Identification—
The IR absorption spectrum of a solution of it containing 50 mg per mL, previously dried at a pressure not exceeding 5 mm of mercury at 60
for 3 hours, in chloroform, determined in a 0.1-mm cell, exhibits maxima only at the same wavelengths as that of a similar preparation of
USP Erythromycin RS, except in the region between 1980 cm
1 and 2050 cm
1.
Limit of thiocyanate—
Standard solutions—
Transfer about 100 mg of potassium thiocyanate, previously dried at 105
for 1 hour, cooled, and accurately weighed, to each of two 50-mL volumetric flasks. Add about 20 mL of methanol to each flask, swirl to dissolve, dilute with methanol to volume, and mix. Transfer 5.0 mL of each of these stock solutions to separate 50-mL volumetric flasks, dilute with methanol to volume, and mix. Transfer 5.0 mL of each of these intermediate solutions to separate 50-mL low-actinic volumetric flasks. To each flask add 1.0 mL of
ferric chloride TS, dilute with methanol to volume, and mix.
[NOTE—Use these
Standard solutions within 30 minutes.
]
Test solution—
Transfer about 100 mg of Erythromycin, accurately weighed, to a 50-mL low-actinic volumetric flask. Add 20 mL of methanol, and swirl to dissolve. Add 1.0 mL of
ferric chloride TS, dilute with methanol to volume, and mix.
[NOTE—Use this
Test solution within 30 minutes.
]
Blank solution—
Transfer 1.0 mL of
ferric chloride TS to a 50-mL low-actinic volumetric flask, dilute with methanol to volume, and mix.
[NOTE—Use this
Blank solution within 30 minutes.
]
Procedure—
Determine the absorbances of each
Standard solution and the
Test solution at the wavelength of maximum absorbance at about 492 nm with a spectrophotometer, using the
Blank solution to zero the instrument. Calculate the suitability value,
S, by the formula:
(A1 / W1)(W2 / A2),
in which
A1 and
A2 are the absorbance values obtained from the respective
Standard solutions; and
W1 and
W2 are the weights, in mg, of the potassium thiocyanate taken to prepare the corresponding
Standard solutions. In a suitable determination, the value,
S, is not less than 0.985 and not more than 1.015. Calculate the percentage of thiocyanate in the Erythromycin taken by the formula:
(58.08/97.18)(AU / WU)(0.5)[(W1 / A1) + (W2 / A2)],
in which 58.08 and 97.18 are the molecular weights of the thiocyanate moiety and of potassium thiocyanate, respectively;
AU is the absorbance of the
Test solution; WU is the weight, in mg, of Erythromycin taken to prepare the
Test solution; and the other terms are as defined above: not more than 0.3% is found.
Limit of related substances—
Using the chromatograms of the
Assay preparation and the
Diluted standard preparation obtained in the
Assay, calculate the percentage of any individual related substance observed having the greatest response, other than erythromycin A, erythromycin B, erythromycin C, and erythromycin A enol ether, in the Erythromycin taken by the formula:
25(CP / W)(ri / rS),
in which
C is the concentration, in mg per mL, of
USP Erythromycin RS in the
Diluted standard preparation; P is the designated percentage of erythromycin A in the
USP Erythromycin RS;
W is the weight, in mg, of Erythromycin taken to prepare the
Assay preparation; ri is the peak response of an individual related substance, other than erythromycin A, erythromycin B, erythromycin C, or erythromycin A enol ether, observed in the chromatogram obtained from the
Assay preparation; and
rS is the erythromycin A peak response in the chromatogram obtained from the
Diluted standard preparation: not more than 3.0% of any individual related substance is found. Calculate the percentage of erythromycin A enol ether in the Erythromycin taken by the formula:
(25 / 11)(CP / W)(rE / rS),
in which 11 is the response factor for erythromycin A enol ether in relation to that of erythromycin A;
rE is the peak response of the erythromycin A enol ether peak observed in the chromatogram obtained from the
Assay preparation; and the other terms are as defined above: not more than 3.0% of erythromycin A enol ether is found. The percentage of erythromycin B obtained in the
Assay is not more than 12.0%; and the percentage of erythromycin C obtained in the
Assay is not more than 5.0%.
