A: Infrared Absorption 197M.

B: It responds to the tests for Sodium 191.

Internal standard solution— Dissolve 75 mg of N,N-diethylaniline in 25 mL of 1 N hydrochloric acid, and dilute quantitatively and stepwise with water to obtain a solution containing about 30 µg per mL.

Standard preparation— Transfer 50.0 mg of N,N-dimethylaniline to a 50-mL volumetric flask, add 25 mL of 1 N hydrochloric acid, swirl to dissolve, dilute with water to volume, and mix. Transfer 2.0 mL of the resulting solution to a 100-mL volumetric flask, dilute with water to volume, and mix. To a suitable centrifuge tube add 1.0 mL of this solution, 1.0 mL of 1.25 N sodium hydroxide, 1.0 mL of Internal standard solution, and 1.0 mL of cyclohexane, shake vigorously for 1 minute, and centrifuge. Use the clear supernatant as the Standard preparation.

Test preparation— Transfer 1.0 g of Ampicillin Sodium to a suitable centrifuge tube, add 2 mL of 1.25 N sodium hydroxide, swirl to dissolve the specimen, add 1.0 mL of Internal standard solution and 1.0 mL of cyclohexane, shake vigorously for 1 minute, and centrifuge. Use the clear supernatant as the Test preparation.

Internal standard solution— Prepare a solution of dioxane in dimethyl sulfoxide containing about 2.1 mg per mL.

Standard solution— Dissolve an accurately weighed quantity of methylene chloride in Internal standard solution to obtain a solution having a known concentration of about 0.33 mg per mL.

Test solution— Dissolve about 500 mg of Ampicillin Sodium, accurately weighed, in 3.0 mL of Internal standard solution.

Chromatographic system (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector and a 1.8-m × 4-mm glass column packed with a 10% phase G39 on unsilanized support S1A. The column temperature is maintained at 65, and the injection port and the detector block temperatures are maintained at about 100 and 260, respectively. Nitrogen is used as the carrier gas at a flow rate of about 60 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 0.5 for methylene chloride and 1.0 for dioxane; the resolution, R, between the methylene chloride peak and the dioxane peak is not less than 4; and the relative standard deviation for replicate injections is not more than 5%.

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 responses for the methylene chloride and dioxane peaks. Calculate the percentage of methylene chloride in the portion of Ampicillin Sodium taken by the formula: in which C is the concentration, in mg per mL, of methylene chloride in the Standard solution; m is the quantity, in mg, of Ampicillin Sodium taken to prepare the Test solution; and RU and RS are the ratios of the response of the methylene chloride peak to that of the dioxane peak obtained from the Test solution and the Standard solution, respectively. The limit is 0.2%.

Mobile phase, Diluent, Standard preparation, Resolution solution, and Chromatographic system— Prepare as directed in the Assay under Ampicillin.

Assay preparation— [note—Ampicillin Sodium is hygroscopic. Minimize exposure to the atmosphere, and weigh promptly.] Transfer an accurately weighed quantity of Ampicillin Sodium, equivalent to about 100 mg of anhydrous ampicillin, to a 100-mL volumetric flask, dissolve in and dilute with Diluent to volume, and mix. Use this solution promptly after preparation.

Procedure— Proceed as directed for Procedure in the Assay under Ampicillin. Calculate the quantity, in µg, of ampicillin (C16H19N3O4S) in each mg of Ampicillin Sodium taken by the formula: in which W is the weight, in mg, of Ampicillin Sodium taken to prepare the Assay preparation; and the other terms are as defined in the Assay under Ampicillin.