Tilmicosin Injection »Tilmicosin Injection is a sterile solution of Tilmicosin in a mixture of Propylene Glycol and Water for Injection,and is solubilized with the aid of Phosphoric Acid.It contains not less than 90.0percent and not more than 110.0percent of the labeled amount of tilmicosin (C46H80N2O13). Packaging and storage— Preserve in light-resistant Containers for Injectionsas described under Injections á1ñ.Store at or below 30. Labeling— Label the Injection to indicate that it is for veterinary use only. USP Reference standards á11ñ USP Endotoxin RS.USP Tilmicosin RS. Identification— The chromatogram of the Assay preparationobtained as directed in the Assayexhibits major peaks for the tilmicosin trans-isomers and the tilmicosin cis-isomers,the retention times of which correspond to those exhibited in the chromatogram of the Standard preparationobtained as directed in the Assay. Bacterial endotoxins á85ñ It contains not more than 0.5USP Endotoxin Unit per mg of tilmicosin. Sterility á71ñ It meets the requirements when tested as directed for Membrane Filtrationunder Test for Sterility of the Product to be Examined,except that the test mixture is prepared as follows.Transfer aseptically 1mLfrom each of 20containers to a vessel containing 200mLof a mixture containing 2mLof polysorbate 20in pH7phosphate buffer prepared as directed for Buffer No.16in the section Phosphate Buffers and Other Solutionsunder Antimicrobial Assays—Antibiotics á81ñ.After that solution has been filtered,wash the filter with three 100-mLportions of the same solution,instead of Diluting Fluid A. pHá791ñ: between 5.5and 6.5. Particulate matter á788ñ Use the procedure under Microscopic Particle Count Test:not more than 50particles per mLthat are equal to or greater than 10µm in effective spherical diameter,and not more than 5particles per mLthat are equal to or greater than 25µm in effective spherical diameter are found. Content of propylene glycol— Internal standard solution— Prepare a solution of pentadecane in acetone containing about 0.5mg per mL. Standard solution— Transfer about 125mg of propylene glycol,accurately weighed,to a 100-mLvolumetric flask,dilute with acetone to volume,and mix.Mix equal,accurately measured volumes of this solution and the Internal standard solution.This solution contains about 0.625mg of propylene glycol per mL. Test solution— Transfer an accurately measured volume of Injection,equivalent to about 250mg of propylene glycol,to a 200-mLvolumetric flask,dilute with acetone to volume,and mix.Mix equal,accurately measured volumes of this solution and the Internal standard solution. Chromatographic system (see Chromatography á621ñ)—The gas chromatograph is equipped with a flame-ionization detector and a 0.53-mm ×15-m fused silica column that has liquid phase G16bonded to the inner surface at a thickness of 1µm.The injection port and the detector block are maintained at about 250,and the column is maintained at a temperature of about 100.Helium is used as the carrier gas at a flow rate of about 15mLper minute.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.6for pentadecane and 1.0for propylene glycol,the resolution,R,between the pentadecane peak and the propylene glycol peak is not less than 7.0,and the relative standard deviation for replicate injections is not more than 2.0%. Procedure— Separately inject equal volumes (about 1µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the area responses for the major peaks.Calculate the quantity,in mg,of propylene glycol in each mLof the Injection taken by the formula: 400(C/V)(RU/RS), in which Cis the concentration,in mg per mL,of propylene glycol in the Standard solution,Vis the volume,in mL,of Injection taken,and RUand RSare the ratios of the propylene glycol peak area response to the pentadecane peak area response obtained from the Test solutionand the Standard solution,respectively.Between 80.0%and 120.0%of the labeled amount of propylene glycol is found. Assay— Dibutylammonium phosphate buffer— To 700mLof water,add 168mLof dibutylamine.Add phosphoric acid slowly until the dibutylamine is just dissolved,stirring vigorously during the addition.Allow to cool,and adjust with phosphoric acid to a pHof 2.55±0.05.Dilute with water to 1000mL,mix,and filter under vacuum. 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 filtered before mixing,or the Mobile phasemay be filtered,minimizing solvent evaporation.Store the Mobile phasein a sealed container when not in use.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ). Diluent— To 700mLof water add 200mLof acetonitrile and 25mLof Dibutylammonium phosphate buffer,dilute with water to 1000mL,and mix. Standard preparation— Quantitatively dissolve an accurately weighed quantity of USP Tilmicosin RSin acetonitrile to obtain a solution having a known concentration of about 2.5mg per mL.Transfer 4.0mLof this solution to a 20-mLvolumetric flask,add 10mLof water,and 0.5mLof Dibutylammonium phosphate buffer,dilute with water to volume,and mix. Assay preparation— Transfer an accurately measured volume of Injection,equivalent to about 300mg of tilmicosin,to a 30-mLvolumetric flask,dilute with Diluentto volume,and mix.Transfer 5.0mLof this solution to a 100-mLvolumetric flask,dilute with Diluentto volume,and mix. 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 1.1mLper 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 1.5%. 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 mg,of each of the tilmicosin isomers in each mLof the Injection taken by the formula: 0.6(CP/V)(rI/rs), in which Cis the concentration,in mg per mL,of USP Tilmicosin RSin the Standard preparation,Pis the potency,in µg per mg,of the relevant (transor cis)tilmicosin isomers in the USP Tilmicosin RS,Vis the volume of Injection taken to prepare the Assay preparation,rIis the peak response of the relevant tilmicosin isomers obtained from the Assay preparation,and rsis the sum of the responses of the tilmicosin trans-isomers and tilmicosin cis-isomers obtained from the Standard preparation.Calculate the quantity,in mg,of C46H80N2O13in each mLof the Injection taken by adding the quantities,in mg per mL,of cis-and trans-isomers found. Auxiliary Information— Staff Liaison:Ian DeVeau,Ph.D.,Senior Scientist Expert Committee:(VET)Veterinary Drugs USP28–NF23Page 1932 Phone Number:1-301-816-8178