Identification—
A:
Transfer a quantity of Ointment, equivalent to about 5 mg of erythromycin, to a separator containing 50 mL of solvent hexane. Shake until dissolved. Extract with three separate 20-mL portions of methanol. Combine the methanol extracts in a beaker, and evaporate to dryness. Dissolve the residue in 2 mL of methanol (test solution). Proceed as directed in the
Identification test under
Erythromycin Delayed-Release Capsules, beginning with “Prepare a Standard solution of
USP Erythromycin RS.”
B:
The retention times of the peaks for erythromycin A, erythromycin B, and erythromycin C in the chromatogram of the Assay preparation correspond to those in the chromatograms of the Standard preparation and Erythromycins B and C standard preparation, as obtained in the Assay.
Assay—
Solution A—
Prepare a degassed mixture of acetonitrile and water (90:10). Store in a reservoir protected from air by sparging with helium.
Solution B—
To 1000 mL of degassed water add 0.5 mL of sodium hydroxide solution (1 in 2), using a suitable syringe and needle to minimize exposure to air. Degas, and store in a reservoir protected from air by sparging with helium.
Solution C—
Use degassed water, and store in a reservoir protected from air by sparging with helium.
Mobile phase—
Using a suitable pumping system, pump
Solution A,
Solution C, and
Solution B from the respective reservoirs in the ratio of 56:37:7. Make any necessary adjustments (see
System Suitability under
Chromatography 
621
).
Diluent—
Prepare a mixture of methanol and water (50:50).
Standard preparation—
Quantitatively prepare a solution of
USP Erythromycin RS in
Diluent having a known concentration of about 0.66 mg per mL.
System suitability solution—
Transfer about 2 mg of
USP Erythromycin Related Compound N RS to a 10-mL volumetric flask, add 0.4 mL of the
Standard preparation and 6 mL of
Erythromycins B and C standard preparation, and mix. Dilute with
Erythromycins B and C standard preparation to volume, and mix.
Assay preparation—
Transfer an accurately weighed portion of Ointment, equivalent to about 60 mg of erythromycin, to a 125-mL separator. Add 50 mL of solvent hexane, and shake until dissolved. Extract with four separate 20-mL portions of Diluent, collecting the extracts in a 100-mL volumetric flask. Dilute the combined extracts with Diluent to volume, mix, and pass a portion of the solution through a filter having a 0.45-µm porosity. Use the clear filtrate as the Assay preparation.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with an electrochemical detector, a glassy carbon electrode fitted with three gaskets, a 4-mm × 5-cm guard column that contains 8-µm packing L50, and a 4-mm × 25-cm analytical column that contains 8-µm packing L50. The electrochemical detector is used in the pulsed integrated amperometric mode with a range of 10 nC, an output of 1 V full scale, a rise time of 0.6 second, positive polarity, E = 0.9 V; t1 = 400 ms; E2 = 0.9 V; t2 = 100 ms; E3 = –0.9 V; t3 = 100 ms. The flow rate is about 1 mL per minute. Chromatograph the
System suitability solution, and record the peak responses as directed for
Procedure: the relative retention times are about 0.4 for erythromycin related compound N, 0.5 for erythromycin C, 1.0 for erythromycin A, and 1.6 for erythromycin B; the resolution,
R, between erythromycin related compound N and erythromycin C is not less than 0.6, between erythromycin C and erythromycin A not less than 2.5, and between erythromycin A and erythromycin B not less than 2.5. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the tailing factor is not more than 2; and the relative standard deviation for replicate injections is not more than 3%.
[NOTE—Turn off the electrochemical detector before stopping the flow of the
Mobile phase.]
Procedure—
Separately inject equal volumes (about 10 µL) of the
Standard preparation,
Erythromycins B and C standard preparation, and the
Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas. Calculate the percentage of erythromycin A in the portion of Ointment taken by the formula:
0.1(CP/W)(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Erythromycin RS in the
Standard preparation; P is the designated percentage of erythromycin A in
USP Erythromycin RS;
W is the quantity, in g, of Ointment taken to prepare the
Assay preparation; and
rU and
rS are the erythromycin A peak areas obtained from the
Assay preparation and the
Standard preparation, respectively. Calculate the percentages of erythromycin B and erythromycin C in the portion of Ointment taken by the formula:
0.0001(CP/W)(rU / rS),
in which
C is the concentration, in µg per mL, of the relevant USP Reference Standard in the
Erythromycins B and C standard preparation; P is the designated percentage of erythromycin B or erythromycin C in the relevant USP Reference Standard;
W is the quantity, in g, of Ointment taken to prepare the
Assay preparation; and
rU and
rS are the peak areas for the relevant analyte obtained from the
Assay preparation and the
Erythromycins B and C standard preparation, respectively. Calculate the percentage content of erythromycin in the Ointment by adding the percentages of erythromycin A, erythromycin B, and erythromycin C found.
