Medium: water;500mL.

Apparatus 2: 50rpm.

Time: 45minutes.

Procedure— Determine the amount of C19H19N7O6dissolved,employing the procedure set forth in the Assay,making any necessary modifications.

Tolerances— Not less than 75%(Q)of the labeled amount of C19H19N7O6is dissolved in 45minutes.

Mobile phase— Transfer 35.1g of sodium perchlorate and 1.40g of monobasic potassium phosphate,accurately weighed,to a 1-Lvolumetric flask,add 7.0mLof 1Npotassium hydroxide and 40mLof methanol,dilute with water to volume,and mix.Adjust with 1Npotassium hydroxide or phosphoric acid to a pHof 7.2.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

System suitability solution— Prepare a solution containing about 0.2mg per mLeach of USP Folic Acid RSand USP Folic Acid Related Compound A RSin an aqueous solvent containing 2mLof ammonium hydroxide and 1g of sodium perchlorate per 100mL.Before use,pass through a filter having a 1-µm or finer porosity.

Standard preparation— Accurately weigh about 30mg of USP Folic Acid RS,corrected for water content,and dissolve in an aqueous solvent containing 2mLof ammonium hydroxide and 1g of sodium perchlorate per 100mL.Using the same solvent,adjust the volume quantitatively to obtain a solution having a known concentration of about 0.20mg per mL.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer a portion of the powder,accurately weighed and equivalent to about 10mg of folic acid,to a 50-mLvolumetric flask with the aid of an aqueous solvent containing 2mLof ammonium hydroxide and 1g of sodium perchlorate per 100mL.Shake gently until the folic acid has dissolved,dilute with the same solvent to volume,mix,and pass through a dry filter,discarding the first portion of the filtrate.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 1mLper minute.Chromatograph the System suitability solutionand the Standard preparation,and record the peak responses as directed for Procedure:the resolution,R,between folic acid related compound A(calcium formyltetrahydrofolate)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 25µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of folic acid (C19H19N7O6)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Folic Acid RSin the Standard preparation;Vis the volume,in mL,of the Assay preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.