Identification—
A:
Thin-Layer Chromatographic Identification Test 
201
—
Test solution—
Transfer a weighed quantity of the Gel, equivalent to 7.5 mg of metronidazole, to a suitable flask, add 15 mL of water, shake to disperse, and sonicate for about 10 minutes. Elute a portion of this solution through a 10-mm × 15-cm chromatographic column containing a 10-cm length of ion-exchange resin with a pledget of glass wool at the bottom and top of the resin, and collect the eluate in a suitable vial.
Standard solution:
0.5 mg per mL.
Application volume:
5 µL.
Developing solvent system:
a mixture of chloroform, methanol, and ammonium hydroxide (6:3:1).
B:
The retention time of the major peak in the chromatogram of the
Assay preparation corresponds to that in the chromatogram of the
Standard preparation, as obtained in the
Assay.
Assay—
Mobile phase—
Dissolve 1.5 g of monobasic potassium phosphate and 1.3 g of dibasic sodium phosphate in 350 mL of water, add 650 mL of methanol, mix, filter, and degas. Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard preparation—
Dissolve an accurately weighed quantity of
USP Metronidazole RS in
Mobile phase, and dilute quantitatively, and stepwise if necessary, with
Mobile phase to obtain a solution having a known concentration of about 0.075 mg per mL.
Assay preparation—
Transfer an accurately weighed quantity of Gel, equivalent to 7.5 mg of metronidazole, to a 100-mL volumetric flask, add 50 mL of Mobile phase, and shake by mechanical means for 20 minutes. Dilute with Mobile phase to volume, and mix. Centrifuge a portion of this solution until clear, and use the supernatant for injection into the chromatograph.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L7. The flow rate is about 1 mL per minute. Chromatograph the
Standard preparation, and record the peak heights as directed for
Procedure: the tailing factor for the metronidazole peak is not more than 2.0; and 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 heights of the major peaks. Calculate the quantity, in mg, of metronidazole (C
6H
9N
3O
3) in the portion of Gel taken by the formula:
100C(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Metronidazole RS in the
Standard preparation; and
rU and
rS are the peak heights of metronidazole obtained from the
Assay preparation and the
Standard preparation, respectively.
