Flumazenil
(floo maz' e nil).
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303.294H-Imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid, 8-fluoro-5,6-dihydro-5-methyl-6-oxo-, ethyl ester.
Ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate
[78755-81-4].» Flumazenil contains not less than 98.0 percent and not more than 102.0 percent of C15H14FN3O3, calculated on the dried basis.
Packaging and storage—
Preserve in tight containers, and store at controlled room temperature.
USP Reference standards 
11
—
11—
USP Flumazenil RS 
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USP Flumazenil Related Compound B RS
Ethyl 8-hydroxy-5,6-dihydro-5-methyl-6-oxo-4H-imidazol-[1,5-a][1,4]benzodiazepine-3-carboxylate.
C15H15N3O4 301.30
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Ethyl 8-hydroxy-5,6-dihydro-5-methyl-6-oxo-4H-imidazol-[1,5-a][1,4]benzodiazepine-3-carboxylate.
C15H15N3O4 301.30
USP Flumazenil Related Compound C RS
N,N-Dimethylformamide diethyl acetal.
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N,N-Dimethylformamide diethyl acetal.
Identification—
B:
The retention time of the major peak for flumazenil in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.
Melting range, Class Ia 741:
between 198
and 202.
741:
between 198 and 202.
Bacterial endotoxins 85—
It contains not more than 25.0 USP Endotoxin Units per mg of flumazenil.
85—
It contains not more than 25.0 USP Endotoxin Units per mg of flumazenil.
Loss on drying 731—
Dry it at 105 for 3 hours: it loses not more than 0.5% of its weight.
731—
Dry it at 105 for 3 hours: it loses not more than 0.5% of its weight.
Residue on ignition 281:
not more than 0.1%.
281:
not more than 0.1%.
Heavy metals, Method II 231:
0.002%.
231:
0.002%.
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).
Adsorbent:
0.25-mm layer of chromatographic silica gel mixture (see Chromatography 621).
621).
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 USP Flumazenil Related Compound C RS 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.
Any spot at an RF value corresponding to flumazenil related compound C 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.
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 |
| Flumazenil related compound C | about 0.04 | Ninhydrin |
test 2—
Diluted phosphoric acid, pH 2.0, Mobile phase, System suitability solution, and Chromatographic system—
Proceed as directed in the Assay.
Standard solution—
Dilute the Standard preparation quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having a known concentration of about 1 µg per mL.
Test solution—
Use the Assay preparation.
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 the 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:
100(CS / CU)(ri / rS)(1/F)
in which CS and CU are the concentrations, in mg per mL, of flumazenil in the Standard solution and the Test solution, respectively; ri is the peak area for any impurity in the Test solution; rS is the peak area for flumazenil in the Standard solution; and F is the relative response factor for each of the known impurities relative to flumazenil. [note—F values are given for all the impurities, along with the corresponding limits, 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).
621).
System suitability solution—
Dissolve appropriate quantities of USP Flumazenil RS and USP Flumazenil Related Compound B 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 of flumazenil.
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 5 µL of the System suitability solution, and record the peak responses: the relative retention times are about 0.8 for flumazenil related compound B and 1.0 for flumazenil; the resolution, R, between flumazenil related compound B and flumazenil is not less than 4.0; 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 5 µL of the Standard preparation, and record the peak responses: the relative standard deviation for replicate injections is not more than 2.0%.
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 5 µL of the System suitability solution, and record the peak responses: the relative retention times are about 0.8 for flumazenil related compound B and 1.0 for flumazenil; the resolution, R, between flumazenil related compound B and flumazenil is not less than 4.0; 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 5 µL of the Standard preparation, and record the peak responses: 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 percentage of C15H14FN3O3 in the portion of Flumazenil taken by the formula:
100(CS /CU)(rU / rS)
in which CS and CU are the concentrations, in mg per mL, of flumazenil in the Standard preparation and the Assay preparation, respectively; and rU and rS are the peak areas obtained from the Assay preparation and the Standard preparation, respectively.
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Ravi Ravichandran, Ph.D.
Principal Scientific Liaison 1-301-816-8330 |
(SM42010) Monographs - Small Molecules 4 |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
|
| 85 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
USP35–NF30 Page 3217
Pharmacopeial Forum: Volume No. 32(1) Page 94