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All entries in this edition are legally effective

Co-amoxiclav Injection
General Notices

Amoxicillin and Potassium Clavulanate Injection

Action and use

Penicillin antibacterial + beta-lactamase inhibitor.

DEFINITION

Co-amoxiclav Injection is a sterile solution of Amoxicillin Sodium and Potassium Clavulanate in Water
for Injections. It is prepared by dissolving Co-amoxiclav for Injection in the requisite amount of Water for
Injections immediately before use.

The injection complies with the requirements stated under Parenteral Preparations.

STORAGE

Co-amoxiclav Injection should be used immediately after preparation.

CO-AMOXICLAV FOR INJECTION

DEFINITION

Co-amoxiclav for Injection is a sterile material consisting of Amoxicillin Sodium and Potassium
Clavulanate with or without excipients. It is supplied in a sealed container.

PRODUCTION
The methods of production, extraction and purification of Potassium Clavulanate used in the formulation
of Co-amoxiclav Injection are such that potassium clavam-2-carboxylate is eliminated or present at a
level not exceeding 0.01%.

The contents of the sealed container comply with the requirements for Powders for Injections or
Infusions stated under Parenteral Preparations and with the following requirements.

Content of amoxicillin, C16 H19 N3 O5 S

90.0 to 107.5% of the stated amount.

Content of clavulanic acid, C8 H9 NO5

90.0 to 107.5% of the stated amount.

IDENTIFICATION

A. Carry out the method for thin-layer chromatography, Appendix III A, using the following solutions.
(1) Shake a quantity of the contents of the sealed container containing the equivalent of 0.4 g of
clavulanic acid in 100 mL of a mixture of 4 volumes of methanol and 6 volumes of 0.1M mixed
phosphate buffer pH 7.0 and filter.
(2) 0.4% w/v of lithium clavulanate EPCRS and 0.8% w/v of amoxicillin trihydrate BPCRS in a
mixture of 4 volumes of methanol and 6 volumes of 0.1M mixed phosphate buffer pH 7.0.

CHROMATOGRAPHIC CONDITIONS

(a) Use as the coating silica gel F254 (Merck silica gel 60 F254 plates are suitable). Impregnate the
plate by spraying it with a 0.1% w/v solution of disodium edetate in mixed phosphate buffer pH 4.0 and
allow to dry overnight. Activate the plate by heating at 105° for 1 hour prior to use.
(b) Use the mobile phase as described below.
(c) Apply 1 µL of each solution.
(d) Develop the plate to 15 cm.
(e) After removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm).

MOBILE PHASE

1 volume of butan-1-ol, 2 volumes of a 0.1% w/v solution of disodium edetate in mixed phosphate
buffer pH 4.0, 6 volumes of glacial acetic acid and 10 volumes of butyl acetate.

CONFIRMATION

The principal spots in the chromatogram obtained with solution (1) are similar in position and colour to
those in the chromatogram obtained with solution (2).
B. In the Assay, the retention time of the two principal peaks in the chromatogram obtained with
solution (1) correspond to those in the chromatogram obtained with solution (2).

TESTS

Alkalinity

pH of a solution containing the equivalent of 10% w/v of amoxicillin, 8.0 to 10.0, Appendix V L.

Clavulanate polymer and other fluorescent impurities

Carry out the method for fluorescence spectrophotometry, Appendix II E, using the following freshly
prepared solutions.

(1) To a quantity of the contents of a sealed container containing the equivalent of 0.1 g of clavulanic
acid add 50 mL of a 0.1M phosphate buffer solution pH 5.0, prepared as described below, shake
vigorously for 1 minute and then shake with the aid of ultrasound for 5 minutes; add sufficient of the
buffer solution to produce 100 mL and filter through a 0.45-µm filter. To prepare the buffer solution
dissolve 15.6 g of sodium dihydrogen orthophosphate in 800 mL of water, adjust the pH to 5.0 using 1M
sodium hydroxide and add sufficient water to produce 1000 mL.
(2) Prepare a solution containing 0.42 µg per mL of quinine sulfate BPCRS in 0.5M sulfuric acid. [
NOTE: The fluorescence of quinine sulfate is 118 times more intense than that of an equivalent
concentration of clavulanate polymer.]

PROCEDURE

Measure the fluorescence of the solutions using an excitation wavelength of 360 nm and an emission
wavelength of 440 nm, using the phosphate buffer solution in the reference cell.

LIMITS

The fluorescence obtained with solution (1) is not more intense than that obtained with solution (2) (5%
w/w, calculated with respect to the content of clavulanic acid).

Related substances

Carry out the method for liquid chromatography, Appendix III D, using the following freshly prepared
solutions.

