Mobile phase— Dissolve 3.0g of ammonium acetate and 1.0g of ammonium chloride in 760mLof water,and mix.Add 5.0mLof tetrahydrofuran and 240mLof acetonitrile,and mix.Pass this solution through a 0.5µm or finer porosity filter.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Transfer about 20mg of USP Natamycin RS,accurately weighed,to a 100-mLvolumetric flask.Add 5.0mLof tetrahydrofuran,and sonicate for 10minutes.Add 60mLof methanol,and swirl to dissolve.Add 25mLof water,and mix.Allow to cool to room temperature.Dilute with water to volume,and mix.Pass this solution through a suitable membrane filter of 0.5µm or finer porosity.

Resolution solution— Dissolve 20mg of Natamycin in a mixture of 99mLof methanol and 1mLof 0.1Nhydrochloric acid,and allow to stand for 2hours.[NOTE—Use this solution within 1hour.]

Assay preparation— Transfer about 20mg of Natamycin,accurately weighed,to a 100-mLvolumetric flask.Proceed as directed under Standard preparation,beginning with “add 5.0mLof tetrahydrofuran.”

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 303-nm detector and a 4.6-mm ×25-cm analytical column that contains packing L1.[NOTE—A3.9-mm ×20-mm pre-column may be used to extend the useful life of the analytical column.]The flow rate is about 3mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed under Procedure:the column efficiency determined from the analyte peak is not less than 3000theoretical plates,the tailing factor is not less than 0.8and not more than 1.3,and the relative standard deviation for replicate injections is not more than 1.0%.Chromatograph the Resolution solution,and record the peak responses as directed under Procedure:the resolution,R,between natamycin and its methyl ester is not less than 2.5.The relative retention times are about 0.7for natamycin and 1.0for its methyl ester.

Procedure— [NOTE—Use peak areas where peak responses are indicated.]Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the percentage of natamycin (C33H47NO13)in the portion of Natamycin taken by the formula: in which WSis the weight,in mg,of USP Natamycin RStaken to prepare the Standard preparation;PSis the stated content,in µg per mg,of USP Natamycin RS;WUis the weight,in mg,of Natamycin taken to prepare the Assay preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.