C15H24O5
Relative molecular mass. 284.4
Chemical name. (3R,5aS,6R,8aS,9R,10S,12R,12aR)-Decahydro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin-10-ol; CAS Reg. No. 81496-81-3.
Other names. Dihydroartemisinin, b-dihydroartemisinin.
Description. Colourless needles or a white or almost white, crystalline powder.
Solubility. Practically insoluble in water; slightly soluble in acetonitrile R, ethanol (~750 g/l) TS and dichloromethane R.
Category. Antimalarial drug.
Storage. Artenimol should be kept in a well-closed container, protected from light.
Requirements
Artenimol contains not less than 97.0% and not more than the equivalent of 102.0% of C15H24O5 using Assay method A, and not less than 98.0% and not more than the equivalent of 102.0% of C15H24O5 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 artenimol RS or with the reference spectrum of artenimol.
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 0.5 ml 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.
Sulfated ash. Not more than 1.0 mg/g.
Loss on drying. Dry over phosphorus pentoxide R under reduced pressure (not exceeding 2.67 kPa or 20 mm of mercury); it loses not more than 10.0 mg/g.
Related substances
• Either test A or test B may be applied.
Prepare fresh solutions and perform the tests without delay.
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). As the mobile phase for gradient elution, use a mixture of 6 volumes of acetonitrile R and 4 volumes of water for the first 17 minutes; then run a gradient, which should reach 100% acetonitrile within 13 minutes.
Prepare the following solutions in methanol R with sonication. For solution (A) use 10 mg of Artenimol per ml and for solution (B) use 50μg of Artenimol per ml.
For the system suitability test prepare solution (C) by dissolving 1.0mg of artemisinin RS per ml and 1.0 mg of artenimol RS per ml in methanol R with sonication.
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 (twin-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 twin 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 twin peak obtained with solution B (0.25%). The sum of the areas of all the peaks, other than the twin peak, is not greater than twice the area of the twin peak obtained with solution B (1.0%). Disregard any peak with an area less than 0.1 times the area of the twin peak in the chromatogram obtained with solution B.
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 10ml of each of the following 5 solutions in toluene R containing (A) 10 mg of Artenimol per ml, (B) 0.05 mg of Artenimol per ml, (C) 0.025 mg of Artenimol per ml, (D) 0.10 mg of Artenimol per ml, and (E) 0.10 mg of artenimol RS per ml. After removing the plate from the chromatographic chamber, allow it to dry in air, and spray with vanillin/ sulfuric acid TS1. 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.
Prepare fresh solutions and perform the tests without delay.
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 Artenimol per ml, and solution (B) 1.0 mg of artenimol RS per ml.
For the system suitability test prepare solution (C) containing 1.0mg of artemisinin RS per ml and 1.0 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 (twin-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 Artenimol, accurately weighed, in sufficient ethanol (~750 g/l) TS to produce 100 ml and dilute 10 ml to 100 ml 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 10ml of ethanol (~750g/l) TS diluted with sufficient sodium hydroxide (0.05mol/l) VS to produce 50ml. Calculate the percentage content of C15H22O5 in the substance being tested by comparison with artenimol RS, similarly and concurrently examined, and with reference to the dried substance.
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