A: Infrared Absorption á197Kñ.

B: The chromatogram of the Assay preparationobtained as directed in the Assayexhibits peaks for the tilmicosin trans-isomer and the tilmicosin cis-isomer,the retention times of which correspond to those exhibited in the chromatogram of the Standard preparationobtained as directed in the Assay.

Dibutylammonium phosphate buffer and Diluent—Prepare as directed in the Assay.

Mobile phase A— To 700mLof water add 25mLof Dibutylammonium phosphate buffer,dilute with water to volume,and mix.Degas before use.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Mobile phase B— Use degassed acetonitrile.

Standard solution— Dissolve an accurately weighed quantity of USP Tilmicosin RSin acetonitrile to obtain a solution having a known concentration of about 0.25mg per mL,sonicating if necessary to achieve dissolution.Transfer 5.0mLof this solution to a 25-mLvolumetric flask,dilute with Diluentto volume,and mix.

Test solution— Transfer about 200mg of Tilmicosin,accurately weighed,to a 50-mLvolumetric flask,add 10mLof acetonitrile,and sonicate briefly to dissolve.Dilute with Diluentto volume,and mix.Use this solution within 24hours.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L1and is programmed for gradient elution by delivering a mixture of Mobile phase Aand Mobile phase Bin a ratio of 82:18initially,and by continuously varying the mixture linearly over a period of 30minutes until the final ratio is 60:40.The flow rate is about 1.1mLper minute.Chromatograph the Standard solution,and record the responses as directed for Procedure:the relative retention times are about 0.8for the tilmicosin trans-isomers (two incompletely resolved peaks),0.9for the tilmicosin cis-isomer,and 1.0for the tilmicosin cis-8-epimer.

Procedure— Separately inject equal volumes (about 10µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the area responses for the major peaks.Calculate the percentage of each related compound in the portion of Tilmicosin taken by the formula: in which Cis the concentration,in mg per mL,of USP Tilmicosin RSin the Standard solution,Pis the designated potency,in µg per mg,of tilmicosin trans-isomers,tilmicosin cis-isomer,and tilmicosin cis-8-epimer,respectively.In the USP Tilmicosin RS,Wis the weight,in mg,of Tilmicosin taken to prepare the Test solution,rcis the area response of the individual related compound peak,other than those obtained for tilmicosin trans-isomers,tilmicosin cis-isomer,and tilmicosin cis-8-epimer,and rsis the sum of the peak area responses for the tilmicosin trans-isomers,the tilmicosin cis-isomer,and the tilmicosin cis-8-epimer obtained from the Standard solution.Not more than 3%of any individual related compound,calculated on the anhydrous basis,is found,and the sum of all the related compounds is not more than 10%,calculated on the anhydrous basis.

Dibutylammonium phosphate buffer— Add,with stirring,70mLof dilute phosphoric acid (1in 10)to 16.8mLof dibutylamine (90:10).Allow to cool,and adjust with phosphoric acid to a pHof 2.5±0.1.Dilute with water to 100mL,and mix.

Mobile phase— To 700mLof water,add 115mLof acetonitrile,55mLof tetrahydrofuran,and 25mLof Dibutylammonium phosphate buffer.Dilute with water to 1000mL,and mix.Each component may be degassed before use,or the Mobile phasemay be sparged with helium for 2minutes before use.Store the Mobile phasein a sealed container when not in use.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).[NOTE—Decreasing the proportion of acetonitrile or tetrahydrofuran increases resolution.]

Diluent— To 900mLof water,add 5.71g of phosphoric acid,adjust with 12.5Nsodium hydroxide to a pHof 2.5±0.1,dilute with water to 1000mL,and mix.

Standard preparation— Transfer about 25mg of USP Tilmicosin RS,accurately weighed,to a 50-mLvolumetric flask,add 10mLof acetonitrile,and sonicate to dissolve.Dilute with Diluentto volume,and mix.Use this solution on the day prepared.

Assay preparation— Transfer about 25mg of Tilmicosin,accurately weighed,to a 50-mLvolumetric flask,add 10mLof acetonitrile,and sonicate to dissolve.Dilute with Diluentto volume,and mix.Use this solution on the day prepared.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L1.The flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the responses as directed for Procedure:the relative retention times are about 0.8for the tilmicosin trans-isomers and 1.0for the tilmicosin cis-isomers,the resolution,R,between the tilmicosin trans-isomers peak and the tilmicosin cis-isomers peak is not less than 1.25,the tailing factors for the peaks are not less than 0.7and not more than 2,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the area responses for the major peaks.Calculate the quantity,in µg,of tilmicosin trans-and cis-isomers in the portion of Tilmicosin taken by the formula: in which Cis the concentration,in mg per mL,of USP Tilmicosin RSin the Standard preparation,Pis the designated potency,in µg per mg,of the relevant (transor cis)tilmicosin isomers in the USP Tilmicosin RS,Wis the weight,in mg,of Tilmicosin taken to prepare the Assay preparation,riis the peak area response for the relevant (transor cis)tilmicosin isomers obtained from the Assay preparation,and rSis the peak area response for the relevant (transor cis)tilmicosin isomers obtained from the Standard preparation.Calculate the percentage of tilmicosin (C46H80N2O13)in the portion of Tilmicosin taken by the formula: in which transand cisare the quantities,in µg per mg,of tilmicosin trans-isomers and tilmicosin cis-isomers in the Tilmicosin,as determined above.Calculate the percentages of tilmicosin trans-isomers and tilmicosin cis-isomers taken by the formula: in which isomeris the quantity,in µg per mg,of either the tilmicosin trans-isomers or the tilmicosin cis-isomers in the Tilmicosin,as determined above.