Assay
Mobile phase
Mix 300 mL of methanol and 10 mL of glacial acetic acid with 690 mL of water. Allow the mixture to cool, and filter, if necessary, through a suitable microporous membrane filter. Degas the solution.
Internal standard solution
Dissolve salicylic acid in methanol, by sonicating, to obtain a solution having a concentration of about 7 mg per mL.
Standard preparation
Dissolve, by sonicating, an accurately weighed quantity of
USP Aminobenzoic Acid RS in methanol, quantitatively dilute with methanol to obtain a solution having a known concentration of about 0.42 mg per mL, and mix. Pipet 5 mL of this solution and 5 mL of the
Internal standard solution into a 50-mL volumetric flask, dilute with methanol to volume, and mix. Pass through 0.6-µm filter paper. Throughout the preparation, protect against actinic light.
Assay preparation
Transfer an accurately weighed quantity of Gel, equivalent to about 4.2 mg of aminobenzoic acid, to a 100-mL volumetric flask, and add 10.0 mL of Internal standard preparation and about 50 mL of methanol. Shake or sonicate, as necessary, dilute with methanol to volume, and mix. Filter, if necessary, through filter paper (Whatman No. 41 or equivalent). Pass through 0.6-µm filter paper. Throughout this preparation, protect against actinic light.
Chromatographic system
(see
Chromatography 621)The liquid chromatograph is equipped with a 280-nm detector and a 3.9-mm × 30-cm column that contains packing L11. The flow rate is about 1.0 mL per minute. Chromatograph replicate 15-µL injections of
Standard preparation until the response ratio variability is within 1.0% of average. The resolution factor is not less than 3.0 between aminobenzoic acid and salicylic acid.
Procedure
Separately inject equal volumes (about 15 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The retention time of salicylic acid is about 3.0 relative to that of aminobenzoic acid as 1.0. Calculate the quantity, in mg, of aminobenzoic acid (C
7H
7NO
2) in the portion of Gel taken by the formula:
100C(RU / RS),
in which
C is the concentration, in mg per mL, of
USP Aminobenzoic Acid RS in the
Standard preparation; and
RU and
RS are the ratios of the peak responses of the aminobenzoic acid peak to the salicylic acid peak obtained from the
Assay preparation and the
Standard preparation, respectively.