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Azithromycin
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C38H72N2O12·xH2O
(anhydrous) 749.00[83905-01-5].

1-Oxa-6-azacyclopentadecan-15-one, 13-[(2,6-dideoxy-3-C-methyl-3-O-methyl--l-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-(dimethylamino)--d-xylo-hexopyranosyl]oxy]- [2R(2R*,3S*,4R*,5R*,8R*,10R*,11R*,12S*,13S*,14R*)].

(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-O-methyl--l-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-(dimethylamino)--d-xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one.

9-Deoxo-9a-aza-9a-methyl-9a-homoerythromycin A.

Monohydrate 767.02 [121479-24-4].

Dihydrate 785.02 [117772-70-0].
Change to read:
» Azithromycin is anhydrous or6 contains one or two molecules of water of hydration. It contains the equivalent of not less than 945 µg and not more than 1030 µg of azithromycin (C38H72N2O12) per mg, calculated on the anhydrous basis.
Packaging and storage— Preserve in tight containers.
Change to read:
Labeling— Label it to indicate whether it is anhydrous, or6 the monohydrate or the dihydrate. The amorphous form is so labeled.6 Where the quantity of azithromycin is indicated in the labeling of any preparation containing Azithromycin, this shall be understood to be in terms of anhydrous azithromycin (C38H72N2O12). The labeling states with which Limit of related substances test the article complies if a test other than Test 1 is used.
USP Reference standards 11—
USP Azaerythromycin A RS.
USP Azithromycin RS.
USP Azithromycin Identity RS.
USP Azithromycin-N-Oxide RS.
USP N-Demethylazithromycin RS.
USP Desosaminylazithromycin RS.
Change to read:
Identification—
A: Infrared Absorption 197K. If a difference appears in the IR spectra of the analyte and the standard, dissolve equal portions of the test specimen and the Reference Standard in equal volumes of methanol. Evaporate the solutions to dryness on a water bath, and dry at 80 for 30 minutes under vacuum. Perform the test on the residues.6
B: The retention time of the azithromycin peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.
Specific rotation 781S: between –45 and –49, (t = 20).
Test solution: 20 mg per mL, in dehydrated alcohol.
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Crystallinity 695: meets the requirements, except, where it is labeled as amorphous, most of the particles do not exhibit birefringence and extinction positions.6
pH 791: between 9.0 and 11.0, in a mixture of methanol and water (1:1) containing 2 mg per mL, prepared by diluting a solution in methanol containing 4 mg per mL with an equal volume of water.
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Water, Method I 921: not more than 2.0% where it is labeled as anhydrous;6 between 4.0% and 5.0% where it is labeled as the dihydrate; between 1.8% and 4.0% where it is labeled as the monohydrate, except that it may be between 4.0% and 6.5% when the requirements of the Loss on drying test are met.
Loss on drying (where it is labeled as Azithromycin monohydrate and has a Water content of between 4.0% and 6.5%) (see Thermal Analysis 891)—[note—The quantity taken for the determination may be adjusted, if necessary, for instrument sensitivity.] Determine the percentage of volatile substances by thermogravimetric analysis in an appropriately calibrated instrument, using about 10 mg of Azithromycin, accurately weighed. Heat the specimen at the rate of 10 per minute between ambient temperature and 150 in an atmosphere of nitrogen at a constant flow rate of about 35 mL per minute. From the thermogram plot the derivatives of the loss on drying (percent loss per minute), identify the inflection points of the two weight loss steps at about 70 and 130: it loses not more than 4.5% of its weight between ambient temperature and the inflection point at about 70, and between 1.8% and 2.6% between the inflection point at about 70 and the inflection point at about 130.
Residue on ignition 281: not more than 0.3%, the charred residue being moistened with 2 mL of nitric acid and 5 drops of sulfuric acid.
Limit of related substances— [note—Perform either Test 1 or Test 2 depending on the manufacturing process used.]
Test 1— [note—Use water that has a resistivity of not less than 18 Mohm-cm.]
Mobile phase— Proceed as directed in the Assay.
pH 7.5 Potassium phosphate buffer— Transfer 2.7 g of monobasic potassium phosphate to a 1000-mL volumetric flask. Dilute with water to volume, and mix. Adjust with 10 N potassium hydroxide to a pH of 7.5 ± 0.1.
Dilution solution— Prepare a mixture of pH 7.5 Potassium phosphate buffer and acetonitrile (750:250).
Standard stock solution— Quantitatively dissolve accurately weighed quantities of USP Desosaminylazithromycin RS, USP N-Demethylazithromycin RS, and USP Azithromycin RS with acetonitrile to obtain a solution having known concentrations of about 45 µg per mL, 105 µg per mL, and 160 µg per mL, respectively.
Standard solution— Transfer 4.0 mL of the Standard stock solution to a 200-mL volumetric flask, dilute with Dilution solution to volume, and mix. This solution contains known concentrations of USP Desosaminylazithromycin RS, USP N-Demethylazithromycin RS, and USP Azithromycin RS of about 0.9 µg per mL, 2.1 µg per mL, and 3.2 µg per mL, respectively.
