Identification—
Proceed as directed in the
Assay, except to inject 50 µL to 200 µL of a mixture of the
Assay preparation and the
Standard preparation (1:1) onto the column, adjusting the response to obtain a response that is between 50% and 90% full scale: a single peak is observed in the chromatogram for the mixed solution.
Quantity delivered per spray—
Prime the spray pump by delivering 10 sprays into a fume hood. Accurately weigh the entire assembly, record the weight, and deliver 8 more sprays into the hood. Again weigh the assembly, and record the weight. Calculate the quantity, in µg, of C
24H
31FO
6 delivered per spray taken by the formula:
[(
W1 
W2) / 8][
A /
D],
in which
W1 and
W2 are the first and second weights, respectively, in g;
A is the quantity, in µg per mL, of C
24H
31FO
6 found in the
Assay; and
D is the density of Nasal Solution, in g per mL. The quantity delivered is between 17 µg and 33 µg per spray.
Assay—
Mobile phase—
Prepare a suitable degassed solution of water, acetonitrile, and glacial acetic acid (69:30:1 to 64:35:1). Adjust the ratio as necessary to obtain suitable chromatographic performance.
Internal standard solution—
Dissolve norethindrone in acetonitrile to obtain a solution containing about 300 µg per mL.
Standard preparation—
Dissolve an accurately weighed quantity of
USP Flunisolide RS in a mixture of acetonitrile and
Mobile phase (1:1) to obtain a solution having a known concentration of about 250 µg per mL. Transfer 1.0 mL of this solution, and 1.0 mL of
Internal standard solution, by means of “to contain” pipets, to a 50-mL volumetric flask. Rinse the pipets with
Mobile phase, adding the rinsings to the flask, dilute with
Mobile phase to volume, and mix. The final concentration of
USP Flunisolide RS is about 5 µg per mL.
Assay preparation—
Transfer an accurately measured volume of Nasal Solution, equivalent to about 250 µg of flunisolide, to a 50-mL volumetric flask, and add 1.0 mL of Internal standard solution by means of “to contain” pipets. Rinse the pipets with Mobile phase, adding the rinsing to the 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-mm × 25-cm column that contains 5- to 10-µm packing L1. The flow rate is about 2 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the resolution,
R, between the analyte and the internal standard is not less than 5.0; and the relative standard deviation for replicate injections is not more than 1.5%.
Procedure—
Separately inject equal volumes (about 50 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The relative retention times are about 0.6 for flunisolide and 1.0 for norethindrone. Calculate the quantity, in mg, of C
24H
31FO
6 in each mL of the Nasal Solution taken by the formula:
(434.51 / 443.51)(50C / V)(RU / RS),
in which 434.51 and 443.51 are the molecular weights of C
24H
31FO
6 and C
24H
31FO
6·½H
2O, respectively;
C is the concentration, in mg per mL, of
USP Flunisolide RS in the
Standard preparation; V is the volume, in mL, of Nasal Solution taken; and
RU and
RS are the peak response ratios of the flunisolide peak and the norethindrone peak obtained from the
Assay preparation and the
Standard preparation, respectively.
