Mobile phase— Prepare a suitable filtered and degassed mixture of water, acetonitrile, 1 M monobasic potassium phosphate, and 1 N acetic acid (909:80:10:1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Diluent— Mix 10 mL of 1 M monobasic potassium phosphate and 1 mL of 1 N acetic acid, dilute with water to 1000 mL, and mix.

Standard preparation— Dissolve a suitable quantity of USP Ampicillin RS, accurately weighed, in Diluent to obtain a solution having a known concentration of about 1 mg per mL, using shaking and sonication, if necessary, to achieve complete dissolution. Use this solution promptly after preparation.

Assay preparation— Transfer an accurately weighed quantity of Ampicillin, equivalent to about 100 mg of anhydrous ampicillin, to a 100-mL volumetric flask, add about 75 mL of Diluent, shake and sonicate, if necessary, to achieve complete dissolution, dilute with Diluent to volume, and mix. Use this solution promptly after preparation.

Resolution solution— Dissolve caffeine in Standard preparation to obtain a solution containing about 0.12 mg per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector, a 4-mm × 5-cm pre-column containing 5- to 10-µm packing L1, and a 4-mm × 30-cm analytical column containing 5- to 10-µm 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 the caffeine and the ampicillin peaks is not less than 2.0. The relative retention times are about 0.5 for ampicillin and 1.0 for caffeine. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the capacity factor, k¢, is not more than 2.5, the tailing factor is not more than 1.4, and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— 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 ampicillin (C13H19NO4S) in each mg of the Ampicillin taken by the formula: in which C is the concentration, in mg per mL, of USP Ampicillin RS in the Standard preparation; P is the potency, in µg of ampicillin per mg, of USP Ampicillin RS; W is the weight, in mg, of Ampicillin taken; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.