Assay—
Standard preparations—
Transfer about 30mg of
USP Dioxybenzone RS,accurately weighed,to a 50-mLvolumetric flask,dissolve in methanol,dilute with the same solvent to volume,and mix.Pipet 2mLof this solution into a 100-mLvolumetric flask,add methanol to volume,and mix.Similarly,prepare a
Standard preparationof
USP Oxybenzone RS,accurately weighed,having a known concentration of about 12µg per mL.
Assay preparation—
Dissolve an accurately weighed portion of Cream,equivalent to about 25mg each of dioxybenzone and oxybenzone,in methanol in a 100-mLvolumetric flask,dilute with methanol to volume,and mix.Pipet 1mLof this solution into a 15-mLconical test tube,evaporate on a water bath just to dryness,using a gentle stream of air,and dissolve the residue in about 200µLof methanol.
Procedure—
Prepare sheets of chromatographic paper (Whatman No.1or equivalent),each measuring about 23×28.5cm,as follows.Immerse the sheets in a 1in 20solution of light mineral oil in solvent hexane,withdraw them immediately,and allow to dry in air.On one sheet mark a starting line about 2.5cm from the long edge,and apply the entire Assay preparationas a uniform streak along the starting line,using a stream of air or an air blower,if necessary,to maintain the width of the streak between 5mm and 10mm.Rinse the conical test tube,which contained the Assay preparation,with about 100µLof methanol,and apply the rinse to the starting line.Similarly,repeat the rinsing and streaking with two additional portions of methanol,and then allow the paper to dry in air for 5minutes.
Staple together the short edges of the paper to form a cylinder,and place it in a 12-×25-cm cylindrical chromatographic chamber containing about 40mLof a mobile solvent consisting of a mixture of equal volumes of acetone and water.Seal the chamber,and allow the chromatogram to develop for 2hours.
Remove the paper from the chamber,air-dry,then remove the staples,and view the chromatogram under short-wavelength (254nm)UVradiation.Mark the two bands representing the separated dioxybenzone and oxybenzone,respectively.[NOTE—Determine the relative position of each benzone on the chromatogram by applying suitable aliquots of each Standard preparation to another prepared chromatographic sheet,and developing the chromatogram in a manner similar to that described for the Assay preparation.]Cut the marked bands from the sheet,and then,keeping the band segments separate,cut each into several pieces to facilitate extraction.Place the pieces from each band in separate glass-stoppered,50-mLconical flasks,add 20.0mLof methanol to each flask,and shake gently for 30minutes.
To provide the chromatographic blank,treat one of the prepared chromatographic sheets in the same manner as described above,but omit the application of the Assay preparation.Cut from the chromatographed paper the areas corresponding to the bands produced by the benzones from the Assay preparation,and in the same manner extract the blank bands for 30minutes with 20.0mLof methanol.
Concomitantly determine the absorbance of each of the 4solutions thus prepared,and of each of the Standard preparations,in a 1-cm cell at the wavelength of maximum absorbance at about 325nm,with a suitable spectrophotometer,using methanol as the blank.Calculate the quantity,in mg,of dioxybenzone (C14H12O4)in the portion of Cream taken by the formula:
2C(AU-AB)/AS,
in which
Cis the concentration,in µg per mL,of
USP Dioxybenzone RSin the dioxybenzone
Standard preparation,and
AU,
AB,and
ASare the absorbances of the dioxybenzone solution from the
Assay preparation,the dioxybenzone chromatographic blank solution,and the dioxybenzone
Standard preparation,respectively.In a similar manner,calculate the quantity,in mg,of oxybenzone (C
14H
12O
3)in the portion of Cream taken,using as
C,
AU,
AB,and
ASthe respective values pertaining to the oxybenzone determination.