Thursday 16 July 2009

Monographs: Pharmaceutical substances: Artemisininum - Artemisinin


C15H22O5

Relative molecular mass. 282.3

Chemical name. (3R,5aS,6R,8aS,9R,12S,12aR)-Octahydro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano[4.3-j]-1,2-benzodioxepin-10(3H)-one; CAS Reg. No. 63968-64-9.

Description. Colourless needles or a white, crystalline powder.

Solubility. Practically insoluble in water; very soluble in dichloromethane R; freely soluble in acetone R and ethyl acetate R; soluble in glacial acetic acid R, methanol R and ethanol (~750 g/l) TS.

Category. Antimalarial drug.

Storage. Artemisinin should be kept in a well-closed container and protected from light.

Requirements

Artemisinin contains not less than 97.0% and not more than the equivalent of 102.0% of C15H22O5 using Assay method A, and not less than 98.0% and not more than the equivalent of 102.0% of C15H22O5 using Assay method B, both calculated with reference to the dried substance.

Identity tests

• Either test A alone or tests B, C, and D 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 artemisinin RS or with the reference spectrum of artemisinin.

B. See the test described below under "Related substances test B". The principal spot obtained with solution D corresponds in position, appearance, and intensity with that obtained with solution E.

C. Dissolve 5 mg in about 0.5 ml of dehydrated ethanol R, add about 0.5ml of hydroxylamine hydrochloride TS2 and 0.25ml of sodium hydroxide (~80 g/l) TS. Heat the mixture in a water-bath to boiling, cool, add 2 drops of hydrochloric acid (~70 g/l) TS and 2 drops of ferric chloride (50 g/l) TS; a deep violet colour is immediately produced.

D. Dissolve 5 mg in about 0.5 ml of dehydrated ethanol R, add 1.0 ml of potassium iodide (80 g/l) TS, 2.5 ml of sulfuric acid (~100 g/l) TS and 4 drops of starch TS; a violet colour is immediately produced.

Melting range. 151 - 154°C.

Specific optical rotation. Use a 10 mg/ml solution in dehydrated ethanol R;

Sulfated ash. Not more than 1.0 mg/g.

Loss on drying. Dry to constant mass at 80 °C; it loses not more than 5.0 mg/g.

Related substances

• Either test A or test B may be applied.

A. Carry out the test as described under 1.14.4 High-performance liquid chromatography, using a stainless steel column (10cm × 4.6mm) packed with particles of silica gel, the surface of which has been modified with chemically bonded octadecylsilyl groups (μmm). The mobile phases for gradient elution consist of a mixture of acetonitrile and water, using the conditions shown in the following table:

Time
(min)

Mobile phase A
(% v/v of acetonitrile)

Mobile phase B
(% v/v of water)

Comment

0 - 17

60

40

Isocratic

17 - 30

60 → 100

40 → 0

Linear gradient

30 - 35

100 → 60

0 → 40

Return to initial conditions

35 - 45

60

40

Isocratic - re-equilibration

Prepare the following solutions. For solution (A) use 10 mg of Artemisinin per ml in a mixture of 8 volumes of acetonitrile R and 2 volumes of water, and for solution (B) use 50μg of Artemisinin per ml in a mixture of 6 volumes of acetonitrile R and 4 volumes of water.

For the system suitability test prepare solution (C) containing 1mg of artemisinin RS per ml and 1 mg of artenimol RS per ml in a mixture of 8 volumes of acetonitrile R and 2 volumes of water.

Operate with a flow rate of 0.6 ml per minute. As a detector use an ultraviolet spectrophotometer set at a wavelength of about 216nm.

Inject alternately 20μl each of solutions A, B, and C.

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, is not greater than that obtained with solution B (0.5%). Not more than one peak is greater than half the area of the principal peak obtained with solution B (0.25%). The sum of the areas of all peaks, other than the principal peak, is not greater than twice the area of the principal peak obtained with solution B (1.0%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with solution B. The test is not valid unless the relative retention of α-artenimol compared with artemisinin is about 0.6, and the resolution between the peaks is not less than 2.0.

B. Carry out the test as described under 1.14.1 Thin-layer chromatography, using silica gel R1 as the coating substance and a mixture of equal volumes of light petroleum R1 and ether R as the mobile phase. Apply separately to the plate 10μl of each of the following 5 solutions in toluene R containing (A) 10 mg of Artemisinin per ml, (B) 0.05 mg of Artemisinin per ml, (C) 0.025 mg of Artemisinin per ml, (D) 0.10 mg of Artemisinin per ml, and (E) 0.10 mg of artemisinin RS per ml. After removing the plate from the chromatographic chamber, allow it to dry in air, spray with anisaldehyde/ sulfuric acid TS, and heat the plate to 105 °C for 7 minutes. Examine the chromatogram in daylight.

Any spot obtained with solution A, other than the principal spot, is not more intense than that obtained with solution B (0.5%). Furthermore, not more than one such spot is more intense than that obtained with solution C (0.25%).

Assay

• Either method A or method B may be applied.

A. Determine by 1.14.4 High-performance liquid chromatography, using a stainless steel column (10cm × 4.6mm) packed with particles of silica gel, the surface of which has been modified with chemically bonded octadecylsilyl groups (3μm). As the mobile phase, use a mixture of 6 volumes of acetonitrile R and 4 volumes of water.

Prepare the following solutions in the mobile phase: solution (A) 1.0mg of Artemisinin per ml; and solution (B) 1.0 mg of artemisinin RS per ml.

For the system suitability test prepare solution (C) containing 1mg of artemisinin RS per ml and 1 mg of artenimol RS per ml in a mixture of 8 volumes of acetonitrile R and 2 volumes of water.

Operate with a flow rate of 0.6 ml per minute. As a detector use an ultraviolet spectrophotometer set at a wavelength of about 216nm.

Inject alternately 20μl each of solutions A, B, and C.

The test is not valid unless the relative retention of α-artenimol compared with artemisinin is about 0.6, and the resolution between the peaks is not less than 2.0.

Measure the areas of the peak responses obtained in the chromatograms from solutions A and B, and calculate the percentage content of C15H22O5 with reference to the dried substance.

B. Dissolve about 0.05 g of Artemisinin, accurately weighed, in sufficient ethanol (~750g/l) TS to produce 100ml, and dilute 10ml to 100ml with the same solvent. Accurately transfer 10 ml to a 50-ml volumetric flask, dilute to volume with sodium hydroxide (0.05 mol/l) VS, mix thoroughly, and warm to 50 °C in a water-bath for 30 minutes. Cool to room temperature.

Measure the absorbance of a 1-cm layer at the maximum at about 292nm against a solvent cell containing a blank prepared with 10 ml of ethanol (~750 g/l) TS diluted with sufficient sodium hydroxide (0.05 mol/l) VS to produce 50 ml. Calculate the percentage content of C15H22O5 in the substance being tested by comparison with artemisinin RS, similarly and concurrently examined, and with reference to the dried substance.

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