrared Absorption 197M.

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.

Mobile phase— Dissolve 5.75 g of monobasic ammonium phosphate in 700 mL of water, add 3.2 mL of a 40% solution of tetrabutylammonium hydroxide, 280 mL of methanol, and 25 mL of tetrahydrofuran, and mix. Adjust with phosphoric acid to a pH of 4.5 ± 0.1, pass through a filter having a 0.5-µm or finer porosity, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Quantitatively dissolve an accurately weighed quantity of USP Cefmetazole RS in Mobile phase to obtain a solution having a known concentration of about 200 µg of cefmetazole (C15H17N7O5S3) per mL. [note—Use this solution within 10 minutes.]

Resolution solution— Prepare a solution of USP Cefmetazole RS in 0.01 N sodium hydroxide containing about 1 mg per mL. Heat at 95 for 10 minutes. To 1 mL of this solution add 2 mL of Standard preparation, and dilute with Mobile phase to obtain 20 mL of solution. This solution contains cefmetazole and cefmetazole lactone (resolution compound).

Assay preparation— Transfer about 20 mg of Cefmetazole, accurately weighed, to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix. [note—Use this solution within 10 minutes.]

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 214-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 2 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the resolution, R, between cefmetazole and cefmetazole lactone is not less than 3.0. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency is not less than 1250 theoretical plates; the tailing factor is not less than 0.94 and not more than 1.6; and the relative standard deviation for replicate injections is not more than 2.0%.

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 µg, of cefmetazole (C15H17N7O5S3) in each mg of Cefmetazole taken by the formula: in which C is the concentration, in µg per mL, of cefmetazole (C15H17N7O5S3) in the Standard preparation; M is the quantity, in mg, of Cefmetazole taken to prepare the Assay preparation; and rU and rS are the cefmetazole peak responses obtained from the Assay preparation and the Standard preparation, respectively.