Test solution— Transfer about 33 mg of Azithromycin, accurately weighed, to a 100-mL volumetric flask, add 5 mL of acetonitrile, and sonicate for about 20 seconds to dissolve. Dilute with Dilution solution to volume, and mix. [note—Use this solution within 6 hours.]
Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with an amperometric electrochemical detector with dual glassy carbon electrodes operated in the oxidative screen mode with electrode 1 set at +0.70 ± 0.05 V and electrode 2 set at +0.85 ± 0.05 V, and the background current optimized to 95 ± 25 nanoamperes, a 4.6-mm × 5-cm guard column that contains 5-µm packing L29, and a 4.6-mm × 15-cm analytical column that contains 5-µm packing L29 or 3-µm packing L49 without the guard column. [note—In general, maintain electrode 1 at 0.12 V less than electrode 2, and maintain the electrodes at a constant temperature of about 26.] The flow rate is about 0.4 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the column efficiency is not less than 1500 theoretical plates for the azithromycin peak; the tailing factor for each of these compounds is not more than 1.5; and the relative standard deviation for replicate injections is not more than 5% for each of these compounds. [note—For the purpose of identification, the relative retention times are about 0.38 for desosaminylazithromycin, 0.54 for N-demethylazithromycin, and 1.0 for azithromycin]
Procedure— Separately inject equal volumes (about 50 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, using an elution period for the Test solution that is 3.3 times the elution time of the azithromycin peak in the chromatogram of the Standard solution, and measure the areas for all of the peaks. Calculate the percentages of desosaminylazithromycin and N-demethylazithromycin in the Azithromycin taken by the formula:
0.1(CP/W)(ri / rS)
in which C is the concentration, in µg per mL, of the appropriate USP Reference Standard in the Standard solution; P is the designated potency, in percentage, of the relevant USP Reference Standard; W is the weight, in mg, of Azithromycin taken to prepare the Test solution; and ri and rS are the peak area responses for the relevant analyte in the chromatograms obtained from the Test solution and the Standard solution, respectively. Calculate the percentages of other related substances in the Azithromycin taken by the formula:
0.01(CP/W)(ri / rS)
in which C is the concentration, in µg per mL, of USP Azithromycin RS in the Standard solution; P is the designated purity, in µg per mg, of USP Azithromycin RS; W is the weight, in mg, of Azithromycin taken to prepare the Test solution; ri is the peak area response for an individual related substance peak in the chromatogram obtained from the Test solution; and rS is the peak area response for the azithromycin peak in the chromatogram obtained from the Standard solution. Not more than 0.3% of desosaminylazithromycin, 0.7% of N-demethylazithromycin, and 1.0% of any other individual related substance is found; and the sum of all related substances is not more than 3.0%.
test 2—
Phosphate buffer solution— Dissolve about 8.7 g of dibasic potassium phosphate in 1 L of water, adjust with 20% phosphoric acid to a pH of 8.2, and mix.
Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and Phosphate buffer solution (6:4). Make adjustments if necessary (see System Suitability under Chromatography 621).
Standard solution 1— Dissolve an accurately weighed quantity of USP Azithromycin RS in Mobile phase, and quantitatively dilute to obtain a solution having a known concentration of about 35 µg per mL.
Standard solution 2— Prepare a solution in Mobile phase containing about 7 mg of USP Azithromycin Identity RS per mL.
Standard solution 3— Prepare a solution in Mobile phase containing about 14 µg of USP Azithromycin-N-Oxide RS per mL.
Test solution— Transfer about 70.0 mg of Azithromycin, accurately weighed, to a 10-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix.
System suitability solution— Prepare a solution in Mobile phase containing about 0.07 mg of USP Azaerythromycin A RS and 7 mg of USP Azithromycin RS per mL.
Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 210-nm detector and a 4.6-mm × 15-cm column that contains 5-µm packing L1. The flow rate is about 0.9 mL per minute. The column temperature is maintained at 30. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the resolution, R, between azaerythromycin A and azithromycin is not less than 8.0; and the tailing factor for the azithromycin peak is not more than 2.5. [note—For the purpose of identification, the relative retention times are about 0.47 for azaerythromycin A and 1.00 for azithromycin.]
