100A/[A+(D+I)],

Mobile phase— Prepare a degassed mixture of methanol,water,and glacial acetic acid (94:6:0.1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Neutralized methanol— Add 1g of sodium bicarbonate to 4liters of methanol,mix,and filter.

Diluent— Prepare a mixture of methanol and water (9:1).

Derivatizing reagent— Dissolve 30g of vanillin in a mixture of methanol and sulfuric acid (950:20)in a container protected from light.[Caution—To avoid splattering,add the sulfuric acid carefully and slowly with a pipet;do not pour.Allow the mixture of methanol and sulfuric acid to cool before adding the vanillin.]Do not filter.

Standard preparations— Dissolve an accurately weighed quantity of USP Narasin RSin Neutralized methanolto obtain a solution having a known concentration of about 1mg per mL.Transfer 1.0mLof this stock solution to a 200-mLvolumetric flask,and transfer 2.0mLand 4.0mLof the stock solution to two separate 100-mLvolumetric flasks,dilute each with Diluentto volume,and mix.These solutions contain about 5,20,and 40µg of USP Narasin RSper mL.

Assay preparation— Transfer about 5g of Narasin Granular,accurately weighed,to a suitable container,add 200.0mLof Diluent,and shake by mechanical means for 1hour.Allow the solids to settle,and dilute an accurately measured volume of the supernatant quantitatively with Diluentto obtain a solution containing about 20µg of narasin per mL.Pass a portion of this solution through a filter having a porosity of 0.5µm or less,and use the filtrate as the Assay preparation.

Resolution solution— Prepare a solution in Neutralized methanolcontaining about 3mg of USP Narasin RSand 1mg of USP Monensin Sodium RSper mL.Transfer 2mLof this solution to a 200-mLvolumetric flask,dilute with Diluentto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 4.6-mm ×25-cm column that contains packing L1,and the outlet of which is attached to a tee,the opposing arm of which is attached to a tube from which is pumped the Derivatizing reagent,and the outlet of which is connected to a 2-mLpostcolumn reaction coil maintained at 98.The outlet of the reaction coil is connected to a detector set at 520nm.The Mobile phaseand the Derivatizing reagentflow at the rate of about 0.7mLper minute.Chromatograph the Resolution solution,and record the peak responses as directed under Procedure:the relative retention times are about 0.7for monensin B,0.75for monensin A,1.0for narasin A,and 1.1for narasin D+I;the resolution,R,between the monensin Bpeak and the monensin Apeak is not less than 1.25;and between the monensin Apeak and the narasin Apeak not less than 3.5.Chromatograph the Standard preparations,and record the peak responses as directed under Procedure:the tailing factor for the narasin Apeak is not more than 1.4when calculated by the formula: in which W0.1is the width of the peak at 10%of peak height,and fis the distance from the peak maximum to the leading edge of the peak,the distance being measured at a point on the baseline at which 10%peak height is reached.The relative standard deviation for replicate injections is not more than 10%.[NOTE—After use,flush the system with methanol.]

Procedure— Separately inject equal volumes (about 200µL)of the Standard preparationsand the Assay preparationinto the chromatograph,and measure the areas of the peak responses for the narasin Aand narasin D+Ipeaks.

(CA+CD+IF)(VE/M),

(1.402D+0.0111I)/(D+I),