TEST 1—
Adsorbent:
0.25-mm layer of chromatographic silica gel mixture.
Test solution—
Prepare a solution of Norethindrone Acetate in chloroform having a concentration of 10 mg per mL.
Standard solutions—
Dilute accurately measured volumes of the Standard stock solution with chloroform to obtain Standard solutions A, B, C, and D having known concentrations of 150 µg per mL, 50 µg per mL, 30 µg per mL, and 10 µg per mL, respectively.
Application volume:
10 µL, as two 5-µL portions.
Developing solvent system:
a mixture of toluene and ethyl acetate (1:1).
Procedure—
Proceed as directed for
Thin-Layer Chromatography under
Chromatography 
621
, except to apply the solutions along a line 2.5 cm from the edge of the plate. Spray the plate with a mixture of methanol and sulfuric acid (7:3), and heat at 100
for 5 minutes. The
Test solution exhibits a principal spot at the same
RF value as the principal spot of
Standard solution A. Any individual secondary spot is not more intense than the spot in the chromatogram obtained from
Standard solution B: not more than 0.5% of any individual impurity is found. The sum of the intensities of all of the secondary spots is not more intense than the spot in the chromatogram obtained from
Standard solution A: not more than 1.5% of total impurities is found.
TEST 2 —
Mobile phase—
Prepare a filtered and degassed mixture of acetonitrile and water (6:4). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Resolution solution—
Dissolve accurately weighed quantities of desoxycorticosterone acetate and
USP Norethindrone Acetate RS in
Mobile phase to obtain a solution having concentrations of about 80 µg of each per mL.
Test solution—
Transfer about 62.5 mg of Norethindrone Acetate, accurately weighed, to a 25-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix.
Diluted test solution—
Transfer 1.0 mL of the Test solution to a 100-mL volumetric flask, dilute with Mobile phase 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 packing L1. The flow rate is about 1 mL per minute. Chromatograph the
Resolution solution, and record the peak responses as directed for
Procedure: the relative retention times are about 0.83 for desoxycorticosterone acetate and 1.0 for norethindrone acetate; and the resolution,
R, between desoxycorticosterone acetate and norethindrone acetate is not less than 3.5.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Diluted test solution and the
Test solution into the chromatograph, record the chromatograms for twice the retention time of norethindrone acetate, and measure all of the peak areas. Calculate the percentage of each impurity in the portion of Norethindrone Acetate taken by the formula:
ri / rs,
in which
ri is the peak area for each impurity obtained from the
Test solution; and
rs is the sum of all the peaks obtained from the
Diluted test solution. [NOTE—Exclude any peak having a response that is less than 0.025% of the norethindrone acetate peak response obtained from the
Test solution.] Not more than 0.5% of any individual impurity is found; and not more than 1.0% of total impurities is found.
).