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

B: A solution of it responds to the tests for Potassium 191.

Mobile phase— Prepare 0.1 M monobasic sodium phosphate, adjust with phosphoric acid to a pH of 4.0 ± 0.1, and pass through a membrane filter having a 0.5-µm or finer porosity. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Dissolve USP Clavam-2-Carboxylate Potassium RS in water to obtain a solution having a known concentration of about 5 µg per mL.

Test solution— Transfer about 100 mg of Clavulanate Potassium, accurately weighed, to a 10-mL volumetric flask, dissolve in and dilute with water to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 210-nm detector and a 4-mm × 30-cm column that contains 3- to 10-µm packing L1. The flow rate is about 0.5 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the column efficiency is not less than 4000 theoretical plates; the tailing factor is not more than 1.5; and the relative standard deviation for replicate injections is not more than 5%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The relative retention times are about 0.7 for clavam-2-carboxylic acid and 1.0 for clavulanic acid. Calculate the percentage of clavam-2-carboxylate potassium in the specimen taken by the formula: in which C is the concentration, in µg per mL, of clavam-2-carboxylate potassium in the Standard solution; W is the quantity, in mg, of Clavulanate Potassium taken to prepare the Test solution; and rU and rS are the clavam-2-carboxylic acid peak responses obtained from the Test solution and the Standard solution, respectively: not more than 0.01% is found.

Internal standard solution— Dissolve 50 µL of 3-methyl-2-pentanone in water, dilute with water to 100.0 mL, and mix.

Test solution— Transfer 1.0 g of Clavulanate Potassium to a centrifuge tube, add 5.0 mL of Internal standard solution, 5.0 mL of 2 N sodium hydroxide, 5.0 mL of isopropyl alcohol, and 5 g of sodium chloride. Shake for 1 minute, and centrifuge to separate the layers. Use the upper layer.

Reference solution— Dissolve 80.0 mg of each of the following amines in 2 N hydrochloric acid: 1,1-dimethylethylamine, diethylamine, tetramethylethylenediamine, 1,1,3,3-tetramethylbutylamine, and N,N¢-diisopropylethylenediamine. Dilute with 2 N hydrochloric acid to 200.0 mL, and mix. Transfer 5.0 mL of this solution to a centrifuge tube. Add 5.0 mL of Internal standard solution, 10.0 mL of 2 N sodium hydroxide, 5.0 mL of isopropyl alcohol, and 5 g of sodium chloride. Shake for 1 minute, and centrifuge to separate the layers. Use the upper layer.

Chromatographic system (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector and a 0.53-mm × 50-m capillary fused silica column that contains a 5-µm film coating of stationary phase G41. The temperatures of the column, the injection port, and the detector block are programmed as follows. The carrier gas is helium flowing at a constant rate of about 8 mL per minute. The split ratio is 1:10. Chromatograph the Reference solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.55 for 1,1-dimethylethylamine, 0.76 for diethylamine, 1.0 for isopropyl alcohol, 1.07 for tetramethylethylenediamine, 1.13 for 1,1,3,3-tetramethylbutylamine, 1.33 for N,N¢-diisopropylethylenediamine, and 1.57 for bis(2-methylamino)ethyl ether.

Procedure— Separately inject equal volumes (about 1 µL) of the Test solution and the Reference solution into the chromatograph, record the chromatograms, and measure the peak area responses. Calculate the percentage of each impurity in the Clavulanate Potassium taken by the formula: in which ri is the peak response for an individual impurity observed in the chromatogram obtained from the Test solution; and rS is the response for the peak corresponding to the relevant analyte in the chromatogram obtained from the Reference solution. Calculate the percentage of any individual impurity for which no relevant reference compound is provided in the Reference solution by the same formula, except for rS use the peak response corresponding to the 1,1-dimethylethylamine peak. The total of all aliphatic amines is not more than 0.2%.

Internal standard solution— Prepare a solution of 3-cyclohexylpropionic acid in cyclohexane containing 1 mg per mL.

Standard solution— Transfer about 75 mg of 2-ethylhexanoic acid, accurately weighed, to a 50-mL volumetric flask, dilute with Internal standard solution to volume, and mix. Transfer 1.0 mL of this solution to a centrifuge tube, and add 4.0 mL of 4 N hydrochloric acid. Shake for 1 minute, and allow the phases to separate, centrifuging if necessary. Withdraw the lower phase, and reserve the upper phase. To the lower phase add 1.0 mL of Internal standard solution, and shake for 1 minute. Allow the phases to separate, centrifuging if necessary. Withdraw the upper phase, and combine with the reserved upper layer.

Test solution— Transfer about 300 mg of Clavulanate Potassium, accurately weighed, to a centrifuge tube. Add 4.0 mL of 4 N hydrochloric acid, and shake with two successive 1.0 mL portions of Internal standard solution. Allow the phases to separate, centrifuging if necessary. Use the combined upper phases.

