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

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

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

pH8.0Buffer— Prepare a solution of dibasic potassium phosphate (2in 100),and adjust with phosphoric acid to a pHof 8.0.

pH9.0Buffer— Prepare a solution of dibasic potassium phosphate (3.5in 100),and adjust with potassium hydroxide TSor diluted phosphoric acid (1in 10),as appropriate,to a pHof 9.0.

pH3.5Buffer— Adjust 20mLof pH8.0Bufferwith phosphoric acid to a pHof 3.5.

Mobile phase— Mix 50mLof pH9.0Bufferwith 400mLof water,add 175mLof tertiary butyl alcohol and 30mLof acetonitrile,dilute with water to 1000mL,and mix.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

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

Standard preparation 1— Transfer about 40mg of USP Erythromycin RS,accurately weighed,to a conical flask,add 5.0mLof methanol,and swirl to dissolve.Add 5.0mLof pH8.0Buffer,and mix.

Standard preparation 2— Transfer about 6mg 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-mLconical flask,add 15.0mLof methanol,and swirl to dissolve.Add 15.0mLof pH8.0Buffer,and mix.

Erythromycin Aenol ether solution— Dissolve about 5mg of USP Erythromycin RSin 1mLof methanol.Add 5mLof pH3.5Buffer,mix,and allow to stand for about 30minutes.

Assay preparation— Transfer about 165mg of Erythromycin Stearate,accurately weighed,to a 100-mLconical flask,add 15.0mLof methanol,and swirl to dissolve.Add 15.0mLof pH8.0Buffer,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 á621ñ)—The liquid chromatograph is equipped with a 215-nm detector and a 4.6-mm ×25-cm column that contains packing L21(1000Å)and is maintained at a constant temperature of about 70.The flow rate is about 2mLper 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 Nand erythromycin Cis not less than 0.8and between erythromycin related compound Nand erythromycin Anot less than 5.5.Chromatograph the Erythromycin Aenol ether solution,and adjust the duration of chromatography to include the erythromycin Aenol ether peak,which has a retention time of about 4.3to 4.7times 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 100µL)of Standard preparation 1,Standard preparation 2,and the Assay preparationinto the chromatograph;record the chromatograms for a period of time that is adequate to include the erythromycin Aenol ether peak,if present;and measure the areas of the peak responses.Calculate the percentage of erythromycin Ain the portion of Erythromycin Stearate taken by the formula: in which CS1is the concentration,in mg per mL,of USP Erythromycin RSin Standard preparation 1;Pis the designated percentage of erythromycin Ain USP Erythromycin RS;Wis the quantity,in mg,of Erythromycin Stearate taken to prepare the Assay preparation;and rUand rS1are the erythromycin Apeak responses in the chromatograms obtained from the Assay preparationand Standard preparation 1,respectively.

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