A: Infrared Absorption 197K.

B: The retention time of the major peak for cefaclor in the chromatogram of the Assay corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.

Solvent— Dissolve 2.4 g of monobasic sodium phosphate in 1000 mL of water, and adjust with phosphoric acid to a pH of 2.5.

Blank solution— Use the Solvent.

Solution A— Dissolve 6.9 g of monobasic sodium phosphate in 1000 mL of water, and adjust with phosphoric acid to a pH of 4.0.

Solution B— Prepare a mixture of Solution A and acetonitrile (550:450), degassing for not more than 2 minutes.

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621). [note—Reducing the acetonitrile content increases the retention time of cefaclor and increases the resolution between cefaclor, delta-3 isomer and cefaclor.]

Standard solution— Dissolve an accurately weighed quantity of USP Cefaclor RS in Solvent to obtain a solution having a known concentration of about 0.05 mg per mL. Sonicate briefly, if necessary, to dissolve, and avoid heating. [note—Use this solution on the day it is prepared.]

System suitability solution— Dissolve a quantity of USP Cefaclor, Delta-3 Isomer RS in the Standard solution to obtain a solution having a concentration of about 0.05 mg per mL.

Test solutions— Transfer about 50 mg of Cefaclor, accurately weighed, to each of two 10-mL volumetric flasks, dilute each with Solvent to volume, and mix. Sonicate briefly, if necessary, to dissolve, and avoid heating. [note—Use these Test solutions within 2 hours when stored at room temperature or within 20 hours when stored under refrigeration.]

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1 mL per minute. The chromatograph is programmed as follows. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the retention time for the cefaclor peak is between 23 and 29 minutes; the resolution, R, between cefaclor, delta-3 isomer and cefaclor is not less than 2.0; and the tailing factor for the cefaclor peak is not more than 1.2. Chromatograph the Blank solution as directed for Procedure. Examine the chromatogram for any extraneous peaks, and disregard any corresponding peaks observed in the chromatogram of the Test solutions. [note—Ensure that any extraneous peaks observed do not represent carryover from previous injections.]

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solutions into the chromatograph, record the chromatograms, and measure all of the peak areas. Calculate the percentage of each cefaclor related compound in the portion of Cefaclor taken by the formula: in which C is the concentration, in mg per mL, of USP Cefaclor RS in the Standard solution; P is the designated potency, in µg per mg, of USP Cefaclor RS; W is the weight, in mg, of the portion of Cefaclor taken to prepare the respective Test solution; ri is the peak response of an individual related compound in the chromatogram obtained from the Test solution; and rS is the peak response for the cefaclor peak in the chromatogram of the Standard solution. Determine the mean values for each cefaclor related compound: not more than 0.5% of any individual cefaclor related compound is found, and not more than 2.0% of total cefaclor related compounds is found. In an acceptable determination, the difference between duplicate determinations of total cefaclor related compounds is not more than 0.2% absolute, or the variation from the mean of the two values is not more than 10%, whichever is greater.

Mobile phase— Dissolve 1 g of sodium 1-pentanesulfonate in a mixture of 780 mL of water and 10 mL of triethylamine. Adjust with phosphoric acid to a pH of 2.5 ± 0.1, add 220 mL of methanol, and mix. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Transfer about 15 mg of USP Cefaclor RS, accurately weighed, to a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix. Sonicate briefly, if necessary, to achieve dissolution, and avoid heating the solution. [note—Use this Standard preparation within 8 hours if stored at room temperature, or within 20 hours if stored under refrigeration.]

Assay preparation— Transfer about 15 mg of Cefaclor, accurately weighed, to a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix. Sonicate briefly, if necessary, to achieve dissolution, and avoid heating the solution. [note—Use this Assay preparation within 8 hours if stored at room temperature, or within 20 hours if stored under refrigeration.]

Resolution solution— Prepare a solution in Mobile phase containing about 0.3 mg of cefaclor and 0.3 mg of USP Cefaclor, Delta-3 Isomer RS per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 265-nm detector and a 4.6-mm × 25-cm column containing 5-µm packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the Resolution solution, and record the responses as directed for Procedure: the relative retention times for cefaclor and cefaclor, delta-3 isomer are about 0.8 and 1.0, the resolution, R, between the cefaclor peak and the cefaclor, delta-3 isomer peak is not less than 2.5, the tailing factor is not more than 1.5, and the relative standard deviation for replicate injections is not more than 2%.

Procedure— [note—Use peak areas where peak responses are indicated.] Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the potency, in µg per mg, of cefaclor (C15H14ClN3O4S) in each mg of the Cefaclor taken by the formula: in which WS and WU are the weights, in mg, of USP Cefaclor RS and of Cefaclor taken to prepare the Standard preparation and the Assay preparation, respectively; P is the designated potency, in µg of cefaclor (C15H14ClN3O4S) per mg, of USP Cefaclor RS; and rU and rS are the peak responses of the cefaclor peaks obtained from the Assay preparation and the Standard preparation, respectively.