(1) Dilute a quantity of the contents of a sealed container containing the equivalent of 0.1 g of
amoxicillin in sufficient mobile phase A to produce 200 mL.
(2) 0.0057% w/v of amoxicillin trihydrate BPCRS in mobile phase A.
(3) 0.05% w/v of amoxicillin impurity standard BPCRS in mobile phase A.

CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (5 cm × 4.6 mm) packed with octadecylsilyl silica gel for
chromatography (3 µm) (Spherisorb S3 ODS2 is suitable).
(b) Use gradient elution and the mobile phase described below.
(c) Use a flow rate of 1.5 mL per minute.
(d) Use a column temperature of 20°.
(e) Use a detection wavelength of 230 nm.
(f) Inject 20 µL of each solution.

MOBILE PHASE

Mobile phase A Dissolve 15.6 g of sodium dihydrogen orthophosphate in 1000 mL of water and
adjust the pH to 4.2 with orthophosphoric acid.

Mobile phase B Mix 10 volumes of mobile phase A with 90 volumes of methanol.

When the chromatograms are recorded under the prescribed conditions, the retention times relative to
Amoxicillin (retention time, about 3 minutes) are: impurity D, about 0.8; impurity J, about 3.0 and 3.2.

SYSTEM SUITABILITY

The test is not valid unless the chromatogram obtained with solution (3) resembles the chromatogram
supplied with amoxicillin impurity standard BPCRS and the resolution between the peaks due to
amoxicillin and impurity D is at least 2.0.

LIMITS

In the chromatogram obtained with solution (1):

the area of any peak due to impurity D is not greater than half the area of the principal peak in the
chromatogram obtained with solution (2) (5%);

the area of any peak due to impurity J (the peak may appear as a doublet) is not greater than half the
area of the principal peak in the chromatogram obtained with solution (2) (5%);

the area of any other secondary peak is not greater than 0.3 times the area of the principal peak in the
chromatogram obtained with solution (2) (3%);

the sum of the areas of all the secondary peaks is not greater than 1.5 times the area of the principal
peak in the chromatogram obtained with solution (2) (15%).
Bacterial endotoxins

Carry out the test for bacterial endotoxins, Appendix XIV C. Dissolve the contents of the sealed
container in water BET to give a solution containing the equivalent of 10 mg per mL of amoxicillin
(solution A). The endotoxin limit concentration of solution A is 2.5 IU of endotoxin per mL.

Water

Not more than 3.5% w/w, Appendix IX C. Use 0.5 g.

ASSAY

Determine the weight of the contents of 10 containers as described in the test for uniformity of weight,
Appendix XII C1, Powders for Parenteral Administration.

Carry out the method for liquid chromatography, Appendix III D, using the following solutions.

(1) Dissolve, with shaking, a quantity of the mixed contents of the 10 containers containing the
equivalent of 0.1 g of amoxicillin in sufficient water to produce 100 mL, mix and filter.
(2) 0.11% w/v of amoxicillin trihydrate BPCRS and 0.02% w/v of lithium clavulanate EPCRS in
water.

CHROMATOGRAPHIC CONDITIONS

(a) Use a stainless steel column (25 cm × 4.6 mm) packed with octadecylsilyl silica gel for
chromatography (5 µm) (Hypersil ODS is suitable).
(b) Use isocratic elution and the mobile phase described below.
(c) Use a flow rate of 1 mL per minute.
(d) Use an ambient column temperature.
(e) Use a detection wavelength of 230 nm.
(f) Inject 10 µL of each solution.

MOBILE PHASE

80 volumes of methanol and 920 volumes of a 1.56% w/v solution of sodium dihydrogen
orthophosphate in water, the pH of which has been adjusted to 4.0 with orthophosphoric acid.

SYSTEM SUITABILITY

The Assay is not valid unless, in the chromatogram obtained with solution (2), the resolution between
the peaks due to amoxicillin and lithium clavulanate is at least 8.0.

DETERMINATION OF CONTENT
Calculate the content of C16 H19 N3 O5 S and C8 H9 NO5 in a container of average content weight using the
declared content of C16 H19 N3 O5 S in amoxicillin trihydrate BPCRS and the declared content of
C8 H8 LiNO5 in lithium clavulanate EPCRS. Each mg of C8 H8 LiNO5 is equivalent to 0.9711 mg of
C8 H9 NO5 .

LABELLING

The label of the sealed container states the quantity of Amoxicillin Sodium contained in it, in terms of the
equivalent amount of amoxicillin, and the quantity of Potassium Clavulanate, in terms of the equivalent
amount of clavulanic acid.

The label of the sealed container states that the preparation contains penicillin.

IMPURITIES

The impurities limited by the requirements of this monograph include impurities D and J listed under
Amoxicillin Sodium.