Identification—
B:
Ultraviolet Absorption 
197U
—
Solution:
20 µg per mL.
Medium:
0.25 N sodium hydroxide.
Absorptivity 6 minutes after preparation, calculated on the dried basis, at 380 nm is between 96.0% and 104.0% of that of
USP Tetracycline Hydrochloride RS, the potency of the Reference Standard being taken into account.
C:
The chromatogram of the
Assay preparation obtained as directed in the
Assay exhibits a major peak for tetracycline, the retention time of which corresponds to that exhibited in the chromatogram of the
Standard preparation obtained as directed in the
Assay.
D:
To 0.5 mg add 2 mL of sulfuric acid: a purplish red color is produced. Add the solution to 1 mL of water: the color becomes yellow.
F:
It responds to the silver nitrate test for
Chloride 191.
Loss on drying 731—
Dry about 100 mg, accurately weighed, in a capillary-stoppered bottle in vacuum at a pressure not exceeding 5 mm of mercury at 60
for 3 hours: it loses not more than 2.0% of its weight.
Limit of 4-epianhydrotetracycline—
Using the
Diluting solvent,
Chromatographic system, and
Procedure set forth in the
Assay, chromatograph a Standard solution prepared by dissolving an accurately weighed quantity of
USP 4-Epianhydrotetracycline Hydrochloride RS in
Diluting solvent to obtain a solution having a known concentration of about 10 µg per mL. Using the chromatogram so obtained and the chromatogram of the
Assay preparation obtained as directed in the
Assay, calculate the percentage of 4-epianhydrotetracycline hydrochloride in the Tetracycline Hydrochloride taken by the formula:
10(CE / W)(rU / rS),
in which
CE is the concentration, in µg per mL, of
USP 4-Epianhydrotetracycline Hydrochloride RS in the Standard solution;
W is the weight, in mg, of Tetracycline Hydrochloride taken to prepare the
Assay preparation; and
rU and
rS are the 4-epianhydrotetracycline peak responses obtained from the
Assay preparation and the Standard solution, respectively: not more than 2.0% is found.
Assay—
Diluting solvent—
Mix 680 mL of 0.1 M ammonium oxalate and 270 mL of dimethylformamide.
Mobile phase—
Mix 680 mL of 0.1
M ammonium oxalate, 270 mL of dimethylformamide, and 50 mL of 0.2
M dibasic ammonium phosphate. Adjust, if necessary, with 3 N ammonium hydroxide or 3 N phosphoric acid to a pH of 7.6 to 7.7. Make any other necessary adjustments (see
System Suitability under
Chromatography 621). Filter through a membrane filter of 0.5-µm or finer porosity.
Standard preparation—
Dissolve an accurately weighed quantity of
USP Tetracycline Hydrochloride RS in
Diluting solvent, and dilute quantitatively with
Diluting solvent to obtain a solution having a known concentration of about 0.5 mg per mL.
Assay preparation—
Transfer about 50 mg of Tetracycline Hydrochloride, accurately weighed, to a 100-mL volumetric flask, dissolve in Diluting solvent, dilute with the same solvent to volume, and mix.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 280-nm detector, a 4.6-mm × 3-cm guard column that contains 10-µm packing L7, and a 4.6-mm × 25-cm analytical column that contains 5- to 10-µm packing L7. The flow rate is about 2 mL per minute. Chromatograph the
Resolution solution, and record the peak responses as directed for
Procedure: the relative retention times are about 0.9 for 4-epianhydrotetracycline and 1.0 for tetracycline; and the resolution,
R, between the 4-epianhydrotetracycline and tetracycline peaks is not less than 1.2. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the relative standard deviation for replicate injections is not more than 2.0%.
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, of C
22H
24N
2O
8·HCl in each mg of the Tetracycline Hydrochloride taken by the formula:
100(CP / W)(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Tetracycline Hydrochloride RS in the
Standard preparation; P is the potency, in µg per mg, of
USP Tetracycline Hydrochloride RS;
W is the weight, in mg, of Tetracycline Hydrochloride taken to prepare the
Assay preparation; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
,4a
,12a
240