Azithromycin for Injection

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Azithromycin for Injection is a sterile, dry mixture of azithromycin and a suitable stabilizing agent. It contains NLT 90.0% and NMT 110.0% of the labeled amount of azithromycin (C₃₈H₇₂N₂O₁₂).

2 IDENTIFICATION

A. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

B. SPECTROSCOPIC IDENTIFICATION TESTS (197), Infrared Spectroscopy: 197A

Standard: 25 mg/mL of USP Azithromycin RS in acetonitrile. Pass the solution through a suitable filter, and remove the solvent by natural evaporation.

Sample: Equivalent to 25 mg/mL of azithromycin from Azithromycin for Injection in acetonitrile. Pass the solution through a suitable filter, and remove the solvent by natural evaporation.

Analysis: Examine the spectra of the Standard and the Sample in the range between 3800 and 650 cm-1.

Acceptance criteria: The Sample exhibits bands at about 900, 995, 1165, 1376, 1456, 1725, and 2936 cm¹ similar to the spectrum from the Standard similarly obtained.

3 ASSAY

3.1 PROCEDURE

Buffer: 6.7 mg/mL of dibasic potassium phosphate in water

Mobile phase: Acetonitrile and Buffer (52:48). Adjust with 10 N potassium hydroxide to a pH of 11.0 ± 0.1.

Diluent: Acetonitrile and water (52:48)

System suitability solution: 1 mg/mL each of USP Azaerythromycin A RS and USP Azithromycin RS in a mixture of acetonitrile and water (52:48). Dissolve first in acetonitrile, and then dilute with water to volume.

Standard solution: 1 mg/mL of USP Azithromycin RS in a mixture of acetonitrile and water (52:48). Dissolve first in acetonitrile, and dilute with water to volume.

Sample solution: Nominally equivalent to 1 mg/mL of azithromycin from Azithromycin for Injection in Diluent prepared as follows. Reconstitute 3 vials individually as directed in the labeling. Mix the contents of all the reconstituted vials. Dilute a portion of the mixture with Diluent.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 215 nm

Columns

Guard: 4.6-mm x 1-cm; 5-µm packing L67

Analytical: 4.6-mm × 15-cm; 5-µm packing L67

Temperatures

Autosampler: 15°

Column: 40°

Flow rate: 1 mL/min

Injection volume: 15 µL

System suitability

Samples: System suitability solution and Standard solution

[NOTE-The relative retention times for azaerythromycin A and azithromycin are 0.68 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 2.5 between azaerythromycin A and azithromycin, System suitability solution

Tailing factor: NLT 0.9 and NMT 1.5, Standard solution

Relative standard deviation: NMT 2%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of azithromycin (C₃₈H₇₂N₂O₁₂) in the portion of Azithromycin for Injection taken:

Result = (r_U/r_S) x (C_S/C_U) x P x F x 100

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution 

C_S = concentration of USP Azithromycin RS in the Standard solution (mg/mL)

C_U = nominal concentration of azithromycin in the Sample solution (mg/mL)

P = potency of USP Azithromycin RS (µg/mg)

F = conversion factor, 0.001 mg/µg

Acceptance criteria: 90.0%-110.0%

4 PERFORMANCE TESTS

UNIFORMITY OF DOSAGE UNITS (905): Meets the requirements

5 IMPURITIES

[NOTE-The test for Limit of Azithromycin N-Oxide, Desosaminylazithromycin, and N-Demethylazithromycin does not quantify aminoazithromycin, formamido analog, methylformamido analog, and 3'-de(dimethylamino)-3'-oxoazithromycin. If these impurities are part of the impurity profile, the Limit of Aminoazithromycin, Formamido Analog, Methylformamido Analog, and 3'-De(dimethylamino)-3'-oxoazithromycin test is recommended in addition to the test for Limit of Azithromycin N-Oxide, Desosaminylazithromycin, and N-Demethylazithromycin.]

5.1 LIMIT OF AZITHROMYCIN N-OXIDE, DESOSAMINYLAZITHROMYCIN, AND N-DEMETHYLAZITHROMYCIN

Buffer: 3.5 g/L of dibasic potassium phosphate

Mobile phase: Acetonitrile and Buffer (23:77). Adjust with 5 N potassium hydroxide to a pH of 10.55 ± 0.05.

Standard stock solution: 0.05 mg/mL of USP Azithromycin N-oxide RS, 45 µg/mL of USP Desosaminylazithromycin RS, and 160 µg/mL each of USP N-Demethylazithromycin RS and USP Azithromycin RS in acetonitrile. Sonicate if necessary to dissolve.

Standard solution: 0.001 mg/mL of azithromycin N-oxide, 0.9 µg/mL of desosaminylazithromycin, and 3.2 µg/mL each of N-demethylazithromycin and azithromycin from Standard stock solution in Mobile phase

Sample solution: Nominally equivalent to 0.3 mg/mL of azithromycin in Mobile phase from Azithromycin for Injection

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: Amperometric electrochemical

Electrodes: Dual series glassy carbon

Mode: Oxidative screen

Electrode 1: +0.70 ± 0.05 V

Electrode 2: +0.82 ± 0.05 V

Column: 4.6-mm x 15-cm; 3-µm packing 149

Autosampler temperature: 5°

Flow rate: 1 mL/min

Injection volume: 50 µL

System suitability

Sample: Standard solution

OFFICI

[NOTE-See Table 1 for relative retention times.]

