á411ñFOLIC ACID ASSAY

USP Reference Standards á11ñ— USP Folic Acid RS.

Mobile Phase— Place 2.0g of monobasic potassium phosphate in a 1-liter volumetric flask,and dissolve in about 650mLof water.Add 12.0mLof a 1in 4solution of tetrabutylammonium hydroxide in methanol,7.0mLof 3Nphosphoric acid,and 240mLof methanol.Cool to room temperature,adjust with either 3Nphosphoric acid or 6Nammonium hydroxide to a pHof 7.0,dilute with water to volume,and mix.Pass through a 0.45-µm filter,and recheck the pHbefore use.[NOTE—The methanol-to-water ratio may be varied by up to 3percent and the pHmay be increased up to 7.15to achieve better separation.]

Diluting Solvent— Prepare as directed under Mobile Phase.Adjust to a pHof 7.0,and bubble nitrogen through the solution for 30minutes before use.

Internal Standard Solution— Dissolve about 25mg of methylparaben in 2.0mLof methanol,dilute with Diluting Solventto 50mL,and mix.

Standard Folic Acid Solution— Transfer about 12mg of USP Folic Acid RS,accurately weighed,to a low-actinic,50-mLvolumetric flask,dissolve in 2mLof ammonium hydroxide,dilute with Diluting Solventto volume,and mix.

Standard Preparation— Transfer 2.0mLof Standard Folic Acid Solutionto a low-actinic,25-mLvolumetric flask,add 2.0mLof Internal Standard Solution,add Diluting Solventto volume,and mix.

Assay Preparation— Transfer an accurately weighed or measured portion of the preparation to be assayed,containing about 1mg of folic acid,to a low-actinic,50-mLvolumetric flask,add 4.0mLof Internal Standard Solution,add Diluting Solventto volume,and mix.

Chromatographic System (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 280-nm detector and a 15-cm ×3.9-mm column that contains packing L1.The flow rate is about 1.0mLper minute.Chromatograph the Standard Preparation,and record the peak responses as directed for Procedure:there is baseline separation of folic acid and methylparaben.

Procedure— Separately inject equal volumes (about 10µL)of Standard Preparationand Assay Preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 0.8for folic acid and 1.0for methylparaben.Calculate the quantity,in µg,of C19H19N7O6in the portion of the preparation taken by the formula: in which Cis the concentration,in µg per mL,of USP Folic Acid RSin the Standard Preparation;and RUand RSare the ratios of the response of the folic acid peak to that of the methylparaben peak obtained from the Assay Preparationand the Standard Preparation,respectively.