Chlordiazepoxide and Amitriptyline Hydrochloride Tablets

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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1 DEFINITION

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Chlordiazepoxide and Amitriptyline Hydrochloride Tablets contain NLT 90.0% and NMT 110.0% of the labeled amount of chlordiazepoxide (C₁₆H₁₄ClN₃O) and an amount of amitriptyline hydrochloride equivalent to NLT 90.0% and NMT 110.0% of the labeled amount of amitriptyline (C₂₀H₂₃N).

Protect all solutions containing chlordiazepoxide from light. (USP 1-Dec-2022)

2 IDENTIFICATION

A. The retention times of the major peaks of the Sample solution correspond to those of the Standard solution, as obtained in the Assay. Add the following:

B. The UV spectra of the chlordiazepoxide and amitriptyline peaks of the Sample solution correspond to those of the Standard solution, as obtained in the Assay.▲ (USP 1-Dec-2022)

3 ASSAY

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3.1 PROCEDURE

Solution A: Transfer 7.0 mL of ammonium hy droxide to an appropriate 1-L flask containing about 500 mL of water. Dilute with water to volume.

Solution B: Acetonitrile Mobile phase: See Table 1.

Table 1

Diluent: Acetonitrile and water (50:50)

Standard solution: 50 μg/mL of USP Chlordiazepoxide RS and 141 μg/mL of USP Amitriptyline Hydrochloride RS (equivalent to 125 μg/mL of amitriptyline) in Diluent

Sample stock solution: Nominally 0.1 mg/mL of chlordiazepoxide and nominally 0.25 mg/mL of amitriptyline from Tablets (NLT 20) prepared as follows. Finely powder Tablets (NLT 20) and transfer a portion of the powder equivalent to 5 mg of chlordiazepoxide and 12.5 mg of amitriptyline to a 50-mL volumetric flask. Add 80% of the flask volume of acetonitrile, sonicate for about 15 min, and then mechanically shake for about 1 h. Dilute with acetonitrile to volume. Centrifuge a portion of the resulting solution and use the supernatant. [NOTE—A centrifuge speed of 3250 rpm for 10 min may be suitable.]

Sample solution: Nominally 50 μg/mL of chlordiazepoxide and nominally 125 μg/mL of amitriptyline from Sample stock solution in water

3.1.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 254 nm. For Identification B, use a diode array detector in the range of 200–400 nm. Column: 2.1-mm × 15-cm; 3.5-μm packing L1

Column temperature: 30° Flow rate: 0.4 mL/min Injection volume: 5 µL

3.1.2 System suitability

Sample: Standard solution

[NOTE—The relative retention times for chlordiazepoxide and amitriptyline are 1.0 and 5.5, respectively.] Suitability requirements

Resolution: NLT 2.0 between chlordiazepoxide and amitriptyline

Tailing factor: NMT 1.5 for chlordiazepoxide; NMT 2.0 for amitriptyline

Relative standard deviation: NMT 1.0% each for chlordiazepoxide and amitriptyline (USP 1-Dec-2022)

3.1.3 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of chlordiazepoxide (C₁₆H₁₄ClN₃O) in the portion of Tablets taken:

Result = (r_u/r_s) × (C_s/C_u) × 100

r_u = peak response of chlordiazepoxide from the Sample solution

r_s = peak response of chlordiazepoxide from the Standard solution

C_s  = concentration of USP Chlordiazepoxide RS in the Standard solution (μg/mL) (USP 1-Dec-2022)

C_u  = nominal concentration of chlordiazepoxide in the Sample solution (μg/mL) (USP 1-Dec-2022)

Calculate the percentage of the labeled amount of amitriptyline (C₂₀H₂₃N) in the portion of Tablets taken:

Result = (r_u/r_s) × (C_s/C_u) × (M_r1/M_r2) × 100

r_u = peak response of amitriptyline from the Sample solution

r_s = peak response of amitriptyline from the Standard solution

C_s = concentration of USP Amitriptyline Hydrochloride RS in the Standard solution (μg/mL) (USP 1-Dec-2022)

C_u = nominal concentration of amitriptyline in the Sample solution (μg/mL) (USP 1-Dec-2022)

M_r1 = molecular weight of amitriptyline, 277.40

M_r2 = molecular weight of amitriptyline hydrochloride, 313.87

Acceptance criteria: 90.0%–110.0% each of the labeled amounts of chlordiazepoxide (C₁₆H₁₄ClN₃O) and amitriptyline (C₂₀H₂₃N)

4 PERFORMANCE TESTS

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4.1 DISSOLUTION 〈711〉

Medium: Simulated gastric fluid TS, prepared without pepsin; 900 mL Apparatus 1: 100 rpm

Time: 30 min

Standard solution A: USP Chlordiazepoxide RS in Medium Standard solution B: USP Amitriptyline Hydrochloride RS in Medium

Sample solution: Pass a portion of solution under test through a suitable filter. Dilute with Medium, if necessary.

