A: Infrared Absorption 197K.

B: The retention time of the tiamulin peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.

Internal standard solution— To a 100-mL volumetric flask add about 90 mL of dimethylformamide and 150 µL of dioxane, dilute with dimethylformamide to volume, and mix.

Standard solution— To a 50-mL volumetric flask add about 40 mL of Internal standard solution, 100 µL of dehydrated alcohol, and 10 µL of toluene. Dilute with Internal standard solution to volume, and mix. Transfer 2 mL of this solution to a 20-mL headspace vial.

Test solution— Transfer about 200 mg of Tiamulin, accurately weighed, to a 20-mL headspace vial. Add 2 mL of Internal standard solution, close the vial, and sonicate to dissolve.

Chromatographic system (see Chromatography 621)— The gas chromatograph is equipped with a headspace injector, a flame-ionization detector, and a 0.530-mm × 30-m capillary column coated with a 1.0-µm film of phase G16 and operated in 1/10 split mode. The carrier gas is helium, flowing at a rate of about 30 mL per minute. The vial is heated to 100, the injector syringe is heated to 130, the injection port temperature is maintained at 200, and the detector temperature is maintained at 250. The column temperature is programmed to be isothermal at 50 for 8 minutes followed by a linear increase of 40 per minute until the temperature has reached 150. Chromatograph the Standard solution, and record the peak areas as directed for Procedure: the relative retention times are about 0.5, 0.9, and 1.0 for alcohol, toluene, and dioxane, respectively; the resolution, R, between toluene and dioxane is not less than 2.0; the tailing factor of toluene and alcohol is not more than 2.0; and the relative standard deviation for triplicate injections, calculated from the ratio between the toluene or alcohol peak relative to the internal standard peak, is not more than 5.0%.

Procedure— Separately inject equal volumes (about 1.0 mL) of the headspace of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses for alcohol, toluene, and dioxane. Calculate the percentages (w/w) of alcohol and toluene in the portion of Tiamulin taken by the formula: in which P is the percent purity of the solvent of interest in the Standard solution; D is the density, in mg per mL, of the solvent of interest used to prepare the Standard solution; V is the volume, in mL, of the solvent of interest used to prepare the Standard solution; WU is the weight, in mg, of Tiamulin taken to prepare the Test solution; and RU and RS are the peak response ratios of the solvents of interest obtained from the Test solution and the Standard solution, respectively: not more than 1.0% of alcohol is found, not more than 0.08% of toluene is found, and not more than 1.0% of alcohol plus toluene is found.

Ammonium carbonate buffer, Mobile phase, and Diluent— Proceed as directed in the Assay.

Standard solution— Use the Standard preparation, prepared as directed in the Assay.

Toluene solution— Transfer 0.1 mL of toluene to a 100-mL volumetric flask, dilute with acetonitrile to volume, and mix. Transfer 0.1 mL of this solution to another 100-mL volumetric flask, dilute with Diluent to volume, and mix.

Test solution 1— Use the Assay preparation.

Test solution 2— Add 1.0 mL of Test solution 1 to a 100-mL volumetric flask, dilute with Diluent to volume, and mix.

Chromatographic system— Proceed as directed in the Assay. Chromatograph the Standard solution, and record the peak areas as directed for Procedure: the retention times of possible tiamulin-related impurities relative to tiamulin are about 0.22 for mutilin, 0.50 for 2-(benzylsulfanyl)-N,N-diethylethanamine, 0.66 for 2,2¢-(disulfane-1,2-diyl)bis(N,N-diethylethanamine), 1.1 for hydroxy-11-oxotiamulin, 1.6 for 1-hydroxy-11-oxotiamulin, and 2.4 for 11-oxotiamulin. [NOTE—Impurities are not limited to those listed above.]

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution, Toluene solution, Test solution 1, and Test solution 2 into the chromatograph, record the chromatograms, identify the peaks, and measure the areas for the major peaks. Disregarding the toluene peak and any peak in the chromatogram of Test solution 1 less than 0.1 times the area of the principal peak in the chromatogram obtained with Test solution 2, calculate the area percentage of each impurity, relative to tiamulin, in the portion of Tiamulin taken by the formula: in which ri is the peak area of each individual impurity obtained from Test solution 1; and rS is the peak area of Tiamulin obtained from Test solution 2: not more than 1.0% of any identified impurity is found; not more than 0.2% of any unidentified impurity is found; and not more than 3.0% of total impurities is found.

(Official January 1, 2007)

Ammonium carbonate buffer— Dissolve 10.0 g of ammonium carbonate in water, add 22 mL of perchloric acid TS, and dilute with water to 1000 mL. Adjust with ammonium hydroxide to a pH of 10.0.

Mobile phase— Mix 490 mL of methanol, 300 mL of Ammonium carbonate buffer, and 210 mL of acetonitrile.

Diluent— Mix 500 mL of Ammonium carbonate buffer and 500 mL of acetonitrile.

Standard preparation— Dissolve an accurately weighed quantity of USP Tiamulin Fumarate RS in Diluent to obtain a solution having a known concentration of about 5.0 mg per mL.

Assay preparation— Dissolve an accurately weighed quantity of Tiamulin in Diluent to obtain a solution having a known concentration of about 4.0 mg per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 212-nm detector and a 4.6-mm × 15-cm column that contains 5-µm packing L1. The flow rate is about 1.0 mL per minute. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the resolution, R, between tiamulin and its subsequent peak is not less than 2.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the percentage of C28H47NO4S in the portion of Tiamulin taken by the formula: in which P is the labeled percentage of tiamulin in USP Tiamulin Fumarate RS; CS and CU are the concentrations, in mg per mL, of the Standard preparation and the Assay preparation, respectively; and rU and rS are the peak areas of tiamulin obtained from the Assay preparation and Standard preparation, respectively.USP29