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Category Beta-lactamase inhibitor.

           Clavulanate Potassium contains the equivalent of not less than 75.5 per cent and not more than 92.0 per cent of C₈H₉NO₅, calculated on the anhydrous basis.

Packaging and storage Clavulanate Potassium shall be kept in tightly closed containers and stored at a temperature between 2º and 8º.

Labelling The label on the container states (1) storage condition; (2) parenteral or non-parenteral grade.

Identification
          A. The chromatogram of the Assay preparation obtained as directed in the Assay exhibits a major peak for clavulanic acid, the retention time of which corresponds to that exhibited in the chromatogram of the Standard preparation obtained as directed in the Assay.
          B. It yields the reactions characteristic of potassium salts (Appendix 5.1).

pH 5.5 to 8.0, in a 1.0 per cent w/v solution (Appendix 4.11).

Water Not more than 1.5 per cent w/w (Karl Fischer Method, Appendix 4.12).

Limit of clavam-2-carboxylate potassium Not more than 0.01 per cent. Carry out the determination as described in the “High-pressure Liquid Chromatography” (Appendix 3.5).
          Mobile phase Prepare 0.1 M sodium dihydrogenphosphate, adjust with phosphoric acid to a pH of 4.0±0.1, and filter through a membrane filter of 0.5 μm or finer porosity. Make adjustments if necessary. Standard solution Dissolve Clavam-2-Carboxylate Potassium RS in water to obtain a solution having a known concentration of about 40 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.
          Resolution solution Dissolve a suitable quantity of Clavulanate Potassium in Standard solution to obtain a solution containing about 1 mg of clavulanate potassium and 30 μg of clavam-2-carboxylate potassium per ml.
          Chromatographic system The chromatographic procedure may be carried out using (a) a stainless steel column (30 cm × 4 mm) packed with octadecylsilane chemically bonded to porous silica or ceramic microparticles (3 to 10 μm) (b) Mobile phase at a flow rate of 0.5 ml per minute, and (c) an ultraviolet photometer set at 210 nm.
          To determine the suitability of the chromatographic system, chromatograph Standard solution, and record the peak responses as directed under Procedure: the symmetry factor is not more than 1.5 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Chromatograph Resolution solution, and record the peak responses as directed under Procedure: the resolution factor between the clavam-2-carboxylic acid and clavulanic acid peaks is not less than 1.0.
          Procedure Separately inject equal volumes (about 20 μl) of Standard solution and 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 

Limit of methanol and tert-butylamine Not more than 0.1 per cent w/w of methanol or not more than 0.2 per cent w/w of tert-butylamine. Carry out the determination as described in the “Gas Chromatography” (Appendix 3.4).
          Standard solution To a 100-ml volumetric flask containing 50.0 ml of 3 M sodium hydroxide add about 6 ml of methanol and 12 ml of tert-butylamine, both accurately weighed, dilute with 3 M sodium hydroxide to
volume, and mix. Transfer 10.0 ml of this stock solution to a second 100-ml volumetric flask, dilute with 3 M sodium hydroxide to volume, and mix. Transfer 10.0 ml of this solution to a third 100-ml volumetric flask, dilute with 3 M sodium hydroxide to volume, and mix. Transfer 7.0 ml of this solution to a fourth 100-ml volumetric flask, add 3.0 ml of 3 M sodium hydroxide, dilute with 4-methyl-2-pentanone to volume, and mix. Allow the phases to separate, and use the clear methyl isobutyl ketone layer as the Standard solution. This solution contains about 36.6 μg of methanol and 63.8 μg of tertbutylamine per ml. Use this solution within 5 hours.
          Test solution Transfer about 3 g of Clavulanate Potassium, accurately weighed, to a 100-ml volumetric flask, add 10.0 ml of 3 M sodium hydroxide, and shake until dissolved. Allow to cool in a cold water-bath, dilute with 4-methyl-2-pentanone to volume, stopper the flask, and shake vigorously for 3 minutes, with occasional venting. Allow the phases to separate, and use the clear methyl isobutyl ketone layer as the Test solution. Use this solution within 5 hours.
          Chromatographic system The chromatographic procedure may be carried out using (a) a glass column (30 m × 0.32 mm) coated with dimethylpolysiloxane oil stationary phase and is maintained at 40º, then programmed to increase at a rate of 55º per minute to 200º, and held at that temperature for 4 minutes, (b) the injection port and the detector block are maintained at 150º, (c) nitrogen as the carrier gas, and (d) a flame ionization detector. To determine the suitability of the chromatographic system, chromatograph Standard solution, and record the methanol, tert-butylamine, and methyl isobutyl ketone peak responses as directed for Procedure: the symmetry factor is not more than 1.6 for the methanol and tert-butylamine peaks; the column efficiency is not less than 25,000 theoretical plates; the resolution factor between the methanol peak and the tert-butylamine peak is not less than 20 and between the tert-butylamine peak and the 4-methyl-2-pentanone peak is not less than 10; and the relative standard deviation for replicate injections is not more than 10 per cent.

