Solution: 10 µg per mL.

Medium: Sodium hydroxide solution (1 in 250).

Ratio: A256 /A365, between 2.80 and 3.00.

3 N phosphoric acid , 6 N ammonium hydroxide, Internal standard solution, Standard stock solution, Standard preparation, and Chromatographic system—Proceed as directed in the Assay.

Test solution— Use the Assay stock solution, prepared as directed in the Assay.

Procedure— Inject about 10 µL of the Test solution into the chromatograph, and allow the Test solution to elute for not less than 2 times the retention time of folic acid. Record the chromatogram, and measure the responses of all the peaks. The sum of the area of all peaks, other than that due to folic acid, is not greater than 2.0%.

3 N phosphoric acid— Dissolve 9.8 g of phosphoric acid in 100 mL of water.

6 N ammonium hydroxide— Dilute 40 mL of ammonium hydroxide with water to 100.0 mL.

Mobile phase— Transfer 2.0 g of monobasic potassium phosphate to a 1000-mL volumetric flask, and dissolve in about 650 mL of water. Add 15.0 mL of solution of 0.5 M tetrabutylammonium hydroxide in methanol, 7.0 mL of 3 N phosphoric acid, and 270 mL of methanol. Cool to room temperature, and adjust with 3 N phosphoric acid or 6 N ammonium hydroxide to a pH of 5.0, dilute with water to volume, mix, and filter. [note—Recheck the pH before use.]

Internal standard solution— Dissolve about 50 mg of methylparaben in 1.0 mL of methanol, dilute with Mobile phase to 25.0 mL, and mix.

Standard stock solution— Prepare a solution of USP Folic Acid RS in Mobile phase having a known concentration of about 1 mg per mL. [note—Use 1 mL of 10% ammonium hydroxide to dissolve the folic acid for every 100 mL of Standard stock solution prepared.]

Standard preparation— Transfer 4.0 mL of Standard stock solution to a 50-mL volumetric flask, add 4.0 mL of Internal standard solution, dilute with Mobile phase to volume, and mix.

Assay stock solution— Transfer an accurately weighed quantity of about 100 mg of Folic Acid to a 100-mL volumetric flask, add about 40 mL of Mobile phase and 1 mL of 10% ammonium hydroxide to dissolve. Dilute with Mobile phase to volume, and mix.

Assay preparation— Transfer 4.0 mL of the Assay stock solution to a 50-mL volumetric flask, add 4.0 mL of Internal standard solution, dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 280-nm detector and a 4.0-mm × 25-cm column that contains packing L1. The flow rate is about 1.2 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the resolution, R, between methylparaben and folic acid is not less than 3.6, and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C19H19N7O6 in the portion of Folic Acid taken by the formula: in which C is the concentration, in mg per mL, of USP Folic Acid RS, on the anhydrous basis, in the Standard preparation; and RU and RS are the ratios of the response of the folic acid peak to that of the methylparaben peak obtained from the Assay preparation and Standard preparation, respectively.