A: Infrared Absorption 197K.

B: The chromatogram of the Assay preparation obtained as directed in the Assay exhibits 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 preparation obtained as directed in the Assay.

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

Mobile phase A— To 700 mL of water add 25 mL of Dibutylammonium phosphate buffer, dilute with water to volume, and mix. Degas before use. Make adjustments if necessary (see System Suitability under Chromatography 621).

Mobile phase B— Use degassed acetonitrile.

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

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

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1 and is programmed for gradient elution by delivering a mixture of Mobile phase A and Mobile phase B in a ratio of 82:18 initially, and by continuously varying the mixture linearly over a period of 30 minutes until the final ratio is 60:40. The flow rate is about 1.1 mL per minute. Chromatograph the Standard solution, and record the responses as directed for Procedure: the relative retention times are about 0.8 for the tilmicosin trans-isomers (two incompletely resolved peaks), 0.9 for the tilmicosin cis-isomer, and 1.0 for the tilmicosin cis-8-epimer.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution into 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 C is the concentration, in mg per mL, of USP Tilmicosin RS in the Standard solution, P is 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, W is the weight, in mg, of Tilmicosin taken to prepare the Test solution, rc is 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 rs is 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.

(Official January 1, 2007)

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

Mobile phase— To 700 mL of water, add 115 mL of acetonitrile, 55 mL of tetrahydrofuran, and 25 mL of Dibutylammonium phosphate buffer. Dilute with water to 1000 mL, and mix. Each component may be degassed before use, or the Mobile phase may be sparged with helium for 2 minutes before use. Store the Mobile phase in a sealed container when not in use. Make adjustments if necessary (see System Suitability under Chromatography 621). [NOTE—Decreasing the proportion of acetonitrile or tetrahydrofuran increases resolution.]

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

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

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

Chromatographic system (see Chromatography 621)—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 mL per minute. Chromatograph the Standard preparation, and record the responses as directed for Procedure: the relative retention times are about 0.8 for the tilmicosin trans-isomers and 1.0 for 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 0.7 and not more than 2, and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into 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 C is the concentration, in mg per mL, of USP Tilmicosin RS in the Standard preparation, P is the designated potency, in µg per mg, of the relevant (trans or cis) tilmicosin isomers in the USP Tilmicosin RS, W is the weight, in mg, of Tilmicosin taken to prepare the Assay preparation, ri is the peak area response for the relevant (trans or cis) tilmicosin isomers obtained from the Assay preparation, and rS is the peak area response for the relevant (trans or 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 trans and cis are 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 isomer is the quantity, in µg per mg, of either the tilmicosin trans-isomers or the tilmicosin cis-isomers in the Tilmicosin, as determined above.