100A/P,

100(A+B)/P,

Mobile phase— Prepare a filtered and 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 3g of vanillin in a mixture of methanol and sulfuric acid (95:2).[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 vanillin.]

Standard preparation— Dissolve an accurately weighed quantity of USP Monensin Sodium RSquantitatively in methanol to obtain a solution containing the equivalent of 1000µg of monensin per mL.Dilute an accurately measured volume of this stock solution quantitatively with Diluentto obtain a solution containing 20.0µg of monensin per mL.

Assay preparation— Transfer about 500mg of Monensin,accurately weighed,to a 250-mLflask,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 monensin per mL.

Resolution solution— Prepare a solution in Neutralized methanolcontaining about 1mg of USP Monensin Sodium RSand 3mg of USP Narasin 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 L1and 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 0.9for monensin B,1.0for monensin A,1.3for narasin A,and 1.5for narasin 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 is not less than 3.5.Chromatograph the Standard preparation,and record the peak responses as directed under Procedure:the tailing factor is not more than 1.4,and the relative standard deviation for replicate injections is not more than 2.0%.[NOTE—After use,flush the system with methanol.]

Procedure— [NOTE—Use peak areas where peak responses are indicated.]Separately inject equal volumes (about 200µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks,including a peak for monensin C/D,if present,at a retention time of about 1.1relative to that of the main monensin Apeak in the chromatogram obtained from the Assay preparation.Calculate the quantity,in µg,of monensin Ain each mg of the Monensin taken by the formula: in which Cis the concentration,in µg per mL,of monensin activity in the Standard preparation,based on the quantity of USP Monensin Sodium RStaken,its designated potency,in µg per mg,and the extent of dilution,Fis the designated percentage of monensin Ain USP Monensin Sodium RS,Dis the dilution factor used in preparing the Assay preparation,Wis the quantity,in g,of Monensin taken to prepare the Assay preparation,and rUand rSare the monensin Apeak responses obtained from the Assay preparationand the Standard preparation,respectively.Calculate the quantity,in µg,of monensin Bin each mg of the Monensin taken by the same formula,except that rUis the monensin Bpeak response obtained from the Assay preparationand rSis the monensin Apeak response obtained from the Standard preparation.Calculate the quantity,in µg,of monensin C/Din each mg of the Monensin taken by the same formula,except that rUis the monensin C/Dpeak response obtained from the Assay preparation.Calculate the potency,in µg of monensin,in each mg of the Monensin taken by the formula: in which Ais the quantity,in µg,of monensin Ain each mg of the Monensin taken,as calculated above,and Bis the quantity,in µg,of monensin Bin each mg of the Monensin taken,and C/Dis the quantity,in µg,of monensin C/Din each mg of Monensin taken,as calculated above.