Procedure— Separately inject equal volumes (about 50 µL) of Standard solution 1, Standard solution 2, Standard solution 3, Test solution, and Mobile phase into the chromatograph, record the chromatograms, identify the peaks in the chromatogram of the Test solution by comparison with the chromatograms obtained from Standard solution 2 and Standard solution 3, and measure the peak area responses. Disregard any peak due to the solvent front and any peak corresponding to those obtained from the Mobile phase. Calculate the percentage of each impurity in the portion of Azithromycin taken by the formula:
(CP/W)(1/F)(ri / rS)
in which C is the concentration, in mg per mL, of USP Azithromycin RS in Standard solution 1; P is the designated purity, in µg per mg, of USP Azithromycin RS; W is the weight, in mg, of Azithromycin taken to prepare the Test solution; F is the relative response factor (see Table 1); ri is the peak area response for each impurity obtained from the Test solution; and rS is the peak area response for azithromycin obtained from Standard solution 1. The impurities meet the limits specified in Table 1.
Table 1
Compound Relative
Retention
Time		 Relative
Response
Factor		 Limit
(w/w, %)
Azithromycin-N-oxide 0.20 0.45 0.40
3¢-(N,N-didemethyl)-3¢-N-formylazithromycin 0.26 1.8 0.30
3¢-N-demethyl-3¢-N-formylazithromycin (rotamer 1) 0.34 4.1 0.15
3¢-N-demethyl-3¢-N-formylazithromycin (rotamer 2) 0.37 4.1 0.15
6-Demethylazithromycin (azaerythromycin A) 0.47 0.67 0.50
3¢-De(dimethylamino)-3¢-oxoazithromycin 0.80 1.9 0.25
2-Desethyl-2-propylazithromycin 1.52 1.0 0.50
3-Deoxyazithromycin (azithromycin B) 1.60 1.0 0.50
3¢-N-demethyl-3¢-N-[(4-methylphenyl)sulfonyl]azithromycin 2.14 7.0 0.50
Individual unknown impurity — 1.0 0.20
Total impurities — — 2.0
Assay— [note—Use water that has a resistivity of not less than 18 Mohm-cm.]
Mobile phase— Dissolve 5.8 g of monobasic potassium phosphate in 2130 mL of water, add 870 mL of acetonitrile, and mix. Adjust with about 6 mL of 10 N potassium hydroxide to a pH of 11.0 ± 0.1, and pass through a filter having a 0.5-µm or finer porosity, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).
Standard stock preparation— Transfer about 16.5 mg of USP Azithromycin RS, accurately weighed, to a 100-mL volumetric flask, add 10 mL of acetonitrile, and dissolve by swirling and with the aid of brief sonication. Dilute with acetonitrile to volume, and mix.
Standard preparation— Transfer 2.0 mL of the Standard stock preparation to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix to obtain a Standard preparation having a known concentration of about 0.0033 mg of USP Azithromycin RS per mL.
Assay preparation— Transfer about 16.5 mg of Azithromycin, accurately weighed, to a 100-mL volumetric flask, add 10 mL of acetonitrile, and dissolve by swirling and with the aid of brief sonication. Dilute with acetonitrile to volume, and mix. Transfer 2.0 mL of the solution so obtained to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix.
Resolution solution— Transfer about 8 mg of USP Azaerythromycin A RS to a 50-mL volumetric flask, add 5 mL of acetonitrile, and dissolve by swirling and with the aid of brief sonication. Dilute with Mobile phase to volume, and mix. Transfer 2.0 mL of the solution so obtained and 2.0 mL of the Standard stock preparation to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix.
Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with an amperometric electrochemical detector with dual glassy carbon electrodes operated in the oxidative screen mode with electrode 1 set at +0.70 ± 0.05 V and electrode 2 set at +0.82 ± 0.05 V, and the background current optimized to 85 ± 15 nanoamperes, a 4.6-mm × 5-cm guard column that contains 5-µm packing L29 and a 4.6-mm × 15-cm analytical column that contains 5-µm packing L29 or 3-µm packing L49 without the guard column. The flow rate is about 1.5 mL per minute. Chromatograph the Resolution solution, and record the responses as directed for Procedure: the relative retention times are about 0.7 for azaerythromycin A and 1.0 for azithromycin with the L29 column and about 0.8 for azaerythromycin A and 1.0 for azithromycin with the L49 column; and the resolution, R, between azaerythromycin A and azithromycin is not less than 2.5. Chromatograph the Standard preparation, and record the responses as directed for Procedure: the tailing factor for the azithromycin peak is not less than 0.9 and not more than 1.5; the column efficiency is not less than 1000 theoretical plates; and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure— Separately inject equal volumes (about 50 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the quantity, in µg, of azithromycin (C38H72N2O12) in each mg of Azithromycin taken by the formula:
(WP/w)(rU / rS)
in which W is the quantity, in mg, of USP Azithromycin RS taken to prepare the Standard preparation; P is the potency, in µg of azithromycin per mg, of USP Azithromycin RS; w is the quantity, in mg, of Azithromycin taken to prepare the Assay preparation; and rU and rS are the azithromycin peak responses obtained from the Assay preparation and the Standard preparation, respectively.
Auxiliary Information— Staff Liaison : Ahalya Wise, M.S. , Scientist
Expert Committee : (MDANT05) Monograph Development-Antibiotics
USP31–NF26 Page 1476
USP31–NF26 Supplement : No. 1 Page 3620
Pharmacopeial Forum : Volume No. 32(2) Page 306
Phone Number : 1-301-816-8161