Internal standard solution— Prepare a solution of pentadecane in acetone containing about 0.5 mg per mL.

Standard solution— Transfer about 125 mg of propylene glycol, accurately weighed, to a 100-mL volumetric 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 0.625 mg of propylene glycol per mL.

Test solution— Transfer an accurately measured volume of Injection, equivalent to about 250 mg of propylene glycol, to a 200-mL volumetric 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 621)—The gas chromatograph is equipped with a flame-ionization detector and a 0.53-mm × 15-m fused silica column that has liquid phase G16 bonded to the inner surface at a thickness of 1 µm. 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 15 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.6 for pentadecane and 1.0 for 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 1 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 quantity, in mg, of propylene glycol in each mL of the Injection taken by the formula: in which C is the concentration, in mg per mL, of propylene glycol in the Standard solution, V is the volume, in mL, of Injection taken, and RU and RS are the ratios of the propylene glycol peak area response to the pentadecane peak area response obtained from the Test solution and the Standard solution, respectively. Between 80.0% and 120.0% of the labeled amount of propylene glycol is found.

Dibutylammonium phosphate buffer— To 700 mL of water, add 168 mL of 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 pH of 2.55 ± 0.05. Dilute with water to 1000 mL, mix, and filter under vacuum.

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 filtered before mixing, or the Mobile phase may be filtered, minimizing solvent evaporation. Store the Mobile phase in a sealed container when not in use. Make adjustments if necessary (see System Suitability under Chromatography 621).

Diluent— To 700 mL of water add 200 mL of acetonitrile and 25 mL of Dibutylammonium phosphate buffer, dilute with water to 1000 mL, and mix.

Standard preparation— Quantitatively dissolve an accurately weighed quantity of USP Tilmicosin RS in acetonitrile to obtain a solution having a known concentration of about 2.5 mg per mL. Transfer 4.0 mL of this solution to a 20-mL volumetric flask, add 10 mL of water, and 0.5 mL of Dibutylammonium phosphate buffer, dilute with water to volume, and mix.

Assay preparation— Transfer an accurately measured volume of Injection, equivalent to about 300 mg of tilmicosin, to a 30-mL volumetric flask, dilute with Diluent to volume, and mix. Transfer 5.0 mL of this solution to a 100-mL volumetric flask, dilute with Diluent to volume, and mix.

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 1.1 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 1.5%.

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 mg, of each of the tilmicosin isomers in each mL of the Injection 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 potency, in µg per mg, of the relevant (trans or cis) tilmicosin isomers in the USP Tilmicosin RS, V is the volume of Injection taken to prepare the Assay preparation, rI is the peak response of the relevant 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 quantity, in mg, of C46H80N2O13 in each mL of the Injection taken by adding the quantities, in mg per mL, of cis- and trans-isomers found.