Wednesday, 15 July 2009

Monographs: Pharmaceutical substances: Amoxicillinum trihydricum - Amoxicillin trihydrate


Relative molecular mass. 419.5

Chemical name. (-)-6-[2-Amino-2-(p-hydroxyphenyl)acetamido]-3,3- dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0]heptane-2-carboxylic acid trihydrate; (2S,5R,6R)-6-[(R)-2-amino-2-(4-hydroxyphenyl)acetamido]-3,3- dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid trihydrate; 6-[[amino(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1- azabicyclo[3.2.0]heptane-2-carboxylic acid trihydrate; CAS Reg. No. 61336-70-7.

Description. A white or almost white, crystalline powder; odourless.

Solubility. Slightly soluble in water and methanol R; very slightly soluble in ethanol (~750 g/l) TS, ether R, and fatty oils; soluble in dilute acids and dilute solutions of alkali hydroxides.

Category. Antibacterial drug.

Storage. Amoxicillin trihydrate should be kept in a tightly closed container.


Amoxicillin trihydrate contains not less than 95.0% and not more than the equivalent of 102.0% of C16H19N3O5S, calculated with reference to the anhydrous substance.

Identity tests

• Either test A alone or tests B and C may be applied.

A. Carry out the examination as described under 1.7 Spectrophotometry in the infrared region. The infrared absorption spectrum is concordant with the spectrum obtained from amoxicillin trihydrate RS or with the reference spectrum of amoxicillin trihydrate.

B. Carry out the test as described under 1.14.1 Thin-layer chromatography, using silanized silica gel R3 as the coating substance and a mixture of 9 volumes of a solution containing 15.4g of ammonium acetate R in 100ml, the pH of which has been adjusted to 5.0 with glacial acetic acid R, and 1 volume of acetone R as the mobile phase. Apply separately to the plate 1 μl of each of 3 solutions in sodium hydrogen carbonate (40 g/l) TS containing (A) 2.5 mg of Amoxicillin trihydrate per ml, (B) 2.5mg of amoxicillin trihydrate RS per ml, and (C) a mixture of 2.5 mg of amoxicillin trihydrate RS and 2.5 mg of ampicillin trihydrate RS per ml. After removing the plate from the chromatographic chamber, allow it to dry in air until the solvents have evaporated. Expose the plate to iodine vapours until the spots appear and examine the chromatogram in daylight.

The principal spot obtained with solution A corresponds in position, appearance, and intensity with that obtained with solution B. The test is valid only if the chromatogram obtained with solution C shows two clearly separated spots.

C. Place about 2 mg in a test-tube (150mm × 15mm), moisten with 1 drop of water, and add about 2ml of sulfuric acid (~1760 g/l) TS. Mix the contents of the tube by swirling; the solution remains practically colourless. Place the tube in a water-bath for 1 minute; a dark yellow colour develops.

Specific optical rotation. Use a 2.0 mg/ml solution in carbon-dioxide-free water R and calculate with reference to the anhydrous substance;

Solution in hydrochloric acid and ammonia. Prepare a solution of 1.0 g in 10ml of hydrochloric acid (0.5 mol/l) VS. Prepare a second solution of 1.0 g in 10ml of ammonia (~100 g/l) TS. Examine both solutions immediately.

Neither of these solutions are more opalescent than opalescence standard TS3.

Heavy metals. Use 1.0 g for the preparation of the test solution as described under 2.2.3 Limit test for heavy metals, Procedure 3; determine the heavy metals content according to Method A; not more than 20μg/g.

Sulfated ash. Not more than 10 mg/g.

Water. Determine as described under 2.8 Determination of water by the Karl Fischer method, Method A, using about 0.1 g of Amoxicillin trihydrate; the water content is not less than 0.115 g/g and not more than 0.145 g/g.

pH value. pH of a 2mg/ml solution in carbon-dioxide-free water R, 3.5-5.5.

Related substances. Carry out the test as described under 1.14.4 High-performance liquid chromatography, using a stainless steel column (25cm × 4.6 mm) packed with particles of silica gel, the surface of which has been modified with chemically bonded octadecylsilyl groups (5μm). Prepare the following pH 5 buffer solution to be used in the mobile phases: to 250 ml of potassium dihydrogen phosphate (27.2 g/l) TS add sodium hydroxide (~80 g/l) TS until a pH of 5.0 is reached, and dilute the solution with sufficient water to produce 1000 ml. As mobile phase A use a mixture of 99 volumes of buffer solution pH 5.0 and 1 volume of acetonitrile R. As mobile phase B use a mixture of 8 volumes of buffer solution pH 5.0 and 2 volumes of acetonitrile R.

Prepare the following solutions in mobile phase A: solution (A) 1.5mg of Amoxicillin trihydrate per ml; solution (B) 0.015 mg of amoxicillin trihydrate RS per ml; and solution (C) 0.15 μg of amoxicillin trihydrate RS per ml.

Operate with a flow rate of 1.0 ml per minute. As a detector use an ultraviolet spectrophotometer set at a wavelength of about 254nm.

Using a 50-μl loop injector, inject solution B. Start the elution isocratically with the mobile phase mixture used for the equilibration. Immediately after elution of the amoxicillin peak start a linear gradient elution to reach a ratio of mobile phase A: B of 0: 100 over a period of 25 minutes. Adjust the sensitivity of the system so that the height of the principal peak is at least 50% of the full scale of the recorder. Continue the chromatography with mobile phase B for 15 minutes, then equilibrate the column for 15 minutes with the mobile phase originally used for the equilibration. The mass distribution ratio for the first peak (amoxicillin) is 1.3-2.5. Inject mobile phase A using the 50-μl loop injector and use the same elution gradient to obtain a blank. Inject solution C using the 50-μl loop injector. Adjust the system to obtain a peak with a signal-tonoise ratio of at least 3.

Using the 50-μl loop injector, inject solution A. Measure the areas of the peak responses obtained in the chromatograms from solutions A and B, and calculate the content of the related substances as a percentage. In the chromatogram obtained with solution A, the area of any peak, other than the principal peak and any peak obtained in the blank chromatogram, is not greater than that of the principal peak obtained with solution B (1%).

Assay. Dissolve about 0.06 g, accurately weighed, in sufficient water to produce 500 ml. Simultaneously, prepare a reference solution containing 0.06 g of amoxicillin trihydrate RS. Transfer 10.0 ml of one solution to a 100-ml volumetric flask and 10.0 ml of the other solution to a second 100-ml volumetric flask. To each add 10 ml of buffer borate, pH 9.0, TS and 1ml of acetic anhydride/ dioxan TS, mix, allow to stand for 5 minutes at room temperature, and dilute to volume with water. Transfer two 2.0 ml aliquots of each solution to separate stoppered test-tubes. To one tube containing the test solution, and to the other, containing the reference solution, add 10 ml of imidazole/mercuric chloride TS, mix, stopper the tubes, and place them in a water-bath at 60°C for exactly 25 minutes. Cool the tubes rapidly to 20 °C (solution A). To the remaining tubes add 10 ml of water and mix (solution B). Without delay, measure the absorbances of a 1-cm layer at the maximum at about 325 nm of both solutions A, using as a blank a mixture of 2.0ml of water and 10ml of imidazole/ mercuric chloride TS placed in the solvent cell. For solutions B use water as a blank placed in the solvent cell.

From the difference between the absorbances of solutions A and solutions B, calculate the percentage content of C16H19N3O5S by comparison with amoxicillin trihydrate RS, with reference to the anhydrous substance.

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