Identification—
Dissolve an amount of Ointment, equivalent to about 60 mg of benzoic acid and 30 mg of salicylic acid, in 25 mL of a solvent consisting of equal volumes of chloroform and methanol. Prepare separate Standard solutions in the same solvent containing, respectively, 2.4 mg of
USP Benzoic Acid RS in each mL and 1.2 mg of
USP Salicylic Acid RS in each mL. Apply 5 µL of each solution at separate points 2.5 cm from the bottom edge of a 20- × 20-cm thin-layer chromatographic plate (see
Chromatography 
621
) coated with a 0.25-mm layer of chromatographic silica gel mixture, and allow the spots to dry. Develop the chromatogram in a solvent system consisting of a mixture of chloroform, acetone, isopropyl alcohol, methanol, and ammonium hydroxide (30:30:15:15:10) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the chromatographic chamber, mark the solvent front, and allow the solvent to evaporate. View the chromatogram under short-wavelength (254 nm) UV radiation: the two major fluorescent spots from the test solution correspond in color and in
RF value to those from the respective Standard solutions.
Assay—
Ferric chloride-urea reagent—
On the day of use, dissolve, without heating, 18 g of urea in a mixture of 2.5 mL of ferric chloride solution (6 in 10) and 12.5 mL of 0.05 N hydrochloric acid.
Column A—
Insert a small pledget of glass wool above the stem constriction of a 20- × 2.5-cm chromatographic tube. Mix 1 g of chromatographic siliceous earth with 0.5 mL of dilute phosphoric acid (3 in 10) to form a uniform, fluffy mixture, transfer to the chromatographic tube, and pack evenly over the glass wool, exerting gentle pressure. Similarly, mix 4 g of chromatographic siliceous earth with 3 mL of Ferric chloride-urea reagent, and pack uniformly over the first layer. Cover the column with a pad of glass wool.
Column B—
Insert a small pledget of glass wool above the stem constriction of a second 20- × 2.5-cm chromatographic tube. Mix 4 g of chromatographic siliceous earth with 2 mL of sodium bicarbonate solution (1 in 12), prepared just prior to use, to a uniform, fluffy mixture, and pack evenly over the glass wool. Cover the column with a pad of glass wool.
Assay preparation—
Transfer an accurately weighed amount of Ointment, equivalent to about 100 mg of benzoic acid and 50 mg of salicylic acid, to a 250-mL volumetric flask, and dissolve in about 150 mL of chloroform by warming on a steam bath. Cool, dilute with chloroform to volume, and mix.
Procedure—
Mount Column A directly over Column B, then pipet 10 mL of Assay preparation onto Column A, and allow it to pass into the column. Wash the columns with two 40-mL portions of chloroform, allowing the first portion to recede to the top of each column before adding the second portion. Discard the eluates, and separate the columns.
Salicylic acid content—
Elute
Column A with 95 mL of a 3 in 100 solution of glacial acetic acid in chloroform, collecting the eluate in a 100-mL volumetric flask. Dilute the contents of the flask with the same solvent to volume, and mix. Dissolve an accurately weighed quantity of
USP Salicylic Acid RS in the same solvent, and dilute quantitatively and stepwise with this solvent to obtain a Standard solution having a known concentration of about 20 µg per mL. Concomitantly determine the absorbances of both solutions in 1-cm cells at the wavelength of maximum absorbance at about 311 nm, with a suitable spectrophotometer, using a 3 in 100 solution of glacial acetic acid in chloroform as the blank. Calculate the quantity, in mg, of salicylic acid (C
7H
6O
3) in the portion of Ointment taken by the formula:
2.5C(AU / AS),
in which
C is the concentration, in µg per mL, of
USP Salicylic Acid RS in the Standard solution; and
AU and
AS are the absorbances of the diluted eluate from
Column A and the Standard solution, respectively.
Benzoic acid content—
Elute
Column B with 95 mL of a 3 in 100 solution of glacial acetic acid in chloroform, collecting the eluate in a 100-mL volumetric flask. Dilute the contents of the flask with the same solvent to volume, and mix. Dissolve an accurately weighed quantity of
USP Benzoic Acid RS in the same solvent, and dilute quantitatively and stepwise with this solvent to obtain a Standard solution having a known concentration of about 40 µg per mL. Concomitantly determine the absorbances of both solutions in 1-cm cells at the wavelength of maximum absorbance at about 275 nm, with a suitable spectrophotometer, using a 3 in 100 solution of glacial acetic acid in chloroform as the blank. Calculate the quantity, in mg, of benzoic acid (C
7H
6O
2) in the portion of Ointment taken by the formula:
2.5C(AU / AS),
in which
C is the concentration, in µg per mL, of
USP Benzoic Acid RS in the Standard solution; and
AU and
AS are the absorbances of the diluted eluate from
Column B and the Standard solution, respectively.
.