30(CS2P/W)(ri / rS2),

(30 / 11)(CS2P / W)(rE / rS2),

(30 / 6.6)(CS2P / W)(rP / rS2),

(Official January 1, 2007)

pH 8.0 Buffer— Prepare a solution of dibasic potassium phosphate (2 in 100), and adjust with phosphoric acid to a pH of 8.0.

pH 9.0 Buffer— Prepare a solution of dibasic potassium phosphate (3.5 in 100), and adjust with potassium hydroxide TS or diluted phosphoric acid (1 in 10), as appropriate, to a pH of 9.0.

pH 3.5 Buffer— Adjust 20 mL of pH 8.0 Buffer with phosphoric acid to a pH of 3.5.

Mobile phase— Mix 50 mL of pH 9.0 Buffer with 400 mL of water, add 175 mL of tertiary butyl alcohol and 30 mL of acetonitrile, dilute with water to 1000 mL, and mix. Make adjustments if necessary (see System Suitability under Chromatography 621).

NOTE—Use the following solutions promptly, or within 1 day if stored in a refrigerator.

Standard preparation 1— Transfer about 40 mg of USP Erythromycin RS, accurately weighed, to a conical flask, add 5.0 mL of methanol, and swirl to dissolve. Add 5.0 mL of pH 8.0 Buffer, and mix.

Standard preparation 2— Transfer about 6 mg each of USP Erythromycin RS, USP Erythromycin B RS, USP Erythromycin C RS, and USP Erythromycin Related Compound N RS, all accurately weighed, to a 50-mL conical flask, add 15.0 mL of methanol, and swirl to dissolve. Add 15.0 mL of pH 8.0 Buffer, and mix.

Erythromycin A enol ether solution— Dissolve about 5 mg of USP Erythromycin RS in 1 mL of methanol. Add 5 mL of pH 3.5 Buffer, mix, and allow to stand for about 30 minutes.

Assay preparation— Transfer about 165 mg of Erythromycin Stearate, accurately weighed, to a 100-mL conical flask, add 15.0 mL of methanol, and swirl to dissolve. Add 15.0 mL of pH 8.0 Buffer, and mix. Allow the resulting suspension to settle, and pass a portion of the supernatant through a filter having a 0.2-µm or finer porosity. Use the clear filtrate.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 215-nm detector and a 4.6-mm × 25-cm column that contains packing L21 (1000 ) and is maintained at a constant temperature of about 70. The flow rate is about 2 mL per minute. Chromatograph Standard preparation 2, and record the peak responses as directed for Procedure: the order of elution of the components is erythromycin related compound N, erythromycin C, erythromycin A, and erythromycin B; and the resolution, R, between erythromycin related compound N and erythromycin C is not less than 0.8 and between erythromycin related compound N and erythromycin A not less than 5.5. Chromatograph the Erythromycin A enol ether solution, and adjust the duration of chromatography to include the erythromycin A enol ether peak, which has a retention time of about 4.3 to 4.7 times that of erythromycin A. Chromatograph Standard preparation 1, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 100 µL) of Standard preparation 1, Standard preparation 2, and the Assay preparation into the chromatograph; record the chromatograms for a period of time that is adequate to include the erythromycin A enol ether peak, if present; and measure the areas of the peak responses. Calculate the percentage of erythromycin A in the portion of Erythromycin Stearate taken by the formula: in which CS1 is the concentration, in mg per mL, of USP Erythromycin RS in Standard preparation 1; P is the designated percentage of erythromycin A in USP Erythromycin RS; W is the quantity, in mg, of Erythromycin Stearate taken to prepare the Assay preparation; and rU and rS1 are the erythromycin A peak responses in the chromatograms obtained from the Assay preparation and Standard preparation 1, respectively.

30(CS2P / W)(rU / rS2),