Suitability requirements

Tailing factor: NMT 2.0 for azithromycin and NMT 2.6 for N-demethylazithromycin

Relative standard deviation: NMT 10.0% for azithromycin N-oxide, desosaminylazithromycin, N-demethylazithromycin, and azithromycin

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each specified impurity in the portion of Azithromycin for Injection taken:

Result = (r_U/r_S) x (C_S/C_U) x P x 100

r_U = peak response of each specified impurity from the Sample solution

r_S = peak response of each specified impurity from the Standard solution 

C_S = concentration of the relevant impurity Reference Standard in the Standard solution (mg/mL) 

C_U = nominal concentration of azithromycin in the Sample solution (mg/mL)

P = potency of the relevant Reference Standard (mg/mg)

Acceptance criteria: See Table 1. The reporting threshold is 0.05%.

Table 1

^a (2R,3S,4R,5R,8R,10R,11R,12S,13S, 14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-(dimethylazinoyl)-β-D-xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one.

^b (2R,3S,4R,5R,8R, 10R,11R,12S, 13S,14R)-2-Ethyl-3,4,10,13-tetrahydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-dimethylamino-ẞ-D-Xylo-hexopyranosyl)oxy]-1-oxa-6-azacyclopentadecan-15-one.

^c Process impurities that are controlled in the drug substance are not to be reported. They are listed here for information only.

^d (3R,4S,5S,6R,7R,9R, 11S,12R,13S,14R,E)-6-[[3,4,6-Trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl)oxy]-14-ethyl-7,12,13-trihydroxy-4-[(2,6-dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl)oxy]-10-(hydroxyimino)-3,5,7,9,11,13-hexamethyloxacyclotetradecan-2-one.

^e (2R,3S,4R,5R,8R,10R,11R,12S,13S, 14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-methylamino-ẞ-D-xylo-hexopyranosyl)oxy]-1-oxa-6-azacyclopentadecan-15-one.

^f 9-Deoxo-9a-aza-9a-homoerythromycin A.

Change to read:

5.2 LIMIT OF AMINOAZITHROMYCIN, FORMAMIDO ANALOG, METHYLFORMAMIDO ANALOG, AND 3'-DE(DIMETHYLAMINO)-3'-OXOAZITHROMYCIN (if present)

Buffer: 3.5 g/L of dibasic potassium phosphate in water

Mobile phase: Acetonitrile and Buffer (46:54). Adjust with 10 N potassium hydroxide to a pH of 11.0 ± 0.1.

Diluent: Acetonitrile and water (46:54)

Standard stock solution: 0.09 mg/mL of USP Desosaminylazithromycin RS, 0.21 mg/mL of USP N-Demethylazithromycin RS, and 0.30 mg/mL of USP Azithromycin RS in acetonitrile

Standard solution: 0.0018 mg/mL of desosaminylazithromycin, 0.0042 mg/mL of N-demethylazithromycin, and 0.006 mg/mL of azithromycin in Diluent

Sample solution: Nominally equivalent to 0.6 mg/mL of azithromycin from Azithromycin for Injection in Diluent. Reconstitute 3 vials individually, as directed in the labeling. Mix the contents of all the reconstituted vials. Dilute a portion of the mixture with Diluent. The Sample solution must be injected immediately after preparation.

Blank: Use the Diluent.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: Amperometric electrochemical

Electrodes: Dual series glassy carbon

Mode: Oxidative screen

Electrode 1: +0.70 ± 0.05 V

Electrode 2: +0.82±0.05 V

Columns

Guard: 4.6-mm x 1-cm; 5-µm packing L67

Analytical: 4.6-mm x 25-cm; 5-µm packing L67

Temperatures

Autosampler: 15°

Column: 40°

Flow rate: 1 mL/min

Injection volume: 25 µL

System suitability

Sample: Standard solution

[NOTE-See Table 2 for relative retention times.]

Suitability requirements

Resolution: NLT 1.5 between desosaminylazithromycin and N-demethylazithromycin

Tailing factor: NMT 1.5 for azithromycin

Relative standard deviation: NMT 5% for azithromycin

Analysis

Samples: Standard solution, Sample solution, and Blank

Disregard any peaks corresponding to those obtained from the Blank.

Calculate the percentage of each impurity in the portion of Azithromycin for Injection taken:

Result = (r_U/r_S) x (C_S/C_U) x P x F x100

r_U = peak response of each impurity from the Sample solution

r_S = peak response of azithromycin from the Standard solution 

C_S = concentration of USP Azithromycin RS in the Standard solution (mg/mL)

C_U = nominal concentration of azithromycin in the Sample solution (mg/mL)

P = potency of USP Azithromycin RS (µg/mg)

F = conversion factor, 0.001 mg/µg

Acceptance criteria: See Table 2.

