Azithromycin Dihydrate USP’s extensive specifications and detailed test protocols ensures quality and compliance for pharmaceutical applications.

Specification – Azithromycin Dihydrate USP

Sr. No.	Test Parameter		Specification
01	Description		White or almost white powder.
02	Solubility		Freely soluble in anhydrous ethanol and in methylene chloride; practically insoluble in water.
03	Identification – A		By IR; The infrared absorption spectrum should be concordant with reference spectrum of that standard.
	Identification – B		By HPLC; The retention time of the azithromycin peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.
04	Optical Rotation		Between – 45°  and  – 49°  at  20◦
05	Crystallinity		Particles shows birefringence and extinction position when microscope stage revolved.
06	pH		Between 9.0  and  11.0
07	Water Determination		Between 4.0  and  5.0%.
08	Residue on Ignition		Not more than 0.3%.
09	Organic Impurities
	Azithromycin N-oxide		Not more than 0.5%
	3’-(N,N-Didemethyl)-3’-N-formylazithromycin		Not more than 0.5%
	Erythromycin A iminoether		Not more than 0.5%
	3’-(N,N-Didemethyl) azithromycin (aminoazithromycin)		Not more than 0.5%
	Azithromycin related compound F		Not more than 0.5%
	Desosaminylazithromycin		Not more than 0.3%
	3’-N-{[4-(Acetylamino)phenyl] sulfonyl}-3’,3’-didemethylazithromycin		Not more than 0.15%
	N-Demethylazithromycin		Not more than 0.7%
	Erythromycin A oxime		Not more than 0.5%
	Azithromycin C (3’ ‘-O-demethylazithromycin)		Not more than 0.5%
	3’-De(dimethylamino)-3’-oxoazithromycin		Not more than 0.5%
	3’-N-{[4-(Acetylamino)phenyl] sulfonyl}-3’-demethylazithromycin		Not more than 0.5%
	Azaerythromycin A		Not more than 0.5%
	Azithromycin impurity P		Not more than 0.2%
	2-Desethyl-2-propylazithromycin		Not more than 0.5%
	3’-N-Demethyl-3’-N-[(4-methylphenyl)sulfonyl]azithromycin		Not more than 0.5%
	3-Deoxyazithromycin (Azithromycin B)		Not more than 1.0%
	Any individual, unidentified impurity		Not more than 0.2%
	Total impurities		Not more than 3.0%
10	Assay  By HPLC#	945.0  to  1030.0 µg/mg of Azithromycin ( C38H72N2O12 ), calculated on the anhydrous basis.

Specification – Azithromycin Dihydrate USP.

STP/MOA – Azithromycin Dihydrate USP

1.0     Description

• • Carry out the test on sample (Spec. White or almost white powder.) . For detailed Description Test Procedure – Refer GTP – Description / Appearance Test.

2.0     Solubility

• • Freely soluble in anhydrous ethanol and in methylene chloride; practically insoluble in water.

3.0     Identification

• • Test A  : By IR;

• • The infrared absorption spectrum should be concordant with reference spectrum of that standard.

• • Test B : By HPLC;

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

4.0     Optical Rotation :

• • Carry out the test on sample as per procedure.

• • Test solution   :   20 mg/ml, in dehydrated alcohol at 20°C.

5.0     Crystallinity :

• • Particles shows birefringence and extinction position when microscope stage revolved.

• • Carry out the test procedure.

6.0     pH

• • Sample Stock solution : 4 mg/ml in methanol.

• • Sample solution : 2mg/ml obtained by mixing equal volume of Sample stock Solution and water. Carry out the test as per procedure. Refer detailed procedure for pH value determination.

7.0     Water Determination

• • Carry out the test method I. Refer the GTP – Water Content Determination

8.0     Residue on Ignition

• • The charred residue being moistened with 2 ml of Nitric acid and 5 drops of  Sulfuric acid.

• • Carry out the test as per procedure.

 9.0     Organic Impurities Test for Azithromycin Dihydrate USP

• • Procedure :  By HPLC.

• • Solution A : 1.8 mg/mL of anhydrous dibasic sodium phosphate in water. Adjust with 1 N sodium hydroxide or 10% phosphoric acid to a pH of 8.9.

• • Solution B : Acetonitrile and methanol (3:1).

• • Solution C : 1.73 mg/mL of monobasic ammonium phosphate. Adjust with ammonia TS to a pH of 10.0 ± 0.05.

• • Solution D : Methanol, acetonitrile, and Solution C (7:6:7)

• • Mobile phase : See Table 1.

Table 1

Time (min)	Solution A (%)	Solution B (%)
0	50	50
25	45	55
30	40	60
80	25	75
81	50	50
93	50	50

• • System Suitability Solution :

• • 0.0165 mg/mL of USP Azithromycin Related Compound F RS and 0.027 mg/mL of USP Desosaminylazithromycin RS in Solution D.

• • Standard Solution : 86 µg/mL of USP Azithromycin RS in Solution D

(Dilution : 86 mg   →   100 ml,       10 ml  →   100 ml)

• • Sample Solution : 8.6 mg/mL of Azithromycin in Solution D

(Dilution : 86 mg  → 10 ml)

• Chromatographic System :

Mode                     :         LC

Detector                :         UV 210 nm

Column                 :         4.6-mm × 25-cm; 5-µm packing L1

Column temperature:      60°

Flow rate               :         1 mL/min.

