Molecular formula. C₂₁H₃₀O₂

Relative molecular mass. 314.5

Graphic formula.

Chemical name. Pregn-4-ene-3,20-dione; CAS Reg. No. 57-83-0.

Description. Colourless crystals or a white to slightly yellowish white, crystalline powder; odourless.

Solubility. Practically insoluble in water; soluble in 8 parts of ethanol (~750 g/l) TS.

Category. Progestational steroid.

Storage. Progesterone should be kept in a well-closed container, protected from light.

Additional information. Progesterone may exist in 2 polymorphic forms, one of which melts at about 130°C, the other at about 121°C.

Requirements

Definition. Progesterone contains not less than 97.0% and not more than 102.0% of C₂₁H₃₀O₂, calculated with reference to the dried substance.

Identity tests

• Either test A or test B 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 progesterone RS or with the reference spectrum of progesterone. If the spectrum obtained from the solid state of the test substance is not concordant with the spectrum obtained from the reference substance, compare the spectra using solutions in chloroform R containing 30 mg/mL and a path length of 0.2 mm.

B. Carry out the test as described under 1.14.1 Chromatography, Thin-layer chromatography, using kieselguhr R1 as the coating substance and a mixture of 10 volumes of propylene glycol R and 90 volumes of acetone R to impregnate the plate, dipping it about 5 mm beneath the surface of the liquid. After the solvent has reached the height of at least 16 cm, remove the plate from the chromatographic chamber and allow it to stand at room temperature until the solvent has completely evaporated. Use the impregnated plate within 2 hours, carrying out the chromatography in the same direction as the impregnation. As the mobile phase, use a mixture of 50 volumes of cyclohexane R and 50 volumes of light petroleum R. Apply separately to the plate 5 μl of each of 2 solutions in a mixture of 9 volumes of chloroform R and 1 volume of methanol R containing (A) 1.0 mg of the test substance per mL and (B) 1.0 mg of progesterone RS per mL. Develop the plate for a distance of 15 cm. After removing the plate from the chromatographic chamber allow it to dry in air until the solvents have evaporated, heat at 120°C for 15 minutes, spray with 4-toluenesulfonic acid/ethanol TS, and then heat at 120°C for 10 minutes. Allow to cool and examine the chromatogram in daylight and in ultraviolet light (365 nm). The principal spot obtained with solution A corresponds in position, appearance, and intensity with that obtained with solution B.

Specific optical rotation. Use a 10 mg/mL solution in dehydrated ethanol R; = +186° to +196°.

Loss on drying. Dry to constant weight at 105°C; it loses not more than 5.0 mg/g.

Related substances. Carry out the test as described under 1.14.1 Chromatography, Thin-layer chromatography, using silica gel R2 as the coating substance and a mixture of 2 parts of chloroform R and 1 part of ethyl acetate R as the mobile phase. Apply separately to the plate 10 μl of each of 2 solutions in a mixture of 1 volume of ethanol (~750 g/l) TS and 1 volume of chloroform R containing (A) 10 mg of the test substance per mL and (B) 0.10 mg of the test substance per mL. After removing the plate from the chromatographic chamber, allow it to dry in air until the solvents have evaporated, and examine the chromatogram in ultraviolet light (254 nm). Any spot obtained with solution A, other than the principal spot, is not more intense than that obtained with solution B.

Assay. Dissolve about 20 mg, accurately weighed, in sufficient methanol R to produce 100 mL; dilute 5.0 mL of this solution to 100 mL with the same solvent. Measure the absorbance of a 1-cm layer of the diluted solution at the maximum at about 240 nm. Calculate the amount of C₂₁H₃₀O₂ in the substance being tested by comparison with progesterone RS, similarly and concurrently examined. In an adequately calibrated spectrophotometer the absorbance of the reference solution should be 0.54 ± 0.03.