Identification—
Insert a pledget of fine glass wool into the base of a chromatographic tube of about 200 × 25 mm. Mix 8.0 mL of nitromethane with 7.0 g of purified siliceous earth in a 150-mL beaker until uniform, and transfer to the chromatographic tube, packing lightly with a suitable tamping rod. Pack a pledget of glass wool on the top of the column. Dilute 1 mL of the Injection with
n-heptane to obtain a solution having a concentration of about 1 mg of progesterone per mL. Transfer 4.0 mL of this solution to the prepared column. Pass 300 mL of
n-heptane through the column, discarding the first 120 mL of the eluate. Collect the subsequent eluate in a 250-mL beaker. Evaporate the solution under a stream of nitrogen on a steam bath to about 50 mL, transfer to a 100-mL beaker, and evaporate to dryness. Remove the last traces of
n-heptane by adding 1 mL of methanol and again drying. Dry the specimen over silica gel for 4 hours: the IR absorption spectrum of a potassium bromide dispersion of the residue so obtained exhibits maxima only at the same wavelengths as that of a similar preparation of
USP Progesterone RS.
Assay—
Mobile phase—
Prepare a degassed mixture of alcohol and water (11:9). Make adjustments if necessary (see
System Suitability under
Chromatography 
621
).
Standard preparation—
Dissolve an accurately weighed quantity of
USP Progesterone RS in 20 mL of tetrahydrofuran, and dilute quantitatively, and stepwise if necessary, with alcohol to obtain a solution having a known concentration of about 0.08 mg per mL.
Assay preparation—
Transfer an accurately measured volume of Injection, equivalent to about 100 mg of progesterone, to a 100-mL volumetric flask, add 20 mL of tetrahydrofuran to dissolve, and dilute with alcohol to volume. Transfer 8 mL of this solution to a 100-mL volumetric flask, dilute with alcohol to volume, and mix.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The flow rate is about 1 mL per minute. The column temperature is maintained at about 40
. Chromatograph a sample of dimethyl sulfoxide, and identify the retention time,
t
, of this nonretarded compound to calculate the capacity factor,
k¢. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the capacity factor,
k¢, for progesterone is not less than 2.0; the tailing factor is not more than 2.0; and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 10 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks.
[NOTE—The run time for the
Assay preparation must be at least twice that of the
Standard preparation.
] Calculate the quantity, in mg, of progesterone (C
21H
30O
2) in each mL of Injection taken by the formula:
1250(C/V)(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Progesterone RS in the
Standard preparation; V is the volume, in mL, of Injection taken; and
rU and
rS are the peak responses for progesterone obtained from the
Assay preparation and the
Standard preparation, respectively.
