á511ñSINGLE-STEROID ASSAY
In the following procedure,the steroid to be assayed is separated from related foreign steroids and excipients by thin-layer chromatography and determined following recovery from the chromatogram.
Preparation of the Plate— Prepare a slurry from 30g of chromatographic silica gel with a suitable fluorescing substance by the gradual addition,with mixing,of about 65mLof a mixture of water and alcohol (5:2).Transfer the slurry to a clean,20-×20-cm plate,spread to make a uniform layer 250µm thick,and allow to dry at room temperature for 15minutes.Heat the plate at 105
for 1hour,and store in a desiccator.
Solvent A— Mix methylene chloride with methanol (180:16).
Solvent B— Mix chloroform with acetone (4:1).
Standard Preparation— Dissolve in a mixture of equal volumes of chloroform and alcohol a suitable quantity of the USP Reference Standard specified in the individual monograph,previously dried as directed (see USP Reference Standards á11ñ)and accurately weighed,to obtain a solution having a known concentration of about 2mg per mL.
Assay Preparation— Prepare as directed in the individual monograph.
Procedure— Divide the area of the chromatographic plate into three equal sections,the left and right sections to be used for the Assay Preparationand the Standard Preparation,respectively,and the center section for the blank.Apply 200µLeach of the Assay Preparationand the Standard Preparationas streaks 2.5cm from the bottom of the appropriate section of the plate.Dry the solution as it is being applied,with the aid of a stream of air.Using the Solventspecified in the individual monograph,develop the chromatogram in a suitable chamber,previously equilibrated and lined with absorbent paper,until the solvent front has moved 15cm above the initial streaks.
Remove the plate,evaporate the solvent,and locate the principal band occupied by the Standard Preparationby viewing under UVlight.Mark this band,as well as corresponding bands in the Assay Preparationand blank sections of the plate.Remove the silica gel from each band separately,either by scraping onto glazed weighing papers or by using a suitable vacuum collecting device,and transfer it to a glass-stoppered,50-mLcentrifuge tube.To each tube add 25.0mLof alcohol,and shake for not less than 2minutes.Centrifuge the tubes for 5minutes,pipet 20mLof the supernatant from each tube into a glass-stoppered,50-mLconical flask,add 2.0mLof a solution prepared by dissolving 50mg of blue tetrazolium in 10mLof methanol,and mix.Proceed as directed for Procedureunder Assay for Steroids á351ñ,beginning with “Then to each flask.”