4.1.1 Instrumental conditions

Mode: UV

Analytical wavelengths: 239 and 309 nm Blank: Medium

4.1.2 Analysis

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

Calculate the percentage of the labeled amount of chlordiazepoxide (C₁₆H₁₄ClN₃O) in the portion of Tablets dissolved: (USP 1-Dec-2022)

Result = (A_u/A_s) × C_SA × V (USP 1-Dec-2022) × D (USP 1-Dec-2022) × (1/L) × 100

A_u = absorbance from the Sample solution, 309 nm

A_s = absorbance from Standard solution A, 309 nm

C_SA = concentration of USP Chlordiazepoxide RS in Standard solution A (mg/mL)

V = volume of Medium, 900 mL (USP 1-Dec-2022)

D = dilution factor of the Sample solution, if needed (USP 1-Dec-2022)

L = label claim of chlordiazepoxide (mg/Tablet)

Calculate the absorbance of amitriptyline in the Sample solution at 239 nm (A ):

Result = A_U239− {A_U309× [(C_S309× A_S239)/(C_S239× A_S309)]}

A_U239 = absorbance from the Sample solution, 239 nm

A_U309 = absorbance from the Sample solution, 309 nm

C_S309 = concentration of chlordiazepoxide from Standard solution A(mg/mL), (USP 1-Dec-2022) 309 nm

A_S239 = absorbance from Standard solution A, 239 nm

C_S239 = concentration of chlordiazepoxide from Standard solution A(mg/mL), (USP 1-Dec-2022) 239 nm

A_S309 = absorbance from Standard solution A, 309 nm

Calculate the percentage of the labeled amount of amitriptyline (C₂₀H₂₃N) in the portion of Tablets dissolved: (USP 1-Dec-2022)

Result = (A_x/A_s) × C_s × V (USP 1-Dec-2022) × D × (M_r1/M_r2) (USP 1-Dec-2022) × (1/L) × 100

A_x = absorbance from the Sample solution, as determined from the previous equation

A_s = absorbance of amitriptyline from Standard solution B, 239 nm

C_s = concentration of USP Amitriptyline Hy drochloride RS in Standard solution B (mg/mL)

V  = volume of Medium, 900 mL (USP 1-Dec-2022)

D = dilution factor of the Sample solution, if needed

M_r1 = molecular weight of amitriptyline, 277.40

M_r2 = molecular weight of amitriptyline hydrochloride, 313.87 (USP 1-Dec-2022)

L = label claim of amitriptyline (mg/Tablet)

[NOTE—All of the chlordiazepoxide measurements may be made with either a single Standard solution or two separate Standard solutions.] Tolerances: NLT 85% (Q ) of the labeled amount of chlordiazepoxide (C₁₆H₁₄ClN₃O), and an amount of amitriptyline hydrochloride equivalent to NLT 85% (Q ) of the labeled amount of amitriptyline (C₂₀H₂₃N) are dissolved.

4.2 UNIFORMITY OF DOSAGE UNITS 〈905〉

Content Uniformity: Meet the requirements for both chlordiazepoxide and amitriptyline

5 IMPURITIES

5.1 ORGANIC IMPURITIES

Standard solution A: 1 mg/mL of USP Chlordiazepoxide Related Compound A RS in acetone Standard solution B: 50 µg/mL of USP 2-Amino-5-chlorobenzophenone RS in acetone

Sample solution: Transfer a portion of finely powdered Tablets equivalent to 25 mg of chlordiazepoxide to a 10-mL conical flask, add 2.5 mL of acetone, and shake. Allow any undissolved particles to settle, and use the supernatant.

5.1.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.) Mode: TLC

Adsorbent: 0.25-mm layer of chromatographic silica gel

Application volumes

Standard solution A: 20 µL

Standard solution B: 10 µL

Sample solution: 50 µL

Developing solvent system: Ethyl acetate

Spray reagent A: 2 N sulfuric acid

Spray reagent B: 1 mg/mL of sodium nitrite in water

Spray reagent C: 5 mg/mL of ammonium sulfamate in water

Spray reagent D: 1 mg/mL of N-(1-naphthyl)ethylenediamine dihydrochloride in water Analysis

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

Develop the chromatogram in a chromatographic chamber (not previously saturated with the Developing solvent system) using the Developing solvent system until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and allow the solvent to evaporate. Locate the spots on the plate by lightly spraying with Spray reagent A, drying at 105° for 15 min, and then spraying in succession with Spray reagent B, Spray reagent C, and Spray reagent D.

Acceptance criteria: Any spots from the Sample solution are not greater in size or intensity than the spots at the respective R values produced by the Standard solutions, corresponding to NMT 4.0% of chlordiazepoxide related compound A, and NMT 0.1% of 2-amino-5- chlorobenzophenone.

6 ADDITIONAL REQUIREMENTS

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PACKAGING AND STORAGE: Preserve in tight, light-resistant containers. Store at controlled room temperature. (USP 1-Dec-2022)

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USP REFERENCE STANDARDS 〈11〉

USP 2-Amino-5-chlorobenzophenone RS 

USP Amitriptyline Hydrochloride RS USP Chlordiazepoxide RS

USP Chlordiazepoxide Related Compound A RS