Assay—
Standard preparations—
Transfer about 30 mg of
USP Dioxybenzone RS, accurately weighed, to a 50-mL volumetric flask, dissolve in methanol, dilute with the same solvent to volume, and mix. Pipet 2 mL of this solution into a 100-mL volumetric flask, add methanol to volume, and mix. Similarly, prepare a
Standard preparation of
USP Oxybenzone RS, accurately weighed, having a known concentration of about 12 µg per mL.
Assay preparation—
Dissolve an accurately weighed portion of Cream, equivalent to about 25 mg each of dioxybenzone and oxybenzone, in methanol in a 100-mL volumetric flask, dilute with methanol to volume, and mix. Pipet 1 mL of this solution into a 15-mL conical test tube, evaporate on a water bath just to dryness, using a gentle stream of air, and dissolve the residue in about 200 µL of methanol.
Procedure—
Prepare sheets of chromatographic paper (Whatman No. 1 or equivalent), each measuring about 23 × 28.5 cm, as follows. Immerse the sheets in a 1 in 20 solution of light mineral oil in solvent hexane, withdraw them immediately, and allow to dry in air. On one sheet mark a starting line about 2.5 cm from the long edge, and apply the entire Assay preparation as 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 5 mm and 10 mm. Rinse the conical test tube, which contained the Assay preparation, with about 100 µL of 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 5 minutes.
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 40 mL of a mobile solvent consisting of a mixture of equal volumes of acetone and water. Seal the chamber, and allow the chromatogram to develop for 2 hours.
Remove the paper from the chamber, air-dry, then remove the staples, and view the chromatogram under short-wavelength (254 nm) UV radiation. 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-mL conical flasks, add 20.0 mL of methanol to each flask, and shake gently for 30 minutes.
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 30 minutes with 20.0 mL of methanol.
Concomitantly determine the absorbance of each of the 4 solutions thus prepared, and of each of the Standard preparations, in a 1-cm cell at the wavelength of maximum absorbance at about 325 nm, 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:
2
C(
AU 
AB)
/ AS,
in which
C is the concentration, in µg per mL, of
USP Dioxybenzone RS in the dioxybenzone
Standard preparation, and
AU,
AB, and
AS are 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
AS the respective values pertaining to the oxybenzone determination.
197U
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