Assay—
Solution A—
Dissolve 1.75 g of dibasic potassium phosphate in 50 mL of water, adjust with diluted phosphoric acid (1 in 10) or 0.2 N sodium hydroxide to a pH of 9.0, add 400 mL of water, 165 mL of tertiary butyl alcohol, and 30 mL of acetonitrile. Dilute with water to 1000 mL, and mix.
Mobile phase—
Prepare a mixture of
Solution A, acetonitrile, and water (5:2:1). Make any necessary adjustments (see
System Suitability under
Chromatography 
621
).
NOTE—Use the following solutions promptly, or within 1 day if stored in a refrigerator.
Standard preparation—
Transfer about 100 mg of
USP Erythromycin RS, accurately weighed, to a 25-mL volumetric flask, add 5 mL of methanol, swirl to dissolve, dilute with
Diluent to volume, and mix.
Diluted standard preparation—
Transfer 3.0 mL of the
Standard preparation to a 100-mL volumetric flask, dilute with
Diluent to volume, and mix. This solution contains about 0.12 mg of
USP Erythromycin RS per mL.
Erythromycins B and C standard solution—
Transfer about 5 mg each of
USP Erythromycin B RS and
USP Erythromycin C RS, both accurately weighed, to a 25-mL volumetric flask, add 5 mL of methanol, swirl to dissolve, dilute with
Diluent to volume, and mix.
Resolution solution—
Transfer about 2 mg of
USP Erythromycin Related Compound N RS to a 10-mL volumetric flask, add 0.4 mL of
Standard preparation, dilute with
Erythromycins B and C standard solution to volume, and mix.
Erythromycin A enol ether retention time solution—
Dissolve about 10 mg of
USP Erythromycin RS in 2 mL of methanol. Add 10 mL of
pH 3.5 Buffer, mix, and allow to stand for about 30 minutes.
Assay preparation—
Transfer about 100 mg of Erythromycin, accurately weighed, to a 25-mL volumetric flask, add 5 mL of methanol, and swirl to dissolve. Dilute with Diluent to volume, and mix.
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 65
. The flow rate is about 2 mL per minute. Chromatograph the
Resolution solution, and record the peak responses as directed for
Procedure: the relative retention times are about 0.56 for erythromycin related compound N (N-demethylerythromycin A), 0.61 for erythromycin C, 1.0 for erythromycin A, and 1.6 for 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 retention time solution, and record the peak responses as directed for
Procedure: the retention time of the erythromycin A enol ether peak is about 3.2 relative to that of the erythromycin A peak as observed in the chromatogram obtained from the
Resolution solution. Chromatograph the
Standard preparation, 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 the
Standard preparation, the
Diluted standard preparation, the
Erythromycins B and C standard solution, 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, as determined in the chromatogram obtained from the
Erythromycin A enol ether retention time solution (about five times the retention time of the main erythromycin A peak). Measure the areas of the peak responses. Calculate the percentage of erythromycin A in the portion of Erythromycin taken by the formula:
25(CAP / W)(rU / rA),
in which
CA 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 mg, of Erythromycin taken to prepare the
Assay preparation; and
rU and
rA are the erythromycin A peak responses in the chromatograms obtained from the
Assay preparation and the
Standard preparation, respectively. Calculate the percentages of erythromycin B and erythromycin C in the portion of Erythromycin taken by the formula:
25(CSP / W)(rU / rS),
in which
CS is the concentration, in mg per mL, of the relevant USP Reference Standard in the
Erythromycins B and C standard solution; P is the designated percentage of erythromycin B or erythromycin C in the relevant USP Reference Standard;
W is the quantity, in mg, of Erythromycin taken to prepare the
Assay preparation; and
rU and
rS are the peak responses of the relevant analyte in the chromatograms obtained from the
Assay preparation and the
Erythromycins B and C standard solution, respectively.
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-L-ribo-hexopyranosyl)oxy]-14-ethyl-7,12,13-trihydroxy-3,5,7,9,11,13-hexamethyl-6-[[3,4,6-trideoxy-3-(dimethylamino)-
-D-xylo-hexopyranosyl]oxy]oxacyclotetradecane-2,10-dione