Estazolam

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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4H-[1,2,4]Triazolo[4,3-a][1,4]benzodiazepine, 8-chloro-6-phenyl-;

8-Chloro-6-phenyl-4H-s-triazolo[4,3-a][1,4]benzodiazepine CAS RN^®: 29975-16-4; UNII: 36S3EQV54C.

1 DEFINITION

Estazolam contains NLT 98.0% and NMT 102.0% of estazolam (C₁₆H₁₁CIN₄), calculated on the dried basis.

2 IDENTIFICATION

Change to read:

A. SPECTROSCOPIC IDENTIFICATION TESTS (197), Infrared Spectroscopy: 197K (CN1-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

PROCEDURE

Buffer: 2.8 g/L of monobasic potassium phosphate in water. Adjust with 1 N sodium hydroxide to a pH of 6.5.

Mobile phase: Acetonitrile, methanol, and Buffer (10:35:55)

Standard stock solution: 0.5 mg/mL of USP Estazolam RS in Mobile phase

Standard solution: 0.02 mg/ml of USP Estazolam RS in water from Standard stock solution

Sample stock solution: 0.5 mg/mL of Estazolam in Mobile phase

Sample solution: 0.02 mg/ml of Estazolam in water from Sample stock solution

Chromatographic system

(See Chromatography (621) System Suitability)

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm x 15 cm; 3-um packing L11

Flow rate: 1 mL/min

Injection volume: 25 μL

Run time: 2.5 times the retention time of the estazolam peak

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 2.0

Relative standard deviation: NMT 2.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of estazolam (C₁₆H₁₁CIN₄) in the portion of Estazolam taken

Result = (r_U/(r_S) × (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 Estazolam RS in the Standard solution (mg/mL)

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

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

IMPURITIES

Residue on Ignition (281): NMT 0.1%

Organic Impurities

Solution A: Acetonitrile

Solution B: Water

Mobile phase: See Table 1.

[Note—The gradient was established on an HPLC system with a dwell volume of approximately 1.0 mL.]

System suitability solution: 1 μg/mL each of USP Estazolam RS, USP Nordazepam RS, and USP Estazolam Related Compound A RS in

acetonitrile

Standard solution: 1 μg/mL of USP Estazolam RS in acetonitrile

Sample solution: 1 mg/mL of Estazolam in acetonitrile

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 15-cm; 3-μm packing L1

Flow rate: 1 mL/min

Injection volume: 20 μL

System suitability

Samples: System suitability solution and Standard solution

Suitability requirements

Resolution: NLT 2.0 between nordazepam and estazolam related compound A, System suitability solution

Tailing factor: NMT 1.2 for estazolarn, Standard solution

Relative standard deviation: NMT 2.0% for estazolam, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of any individual impurity in the portion of Estazolam taken:

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

r_U = peak response of each individual impurity from the Sample solution

r_S = peak response of estazolam from the Standard solution

C_S = concentration of USP Estazolam RS in the Standard solution (μg/mL)

C_U = concentration of Estazolam in the Sample solution (μg/mL)

F = relative response factor (see Table 2)

Acceptance criteria: See Table 2.

^a 7-Chloro-1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one.

^b 5-Chloro-2-(3-chloromethyl-4H-1,2,4-triazol-4-yl)-benzophenone.

^c N-(2-Benzoyl-4-chlorophenyl) formamide.

^d 2-(N-Chloroacetyl-N-chloroacetylhydrazonomethyl)amino-5-chlorobenzophenone.

^e (2-Amino-5-chlorophenyl) phenyl-methanone.

^f N-(2-Benzoyl-4-chlorophenyl)-2-chloroacetamide.

4 SPECIFIC TESTS

Loss on Drying (731)

Analysis: Dry at 105° for 4 h.

Acceptance criteria: NMT 1.0%

5 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight, light-resistant containers. Store at controlled room temperature.