          Procedure Separately inject equal volumes (about 1 μl) of Standard solution and Test solution into the chromatograph, record the chromatograms, and measure the areas of the responses of the methanol and tert-butylamine peaks.
          Calculation Calculate the percentages of methanol and of tert-butylamine in the Clavulanate Potassium.
Chromatographic purity Carry out the determination as described in the “High-pressure Liquid Chromatography” (Appendix 3.5).
          Solution A Prepare a solution of 0.05 M sodium dihydrogenphosphate, adjust with phosphoric acid to a pH of 4.0±0.1, and filter through a filter of 0.5-μm or finer porosity.
          Solution B Prepare a mixture of equal volumes of Solution A and methanol.
          Mobile phase Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments to either solution as necessary.
          Standard solution Prepare a solution of 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 Clavulanate Lithium RS and amoxicillin in Solution A containing 0.1 mg of each per ml.
          Chromatographic system The chromatographic procedure may be carried out using (a) a stainless steel column (10 cm × 4.6 mm) packed with octadecylsilane chemically bonded to porous silica or ceramic microparticles (3 to 10 μm) and is maintained at a constant temperature of about 40º, (b) Mobile phase at a flow rate of about 1 ml per minute, and (c) an ultraviolet photometer set at 230 nm.
          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 per cent 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 per cent over a period of 11 minutes. The system is held at that composition for 3 minutes, and is then changed to 100 per cent Solution A for 6 minutes. To determine the suitability of the chromatographic system, chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the relative retention times are about 2.5 for amoxicillin and 1.0 for clavulanic acid; the symmetry 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 factor between the clavulanic acid peak and the amoxicillin peak is not less than 13. Chromatograph Standard solution, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2 per cent.
          Procedure Separately inject equal volumes (about 20 μl) of Standard solution and Test solution into the chromatograph, record the chromatograms, and measure the peak responses.
          Calculation Calculate the percentage, in terms of clavulanate potassium equivalent, of each impurity in the Clavulanate Potassium taken by the expression:

10(237.3/205.1)(C)(r_i/r_S),

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 Clavulanate Potassium RS in Standard solution; ri is the peak response of an individual impurity peak in the chromatogram obtained from Test solution; and rS is the clavulanic acid peak response in the chromatogram obtained from Standard solution. The sum of all the impurity peaks is not more than 2 per cent.

Assay Carry out the determination as described in the “High-pressure Liquid Chromatography” (Appendix 3.5).
          pH 4.4 Sodium phosphate buffer Dissolve 7.8 g of sodium dihydrogenphosphate in 900 ml of water, adjust with phosphoric acid or 10 M 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 95 volumes of pH 4.4 sodium phosphate buffer and 5 volumes of methanol. Make adjustments if necessary.
          Standard preparation Dissolve an accurately weighed quantities of Clavulanate Lithium RS in water to obtain a solution having a known concentration of about 250 μg 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 250 μg of clavulanate lithium per ml.
          Chromatographic system The chromatographic procedure may be carried out using (a) a stainless steel column (30 cm × 4 mm) packed with octadecylsilane chemically bonded to porous silica or ceramic microparticles (3 to 10 μm), (b) Mobile phase at a flow rate of about 2 ml per minute, and (c) an ultraviolet photometer set at 220 nm.
          To determine the suitability of the chromatographic system, chromatograph Resolution solution, and record the peak responses as directed under Procedure: the resolution factor between the amoxicillin and clavulanic acid peaks is not less than 3.5 and the relative retention times are about 0.5 for clavulanic acid and 1.0 for amoxicillin. Chromatograph Standard preparation, and record the peak responses as directed under Procedure: the symmetry factor for clavulanic acid peak is not more than 1.5 and the relative standard deviation for replicate injections is not more than 2.0 per cent.
          Procedure Separately inject equal volumes (about 20 μl) of Standard preparation and Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks.
          Calculation Calculate the content of C₈H₉NO₅ in the Clavulanate Potassium taken, using the declared content of C₈H₉NO₅ in Clavulanate Lithium RS.