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
.
Limit of related substances—
[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, 105, and 160 µg per mL, respectively.
Standard solution—
Transfer 4.0 mL of
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, 2.1, 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 relative retention times are about 0.38 for desosaminylazithromycin, 0.54 for
N-demethylazithromycin, and 1.0 for azithromycin; 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.
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%.
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 (C
38H
72N
2O
12) 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.
;)
-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*)].