Diluent— Prepare a mixture of acetonitrile and water (1:1).

Internal standard solution— Pipet 4.0 mL of dehydrated methanol into a 100-mL volumetric flask. Dilute with water to volume, and mix.

Standard solution— Accurately weigh, by difference, about 1.6 mL of dehydrated alcohol into a tared 50-mL volumetric flask containing about 15 mL of water. Dilute with Diluent to volume, and mix. Pipet 10.0 mL of this solution into a 50-mL volumetric flask, dilute with Diluent to volume, and mix. Pipet 25.0 mL of this solution into a 50-mL volumetric flask, add 5.0 mL of Internal standard solution, dilute with water to volume, and mix.

Test solutions— Prepare as directed for Assay preparations, with the following changes. Pipet 25.0 mL of each solution into individual 50-mL volumetric flasks. Add 5.0 mL of Internal standard solution, dilute with water to volume, and mix.

Chromatographic system (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector and a 2-mm × 2-m glass column that contains support S2. The carrier gas is helium, flowing at a rate of 30 mL per minute. The column temperature is 100. The injection port temperature and the detector temperature are maintained at 200. Chromatograph the Standard solution, and record the peak areas as directed for Procedure: the relative retention times are about 0.4 for the internal standard and about 1.0 for alcohol; and the relative standard deviation for replicate injections, determined from the ratios of alcohol peak areas to those of the internal standard, is not more than 1.5%.

Procedure— Separately inject equal volumes (about 2 µL) of the Standard solution and each of the Test solutions into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the amount of alcohol, in mg, in each Transdermal System taken by the formula: in which C is the concentration, in mg per mL, of dehydrated alcohol in the Standard solution; and RU and RS are the ratios of the peak responses of alcohol to the internal standard obtained from the Test solutions and the Standard solution, respectively. Calculate the average amount of alcohol in the Test solution taken: between 80% and 120% of the labeled amount of C2H5OH is found.

Diluent— Prepare a mixture of acetonitrile and water (1:1).

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and water (55:45). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Estradiol RS in Diluent. Dilute quantitatively, and stepwise if necessary, with Diluent to obtain a solution having a known concentration of about 0.1 mg per mL.

Assay preparations— Cut 10 Transdermal Systems into pieces, keeping the pieces from each system separate. Remove the protective liners, if any, from the strips, and discard. Transfer the pieces of each system into separate stoppered flasks of suitable size, and add an accurately measured volume of Diluent to each flask to obtain solutions having a concentration of about 0.1 mg of estradiol per mL. Shake by mechanical means for about 3 hours, and sonicate for 15 minutes.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm × 15-cm column that contains packing L1. The column temperature is maintained at 35. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the tailing factor is between 0.9 and 1.6; and the relative standard deviation for replicate injections is not more than 2.5%.

Procedure— Separately inject equal volumes (about 25 µL) of the Standard preparation and the Assay preparations into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of estradiol (C18H24O2) in each Transdermal System taken by the formula: in which V is the volume, in mL, of Diluent used to prepare the Assay preparation; C is the concentration, in mg per mL, of USP Estradiol RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively. Calculate the average quantity, in mg, of estradiol in each Transdermal System. Use the individual assays to determine the Uniformity of dosage units.