Table 2

^a Process impurities that are controlled in the drug substance are not to be reported. They are listed here for information only.

^b (3R,4R,5S,6R,9R,10S, 11S,12R,13S, 15R,Z)-12-[[3,4,6-Trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl] oxy]-6-ethyl-4,5-dihydroxy-10-[(2,6-dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl)oxy]-3,5,9,11,13,15-hexamethyl-7,16-dioxa-2-azabicyclo[11.2.1] hexadec-1-en-8-one.

^c (2R,3S,4R,5R,8R, 10R,11R,12S, 13S, 14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3-amino-3,4,6-trideoxy-ẞ-D-xylo-hexopyranosyl)oxy]-1-oxa-6-azacyclopentadecan-15-one.

^d (2R,3S,4R,5R,8R,10R,11R,12S, 13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3-formamido-3,4,6-trideoxy-ẞ-D-xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one.

^e (2R,3S,4R,5R,8R,10R,11R,12S, 13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12-hexamethyl-14-hydroxymethyl-11-[[3-dimethylamino-3,4,6-trideoxy-ẞ-D-xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one.

^f These impurities are controlled using the Limit of Azithromycin N-Oxide, Desosaminylazithromycin, and N-Demethylazithromycin test. They are listed here for information only.

^g (2R,3S,4R,5R,8R, 10R,11R,12S,13S,14R)-2-Ethyl-3,4,10,13-tetrahydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-dimethylamino-ẞ-D-xylo-hexopyranosyl)oxy]-1-oxa-6-azacyclopentadecan-15-one.

^h (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3-(N-methyl) formamido-3,4,6-trideoxy-ẞ-D-xylo-hexopyranosyl] oxy]-1-oxa-6-azacyclopentadecan-15-one.

ⁱ (2R,3S,4R,5R,8R, 10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-methylamino-ẞ-D-xylo-hexopyranosyl)oxy]-1-oxa-6-azacyclopentadecan-15-one.

^j (3R,4S,5S,6R,7R,9R, 11S,12R,13S,14R,E)-6-[[3,4,6-Trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl)oxy]-14-ethyl-7,12,13-trihydroxy-4-[(2,6-dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl)oxy]-10-(hydroxyimino)-3,5,7,9,11,13-hexamethyloxacyclotetradecan-2-one.

^k 9-Deoxo-9a-aza-9a-homoerythromycin A.

^l (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3,3-dimethyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-oxo-β-D-xylo-hexopyranosyl] oxy]-1-oxa-6-azacyclopentadecan-15-one.

^m (2R,3S,4R,5R,8R,10R,11R,12S, 13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-m 3,5,6,8,10,12,14-heptamethyl-11-[[3-[N-(4-methylphenylsulfonyl)-N-methylamino]-3,4,6-trideoxy-B-D-xylo-hexopyranosyl] oxy]-1-oxa-6-azacyclopentadecan-15-one (ERR 1-Aug-2023)

ⁿ (2R,3R,4S,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-4,10-dihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-(dimethylamino)-ẞ-D-xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one.

^o Total impurities include desosaminylazithromycin and N-demethylazithromycin.

6 SPECIFIC TESTS

BACTERIAL ENDOTOXINS TEST (85): Meets the requirements

STERILITY TESTS (71): Meets the requirements

PARTICULATE MATTER IN INJECTIONS (788): Meets the requirements

PH (791): 6.4-6.8, determined in a solution constituted as directed in the labeling

WATER DETERMINATION (921), Method I: NMT 2.0%

OTHER REQUIREMENTS: It meets the requirements under Injections and Implanted Drug Products (1).

7 ADDITIONAL REQUIREMENTS

7.1 PACKAGING AND STORAGE

Preserve as described under Packaging and Storage Requirements (659), Injection Packaging, Packaging for Constitution. Store at controlled room temperature.

7.2 LABELING

It meets the requirements for Labeling (7), Labels and Labeling for Injectable Products.

Change to read:

7.3 USP REFERENCE STANDARDS (11)

USP Azaerythromycin A RS

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

C₃₇H₇₀N₂O₁₂  734.97 (ERR 1-Aug-2023)

USP Azithromycin RS

USP Azithromycin N-oxide RS

(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-(dimethylazinoyl)-ẞ-D-xylo-hexopyranosyl)oxy]-1-oxa-6-azacyclopentadecan-15-one.

C₃₈H₇₂N₂O₁₃  765.00 (ERR 1-Aug-2023)

USP N-Demethylazithromycin RS

(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-methylamino-ẞ-D-xylo-hexopyranosyl)oxy]-1-oxa-6-azacyclopentadecan-15-one.

C₃₇H₇₀N₂O₁₂  734.97 (ERR 1-Aug-2023)

USP Desosaminylazithromycin RS

(2R,3S,4R,5R,8R, 10R,11R,12S, 13S,14R)-2-Ethyl-3,4,10,13-tetrahydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one.

C₃₀H₅₈N₂O₉  590.80 (ERR 1-Aug-2023)