Identification—
Transfer a quantity of Lotion,equivalent to about 5mg of hydrocortisone,to a separator containing 10mLof methylene chloride,shake for 1minute,and allow the layers to separate.Filter the methylene chloride extract onto a suitable chromatographic column that has been packed with 2g of activated magnesium silicate.Wash the column with 25mLof methylene chloride with the aid of slight air pressure,discarding the washings,and elute the hydrocortisone with 10mLof methanol.Using
USP Hydrocortisone RSto prepare a Standard solution having a concentration of 500µg per mL,and using as the solvent system a mixture of 180volumes of chloroform,15volumes of methanol,and 1volume of water,proceed as directed under
Thin-layer Chromatographic Identification Test á201ñ.
Assay—
Mobile phase—
Prepare a filtered and degassed mixture of water,acetonitrile,and methanol (58:21:21).Make adjustments if necessary (see
System Suitabilityunder
Chromatography á621ñ).
Standard preparation—
Dissolve an accurately weighed quantity of
USP Hydrocortisone RSin alcohol and dilute quantitatively,and stepwise if necessary,with alcohol to obtain a solution having a known concentration of about 0.1mg per mL.Quantitatively dilute a known volume of the final solution with an equal volume of water to obtain the
Standard preparation.
Assay preparation—
In a tared,100-mLvolumetric flask,weigh 100mLof Lotion that previously has been shaken to ensure homogeneity,allow to stand until the entrapped air rises,and finally invert carefully just prior to transfer to the volumetric flask.Transfer an accurately weighed quantity of Lotion,freshly mixed but free from air bubbles,equivalent to about 10mg of hydrocortisone,to a 40-mLbeaker,and add about 30mLof alcohol.Warm gently until the Lotion is dispersed,and cool to room temperature.Quantitatively transfer the mixture,filter through a pledget of cotton previously moistened with alcohol to a 100-mLvolumetric flask,rinse the beaker with two 20-mLportions of alcohol,and collect the washings in the same volumetric flask.Dilute with alcohol to volume,and mix.Quantitatively dilute a known volume of this solution with an equal volume of water to obtain the Assay preparation.
System suitability preparation—
Dissolve about 5mg of propylparaben in 100mLof alcohol.Dilute 1mLof this solution with the Standard preparationto 10mL,and mix.
Chromatographic system
(see
Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1mLper minute.Chromatograph the
Standard preparationand the
System suitability preparation,and record the peak responses as directed for
Procedure:the column efficiency determined from the analyte peak is not less than 1000theoretical plates,the tailing factor for the analyte peak is not more than 1.2,the resolution,
R,between the propylparaben and hydrocortisone peaks is not less than 2.0,and the relative standard deviation for replicate injections of the
Standard preparationis not more than 3.0%.
Procedure—
Separately inject equal volumes (about 20µL)of the
Standard preparationand the
Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of hydrocortisone (C
21H
30O
5)in the portion of Lotion taken by the formula:
200C(rU/rS),
in which
Cis the concentration,in mg per mL,of
USP Hydrocortisone RSin the
Standard preparation;and
rUand the
rSare the peak responses obtained from the
Assay preparationand the
Standard preparation,respectively.From the observed weight of 100mLof the Lotion,calculate the quantity of C
21H
30O
5in each 100mL.