Chromatographic system (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector and a 0.53-mm × 25-m capillary fused silica column that contains a 1-µm film coating of stationary phase G35. The temperatures of the column, the injection port, and the detector block are programmed as follows. The carrier gas is hydrogen flowing at a constant rate of about 100 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the resolution, R, between the 2-ethylhexanoic acid peak and the 3-cyclohexylpropionic acid peak is not less than 2.0.

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 peak area responses. Calculate the percentage of 2-ethylhexanoic acid in the Clavulanate Potassium taken by the formula: in which WS is the weight, in mg, of 2-ethylhexanoic acid taken to prepare the Standard solution; WU is the weight, in mg, of Clavulanate Potassium taken to prepare the Test solution; and RU and RS are the ratios of the 2-ethylhexanoic acid peak to the 3-cyclohexylpropionic acid peak observed in the chromatograms obtained from the Test solution and the Standard solution, respectively. Not more than 0.8% is found.

Solution A— Prepare a solution of 0.05 M monobasic sodium phosphate, adjust with phosphoric acid to a pH of 4.0 ± 0.1, and pass through a filter having a 0.5-µm or finer porosity.

Solution B— Prepare a mixture of Solution A and methanol (50:50).

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments to either Solution as necessary (see System Suitability under Chromatography 621).

Standard solution— Prepare a solution of USP Clavulanate Lithium RS in Solution A having a known concentration of about 0.1 mg per mL.

Test solution— Prepare a solution of Clavulanate Potassium in Solution A containing 10.0 mg per mL.

Resolution solution— Prepare a solution of USP Clavulanate Lithium RS and amoxicillin in Solution A containing 0.1 mg of each per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 230-nm detector and a 4.6-mm × 10-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. The system is programmed to provide a mobile phase consisting of variable mixtures of Solution A and Solution B. The system is equilibrated for 15 minutes with 100% Solution A, and held at that composition for 4 minutes after injection of the solution under test, after which the proportion of Solution B is increased linearly from 0% to 50% over a period of 11 minutes. The system is held at that composition for 3 minutes, and is then changed to 100% Solution A for 6 minutes. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the relative retention times are about 1.0 for clavulanic acid and 2.5 for amoxicillin; the tailing factor for the clavulanic acid peak is not more than 2.0; the column efficiency determined from the clavulanic acid peak is not less than 2000 theoretical plates; and the resolution, R, between the clavulanic acid peak and the amoxicillin peak is not less than 13. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage, in terms of clavulanate potassium equivalent, of each impurity in the Clavulanate Potassium taken by the formula: in which 237.3 is the molecular weight of clavulanate potassium; 205.1 is the molecular weight of clavulanate lithium; C is the concentration, in mg per mL, of USP Clavulanate Potassium RS in the Standard solution; ri is the peak response of an individual impurity peak in the chromatogram obtained from the Test solution; and rS is the clavulanic acid peak response in the chromatogram obtained from the Standard solution. The sum of all the impurity peaks is not greater than 2%.

(Official January 1, 2007)

pH 4.4 sodium phosphate buffer— Dissolve 7.8 g of monobasic sodium phosphate in 900 mL of water, adjust with phosphoric acid or 10 N sodium hydroxide to a pH of 4.4 ± 0.1, dilute with water to make 1000 mL, and mix.

Mobile phase— Prepare a suitable mixture of pH 4.4 sodium phosphate buffer and methanol (95:5), and pass through a membrane filter having a 0.5-µm or finer porosity. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Clavulanate Lithium RS in water to obtain a solution having a known concentration of about 0.25 mg per mL.

Assay preparation— Transfer about 50 mg of Clavulanate Potassium, accurately weighed, to a 200-mL volumetric flask, dissolve in and dilute with water to volume, and mix.

Resolution solution— Dissolve a suitable quantity of amoxicillin in Standard preparation to obtain a solution containing about 0.5 mg of amoxicillin and 0.25 mg of clavulanate lithium per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 220-nm detector and a 4-mm × 30-cm column that contains 3- to 10-µm packing L1. The flow rate is about 2 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency determined from the analyte peak is not less than 550 theoretical plates; the tailing factor for the analyte peak is not more than 1.5; and the relative standard deviation for replicate injections is not more than 2.0%. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.5 for clavulanic acid and 1.0 for amoxicillin; and the resolution, R, between the amoxicillin and clavulanic acid peaks is not less than 3.5.

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 clavulanic acid (C8H9NO5) in each mg of the Clavulanate Potassium taken by the formula: in which C is the concentration, in mg per mL, of USP Clavulanate Lithium RS in the Standard preparation; P is the designated potency, in µg of clavulanic acid per mg, of USP Clavulanate Lithium RS; W is the quantity, in mg, of Clavulanate Potassium taken to prepare the Assay preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.