Identification—
Filter a volume of well-shaken Injectable Suspension, equivalent to not less than 100 mg of progesterone, through a medium-porosity, sintered-glass crucible, filtering again through the same crucible if the fluid is not clear. Wash with several 5-mL portions of water until 2 mL of the last washing, evaporated on a steam bath, leaves no weighable residue: the washed solid, dried at 105
to constant weight, melts between 126
and 131
, and responds to
Identification test
A under
Progesterone.
Assay—
Mobile phase—
Prepare a filtered and degassed mixture of water and isopropyl alcohol (72:28). Make adjustments if necessary (see
System Suitability under
Chromatography 
621
.)
Diluent—
Prepare a mixture of alcohol and water (85 in 100).
Internal standard solution—
Dissolve an accurately weighed quantity of
USP Methyltestosterone RS in
Diluent to obtain a solution containing about 6.6 mg per mL.
Standard preparation—
Dissolve an accurately weighed quantity of
USP Progesterone RS in
Diluent, and dilute quantitatively, and stepwise if necessary, with
Diluent to obtain a solution having a known concentration of about 2.5 mg per mL.
Standard curve—
To four separate polytef-lined, screw-capped centrifuge tubes, pipet 3-, 4-, 5-, and 6-mL portions, respectively, of the Standard preparation. Add Diluent to each tube to make about 8 mL of solution. Transfer 1.0 mL of Internal standard solution to each tube, and mix.
Assay preparation—
Transfer an accurately measured volume of Injectable Suspension, equivalent to about 25 mg progesterone, to a polytef-lined, screw-capped, 25 mL test tube. Add 16 mL of Diluent, and shake until clear. Add 2.0 mL of Internal standard solution, and mix.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 254-nm detector and a 2.1-mm × 1-m column that contains packing L2. The chromatographic conditions are such that retention times of the internal standard and progesterone are between 3 and 6 minutes and 7 and 11 minutes, respectively. Chromatograph any solution of the
Standard curve, and record the peak areas as directed for
Procedure: the resolution,
R, between progesterone and methyltestosterone is not less than 3.5; and the relative standard deviation for replicate injections is not more than 1.5%.
Procedure—
Separately inject equal volumes (5.0 µL) of each solution of the
Standard curve and the
Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas. Plot the progesterone to methyltestosterone peak ratio versus the concentration, in mg per mL, of progesterone in the
Standard curve solutions. Extrapolate the progesterone to methyltestosterone peak ratio of the
Assay preparation in the
Standard curve so obtained, and determine the progesterone concentration,
C, in mg per mL, in the
Assay preparation. Calculate the amount, in mg, of progesterone (C
21H
30O
2) in each mL of the Injectable Suspension taken by the formula:
V/A(C),
in which
V is the total volume of the
Assay preparation; A is the volume in mL of Injectable Suspension taken; and
C is the concentration of progesterone in the
Assay preparation calculated above.
