Related compounds—
TEST 1—
Ninhydrin solution—
Dissolve 0.5 g of ninhydrin in 90 mL of alcohol, and add 10 mL of glacial acetic acid.
Diluent—
Prepare a mixture of alcohol and chloroform (1:1).
Test solution—
Transfer about 250 mg of Flumazenil, accurately weighed, to a 5-mL volumetric flask. Dissolve in and dilute with Diluent to volume, and mix.
Standard solution 1—
Prepare a solution of
USP Flumazenil RS and
N,N-dimethylformamide diethyl acetal in
Diluent having known concentrations of about 0.5 mg per mL and about 0.6 µL per mL, respectively.
Standard solution 2—
Dilute 2.0 mL of Standard solution 1 with Diluent to 10.0 mL.
Application volume:
10 µL.
Developing solvent system:
a mixture of chloroform, glacial acetic acid, alcohol, and water (75:15:7.5:2.5).
Procedure—
Proceed as directed for
Thin-Layer Chromatography under
Chromatography 
621
. Dry the plate for 10 minutes in a current of cold air, and view under short-wavelength UV light. Spray the plate with
Ninhydrin solution, and heat at 105
for 15 minutes. The
RF values of analytes are as follows.
| Compound |
 RF |
Detection |
| Flumazenil |
about 0.8 |
UV |
N,N-Dimethylformamide
diethyl acetal |
about 0.04 |
Ninhydrin |
Any spot at an
RF value corresponding to
N,N-dimethylformamide diethyl acetal in the chromatogram obtained from the
Test solution is not more intense than the corresponding spot in the chromatogram obtained from
Standard solution 2: not more than 0.2% is found.
TEST 2—
Diluted phosphoric acid, pH 2.0, Mobile phase, System suitability solution, and Chromatographic system—
Proceed as directed in the Assay.
Standard solution—
Dissolve an accurately weighed quantity of
USP Flumazenil RS in
Mobile phase, and dilute quantitatively, and stepwise if necessary, with
Mobile phase to obtain a solution having a known concentration of about 1 µg per mL.
Test solution—
Transfer about 25.0 mg of Flumazenil, accurately weighed, to a 25-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix.
Procedure—
Separately inject equal volumes (about 5 µL) of the
Standard solution and the
Test solution into the chromatograph, record the chromatograms for at least three times the retention time of flumazenil peak, and measure the areas for the major peaks. Calculate the percentage of any impurity in the portion of Flumazenil taken by the formula:
2500(C/W)(1/F)(ri / rS),
in which
C is the concentration, in mg per mL, of
USP Flumazenil RS in the
Standard solution; F is the relative response factor according to the
Table below;
W is the weight, in mg, of Flumazenil, on the dried basis, used to prepare the
Test solution; ri is the peak area for any impurity in the
Test solution; and
rS is the peak area for flumazenil in the
Standard solution: the impurities meet the requirements given in the
Table below.
| Compound Name |
Relative Retention
Time |
Relative Response
Factor |
Limit (%) |
| Flumazenil related compound A |
about 0.4 |
1.1 |
0.2 |
7-Fluoro-4-methyl-3,4-dihydro-2,5H-1,4-
benzodiazepine-2,5-dione |
about 0.5 |
1.5 |
0.2 |
Ethyl 5,6-dihydro-5-methyl-6-oxo-4H-imidazo-
[1,5-a][1,4]benzodiazepine-3-carboxylate |
about 0.7 |
1.3 |
0.2 |
| Flumazenil related compound B |
about 0.8 |
1.1 |
0.2 |
| Flumazenil |
1.0 |
— |
— |
Ethyl 8-chloro-5,6-dihydro-5-methyl-6-oxo-4H-
imidazo-[1,5-a][1,4]benzodiazepine-3-carboxylate |
about 2.2 |
1.1 |
0.2 |
| Any individual unknown impurity |
— |
1.0 |
0.1 |
| Total |
— |
— |
0.5 |
Assay—
Diluted phosphoric acid, pH 2.0—
Adjust 800 mL of water with phosphoric acid to a pH of 2.0 ± 0.05.
Mobile phase—
Prepare a filtered and degassed mixture of
Diluted phosphoric acid, pH 2.0, methanol, and tetrahydrofuran (80:13:7). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
System suitability solution—
Dissolve appropriate quantities of chlordiazepoxide and
USP Flumazenil RS in
Mobile phase, and dilute stepwise if necessary, with
Mobile phase to obtain a solution having a known concentration of about 6.4 µg per mL of each compound.
Standard preparation—
Dissolve an accurately weighed quantity of
USP Flumazenil RS in
Mobile phase, and dilute quantitatively, and stepwise if necessary, with
Mobile phase to obtain a solution having a known concentration of about 1.0 mg per mL.
Assay preparation—
Transfer about 25.0 mg of Flumazenil, accurately weighed, to a 25-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 230-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1.0 mL per minute. Chromatograph the
System suitability solution, and record the peak responses as directed for
Procedure: the relative retention times are about 0.82 for chlordiazepoxide and 1.0 for flumazenil; the resolution,
R, between chlordiazepoxide and flumazenil is not less than 2.0; the column efficiency is not less than 1500 theoretical plates for the flumazenil peak; and the tailing factor is not more than 1.5 for the flumazenil peak. 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 5 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the flumazenil peaks. Calculate the quantity, in mg, of C
15H
14FN
3O
3 in the portion of Flumazenil taken by the formula:
25C(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Flumazenil RS in the
Standard preparation; and
rU and
rS are the peak areas obtained from the
Assay preparation and the
Standard preparation, respectively.
;)
