A: Ultraviolet Absorption 197U—

Solution: 25 µg per mL.

Medium: water.

B: It responds to the tests for Sodium 191.

Test solution: a known amount of specimen, equivalent to about 50 mg of cephalothin, per mL, in water.

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

Standard solution— Use the Standard preparation, prepared as directed in the Assay, transfer 1.0 mL to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Test solution— Use the Assay preparation prepared as directed in the Assay.

Procedure— Proceed as directed for the Procedure in the Assay, except to inject equal volumes (about 20 µL) of the Standard solution and the Test solution and to continue the chromatography of the Test solution for at least 4 times the retention time of the main cephalothin peak. The area of any peak in the chromatogram obtained from the Test solution, except the main peak, is not greater than the area of the main peak in the chromatogram obtained from the Standard solution (1.0%), and the sum of the areas of any such peaks is not greater than 3 times the area of the main peak in the chromatogram obtained from the Standard solution (3.0%). [note—Any peak in the chromatogram obtained from the Test solution with an area less than one-tenth that of the main peak in the chromatogram obtained from the Standard solution is disregarded.]

Mobile phase— Dissolve 17 g of sodium acetate in 790 mL of water, add 0.6 mL of glacial acetic acid, and if necessary adjust with 0.1 N sodium hydroxide or glacial acetic acid to a pH of 5.9 ± 0.1. Add 150 mL of acetonitrile and 70 mL of alcohol, and mix. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Cephalothin Sodium RS quantitatively in Mobile phase to obtain a solution having a known concentration of about 1 mg per mL.

Resolution solution— Heat a 5-mL portion of the Standard preparation in a water bath at 90 for 10 minutes. Cool the solution, and immediately inject a portion of it into the chromatograph as directed under Chromatographic system.

Assay preparation— Transfer about 25 mg of Cephalothin Sodium, accurately weighed, to a 25-mL volumetric flask, add about 15 mL of Mobile phase, swirl to dissolve, 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 4.6-mm × 25-cm column that contains 5 µm packing L1 and is maintained at a constant temperature of about 40. The flow rate is about 1 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed under Procedure: the resolution between the two principal peaks is not less than 9.0. Chromatograph the Standard preparation, and record the peak responses as directed under Procedure: the tailing factor is not more than 1.8, and the relative standard deviation for replicate injections is not more than 1.0%.

Procedure— [note—Use peak areas where peak responses are indicated.] Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in µg, of cephalothin (C16H16N2O6S2) in each mg of Cephalothin Sodium taken by the formula: in which C is the concentration, in mg per mL, of USP Cephalothin Sodium RS in the Standard preparation; P is the assigned potency, in µg of cephalothin per mg, of USP Cephalothin Sodium RS; W is the quantity, in mg, of Cephalothin Sodium taken to prepare the Assay preparation; and rU and rS are the cephalothin peak responses obtained from the Assay preparation and the Standard preparation, respectively.