Flumazenil

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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C₁₅H₁₄FN₃O₃ 303.29

4H-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

CAS RN^®: 78755-81-4; UNII: 40P7XK9392.

1 DEFINITION

Flumazenil contains NLT 98.0% and NMT 102.0% of flumazenil (C₁₅H₁₄FN₃O₃), calculated on the dried basis.

2 IDENTIFICATION

Change to read:

A. SPECTROSCOPIC IDENTIFICATION TESTS (197), Infrared Spectroscopy: 197K (CN 1-MAY-2020)

B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

3 ASSAY

3.1 PROCEDURE

Solution A: 800 mL of water, adjusted with phosphoric acid to a pH of 2.0 ± 0.05

Mobile phase: Methanol, tetrahydrofuran, and Solution A (13:7:80)

System suitability solution: 6.4 µg/mL each of USP Flumazenil RS and USP Flumazenil Related Compound B RS in Mobile phase

Standard solution: 1.0 mg/mL of USP Flumazenil RS in Mobile phase

Sample solution: 1.0 mg/mL of Flumazenil in Mobile phase

3.2 Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 230 nm

Column: 4.6-mm x 25-cm; 5-µm packing L1

Flow rate: 1 mL/min

Injection volume: 5 µL

Run time: NLT 1.3 times the

3.3 System suitability

Samples: System suitability solution and Standard solution

[NOTE-See Table 2 for the relative retention times.]

Suitability requirements

Resolution: NLT 4.0 between flumazenil related compound B and flumazenil, System suitability solution

Tailing factor: NMT 1.5 for flumazenil, System suitability solution

Relative standard deviation: NMT 2.0%, Standard solution

3.4 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of flumazenil (C₁₅H₁₄FN₃O₃) in the portion of Flumazenil taken:

Result = (r_U/r_S) x (C_S/C_U) x 100

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution

C_S = concentration of USP Flumazenil RS in the Standard solution (mg/mL)

C_U = concentration of Flumazenil in the Sample solution (mg/mL)

Acceptance criteria: 98.0%-102.0% on the dried basis

4 IMPURITIES

4.1 RESIDUE ON IGNITION (281)

NMT 0.1%

Change to read:

4.2 LIMIT OF FLUMAZENIL RELATED COMPOUND C

Diluent: Alcohol and chloroform (50:50)

Standard solution A: 0.5 mg/mL of USP Flumazenil RS and 0.6 µL/mL of USP Flumazenil Related Compound C RS in Diluent

Standard solution B: 0.1 mg/mL of USP Flumazenil RS and 0.12 µL/mL (ERR 1-Jun-2018) of USP Flumazenil Related Compound C RS from Standard solution A in Diluent

Sample solution: 50 mg/mL of Flumazenil in Diluent

Chromatographic system

(See Chromatography (621), Thin-Layer Chromatography.)

Mode: TLC

Adsorbent: 0.25-mm layer of chromatographic silica gel mixture

Application volume: 10 µL

Developing solvent system: Chloroform, glacial acetic acid, alcohol, and water (75:15:7.5:2.5)

Spray reagent: Dissolve 0.5 g of ninhydrin in 90 mL of alcohol, and add 10 mL of glacial acetic acid.

Analysis

Samples: Standard solution A, Standard solution B, and Sample solution

Proceed as directed in the chapter. Dry the plate for 10 min in a current of cold air, and view under short-wavelength UV light. Spray the plate with Spray reagent, and heat at 105° for 15 min. The R_F values of the analytes with their corresponding methods of detection are listed in Table 1.

Table 1

Acceptance criteria: Any spot at an R, value corresponding to flumazenil related compound C of the Sample solution is not more intense than the corresponding spot of Standard solution B (NMT 0.2%).

4.3 ORGANIC IMPURITIES

Solution A, Mobile phase, System suitability solution, Sample solution, Chromatographic system, and System suitability: Proceed as directed in the Assay except use a Run time of NLT 3 times the retention time of flumazenil.

Standard solution: 1 µg/mL of USP Flumazenil RS in Mobile phase

Analysis

Samples: Sample solution and Standard solution

Calculate the percentage of each impurity in the portion of Flumazenil taken:

Result = (r_U/r_S) x (C_S/C_U) x (1/F) x 100

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution

C_S = concentration of USP Flumazenil RS in the Standard solution (mg/mL)

C_U = concentration of Flumazenil in the Sample solution (mg/mL)

F = relative response factor (see Table 2)

Acceptance criteria: See Table 2.

Table 2

^a 8-Fluoro-5-methyl-6-oxo-5,6-dihydro-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylic acid; also known as 8-Fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazol-[1,5-a][1,4]benzodiazepine-3-carboxylic acid.

^b 7-Fluoro-4-methyl-3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione.

^c Ethyl 5-methyl-6-oxo-5,6-dihydro-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate.

^d Ethyl 8-chloro-5-methyl-6-oxo-5,6-dihydro-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate.

5 SPECIFIC TESTS

5.1 BACTERIAL ENDOTOXINS TEST (85)

NMT 25.0 USP Endotoxin Units/mg of flumazenil

5.2 LOSS ON DRYING (731)

Analysis: Dry at 105° for 3 h.

Acceptance criteria: NMT 0.5%

6 ADDITIONAL REQUIREMENTS

6.1 PACKAGING AND STORAGE

Preserve in tight containers, and store at controlled room temperature.

6.2 USP REFERENCE STANDARDS (11)

USP Flumazenil RS

USP Flumazenil Related Compound B RS

Ethyl 8-hydroxy-5-methyl-6-oxo-5,6-dihydro-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate; also known as Ethyl 8-hydroxy-5,6-dihydro-5-methyl-6-oxo-4H-imidazol-[1,5-a] [1,4]benzodiazepine-3-carboxylate.

C₁₅H₁₅N₃O₄ 301.30

USP Flumazenill Related Compound C RS

1,1-Diethyoxy-N,N-dimethylmethanamine;

also known as N,N-Dimethylformamide diethyl acetal.

C₇H₁₇NO₂ 147.22