Injection volume    :         50 µL

• System Suitability :

• • Samples      :         System suitability solution and Standard solution

• • Suitability Requirements : Peak-to-valley ratio          :         NLT 1.4,

• • System suitability solution. Calculate the peak-to-Valley ratio as follows :

Result   =   H P / H V

H P	=	height above the baseline of the desosaminylazithromycin peak
H V	=	height above the baseline of the lowest point of the curve separating the desosaminylazithromycin and azithromycin related compound F peaks Tailing factor : 0.8 – 1.5, standard solution

• Analysis

• • Samples : Standard solution and Sample solution

• • Calculate the percentage of each impurity in the portion of Azithromycin taken :

• • Result = ( r U / r S ) × ( C S / C U ) × P × F 1 × (100/ F 2 )

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	=	concentration of Azithromycin in the Sample solution (mg/mL)
P	=	potency of USP Azithromycin RS (µg/mg of azithromycin)
F 1	=	conversion factor, 0.001 mg/µg
F 2	=	relative response factor

• • Acceptance Criteria : See Table 2.

• • Disregard peaks eluting before azithromycin USP, N-oxide and after 3-deoxyazithromycin (azithromycin B). Disregard peaks with a response less than 0.1 times the response of the azithromycin peak in the Standard solution (0.1%).

Table 2

Name	Relative Retention Time	Relative Response Factor	Acceptance Criteria, NMT (%)
Azithromycin N-oxidea	0.29	0.43	0.5
3-(N,N-Didemethyl)-3-N-formylazithromycinb	0.37	1.7	0.5
Erythromycin A iminoetherd	0.42	1.0	0.5
3-N,N-Didemethyl) azithromycin (aminoazithromycin)e	0.43	1.0	0.5
Azithromycin related compound Fcf	0.51	3.8	0.5
Desosaminylazithromycing	0.54	1.0	0.3
3-N-{[4-(Acetylamino)phenyl]sulfonyl}-3,3-didemethylazithromycinh	0.55	12	0.15
N-Demethylazithromycini	0.61	1.0	0.7
Erythromycin A oximej	0.64	1.0	0.5
Azithromycin C (3-O-demethylazithromycin)k	0.73	1.0	0.5
3-De(dimethylamino)-3-oxoazithromycinl	0.76	1.5	0.5
3-N-{[4-(Acetylamino)phenyl]sulfonyl}-3-demethylazithromycinm	0.79	10	0.5
Azaerythromycin An	0.83	1.0	0.5
Azithromycin impurity Po	0.92	1.0	0.2
Azithromycin	1.0	—	—
2-Desethyl-2-propylazithromycinp	1.23	1.0	0.5
3-N-Demethyl-3-N-[(4-methylphenyl) sulfonyl] azithromycinq	1.26	5.0	0.5
3-Deoxyazithromycin (azithromycin B)r	1.31	1.0	1.0
Any individual, unidentified impurity	—	1.0	0.2
Total impurities	—	—	3.0

10.0     Assay of Azithromycin Dihydrate

• • Solution A : 10 M Potassium hydroxide.

• • Solution B : 6.7 g/L of dibasic potassium phosphate adjusted with Solution A to a pH of 11.0

• • Solution C : 6.7 g/L of dibasic potassium phosphate adjusted with phosphoric acid to a pH of 8.0

• • Mobile phase : Acetonitrile and Solution B (60 : 40)

• • Diluent  :  Acetonitrile and Solution C (60 : 40)

• • System Suitability Solution : 0.5 mg/mL each of USP Azithromycin RS and USP Azaerythromycin A RS prepared as follows. Dissolve USP Azithromycin RS and USP Azaerythromycin A RS first in acetonitrile, using 5% of the final volume, and then dilute with Diluent to volume.

• • Standard Solution : 0.53 mg/mL of USP Azithromycin RS prepared as follows.

(Dilution : 53 mg → 100 ml)

• • Dissolve USP Azithromycin RS first in acetonitrile, using 2% of the final volume, and then dilute with Diluent to volume.

• • Sample Solution : 0.53 mg/mL of Azithromycin prepared as follows.

(Dilution : 53 mg → 100 ml)

• • Dissolve Azithromycin first in acetonitrile, using 2% of the final volume, and then dilute with Diluent to volume.

• • Chromatographic System :

Mode                     :         LC

Detector                :         UV 210 nm

Column                 :         4.6-mm × 25-cm; 5-µm packing L67

Column temperature :     40°

Flow rate               :         1 mL/min.

Injection volume    :         10 µL

• • System Suitability :

• • Samples  : System suitability solution and Standard solution

[Note:- The relative retention times for azaerythromycin A and azithromycin are 0.7 and 1.0, respectively]

• • Suitability Requirements :

• • Resolution : NLT 3.0 between azaerythromycin A and azithromycin, System suitability solution.

• • Tailing factor  :  0.8 – 1.5 for azithromycin, Standard solution

• • Relative Standard Deviation : NMT 1.10% for azithromycin, Standard solution

• • Analysis

• • Samples  :  Standard solution and Sample solution

• • Calculate the quantity, in µg, of azithromycin (C38H72N2O12 ) in each mg of Azithromycin taken:

Result = ( r U / r S ) × ( C S / C U ) × P

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
C U	=	concentration of Azithromycin in the Sample solution
P	=	potency of USP Azithromycin RS (µg/mg of azithromycin)

Method of Analysis – Azithromycin Dihydrate USP