Chromatographic purity
Mobile phase, Resolution solution, and Chromatographic system
Proceed as directed in the Assay.
Test solution 1
Transfer about 25 mg of Minocycline Hydrochloride, accurately weighed, to a 100-mL volumetric flask, dilute with water to volume, and mix. Protect this solution from light, store in a refrigerator, and use within 3 hours.
Test solution 2
Transfer 1.0 mL of Test solution 1 to a 50-mL volumetric flask, dilute with water to volume, and mix. Protect this solution from light, store in a refrigerator, and use within 3 hours.
Test solution 3
Transfer 6.0 mL of Test solution 2 to a 10-mL volumetric flask, dilute with water to volume, and mix. Protect this solution from light, store in a refrigerator, and use within 3 hours.
Procedure
Separately inject equal volumes (about 20 µL) of
Test solution 1,
Test solution 2, and
Test solution 3 into the chromatograph, record the chromatograms, and measure the areas for all the peaks.
[NOTERecord the chromatogram of
Test solution 1 for a period of time that is about 2.6 times the retention time of minocycline.
] Calculate the percentage of epiminocycline in the portion of Minocycline Hydrochloride taken by the formula:
1.2rE1/rM3,
in which
rE1 is the peak response for epiminocycline obtained from
Test solution 1; and
rM3 is the peak response for minocycline obtained from
Test solution 3. Not more than 1.2% is found. Calculate the total percentage of impurities other than epiminocycline in the portion of Minocycline Hydrochloride taken by the formula:
2rs / rM2,
in which
rs is the sum of the responses of all impurity peaks other than epiminocycline obtained from
Test solution 1; and
rM2 is the peak response for minocycline obtained from
Test solution 2. Not more than 2.0% of other impurities is found.
Other requirements
Where the label states that Minocycline Hydrochloride is sterile, it meets the requirements for
Sterility Tests 71 and for
Bacterial endotoxins under
Minocycline for Injection. Where the label states that Minocycline Hydrochloride must be subjected to further processing during the preparation of injectable dosage forms, it meets the requirements for
Bacterial endotoxins under
Minocycline for Injection.
Assay
Mobile phase
Prepare a mixture of 0.2 M ammonium oxalate, 0.01 M edetate disodium, dimethylformamide, and tetrahydrofuran (600:180:120:80). Adjust with ammonium hydroxide to a pH of 7.2, and pass through a filter having a 0.5-µm or finer porosity. Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard preparation
Dissolve an accurately weighed quantity of
USP Minocycline Hydrochloride RS in water to obtain a solution having a known concentration of about 500 µg of minocycline (C
23H
27N
3O
7) per mL. Use this solution within 3 hours.
Resolution solution
Transfer 10 mg of
USP Minocycline Hydrochloride RS to a 25-mL volumetric flask, add 20 mL of 0.2 M ammonium oxalate, and swirl to dissolve. Heat on a water bath at 60
for 180 minutes, and allow to cool. Dilute with water to volume, and mix.
Assay preparation
Transfer an accurately weighed quantity of Minocycline Hydrochloride, equivalent to about 50 mg of minocycline (C23H27N3O7), to a 100-mL volumetric flask, add water to volume, and mix.
Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1, and is maintained at a constant temperature of about 40
. The flow rate is about 1.5 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the capacity factor,
k¢, is not less than 5.0 and not more than 11.5; the tailing factor for the analyte peak is not less than 0.9 and not more than 2.0; and the relative standard deviation for replicate injections is not more than 2.0%. Chromatograph the
Resolution solution and record the peak responses as directed for
Procedure: the relative retention times are about 0.7 for epiminocycline and 1.0 for minocycline; and the resolution,
R, between epiminocycline and minocycline is not less than 4.6.
Procedure
Separately inject equal volumes (about 20 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in µg per mg, of minocycline (C
23H
27N
3O
7), in the portion of Minocycline Hydrochloride taken by the formula:
100(C/W)(rU / rS),
in which
C is the concentration, in µg per mL, of minocycline (C
23H
27N
3O
7) in the
Standard preparation; W is the weight, in mg, of Minocycline Hydrochloride taken; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.