Ethambutol Hydrochloride

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

Ethambutol Hydrochloride contains NLT 98.0% and NMT 100.5% of ethambutol hydrochloride (C₁₀H₂₄N₂O₂ · 2HCl), calculated on the dried basis.

2 IDENTIFICATION

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197A or 197K

B. Identification Tests—General 〈191〉, Chemical Identification Tests, Chloride

Sample solution: 100 mg/mL in water

Acceptance criteria: Meets the requirements

3 ASSAY

Procedure

Sample solution: 200 mg of Ethambutol Hydrochloride in a mixture of 100 mL of glacial acetic acid and 5 mL of mercuric acetate TS. Add crystal violet TS.

Analysis: Titrate with 0.1 N perchloric acid VS (the color change at the endpoint is from blue to blue-green). Perform a blank determination, and make any necessary corrections (see Titrimetry 〈541〉). Each milliliter of 0.1 N perchloric acid is equivalent to 13.86 mg of ethambutol hydrochloride (C₁₀H₂₄N₂O₂ · 2HCl).

Acceptance criteria: 98.0%–100.5% on the dried basis

4 IMPURITIES

Organic Impurities

Protect all solutions containing ethambutol from light.

Solution A: Methanol and water (50:50)

Solution B: Methanol

Mobile phase: See Table 1.

Table 1

Impurity stock solution A: To 4 mg of USP Ethambutol Related Compound A RS, add 4 mL of acetonitrile and 0.1 mL of triethylamine. Sonicate for 5 min and add 15 μL of (R)-(+)-alpha-methylbenzyl isocyanate. Heat the mixture at 70° for 20 min.

Impurity stock solution B: To 4 mg of USP Ethambutol Related Compound B RS, add 4 mL of acetonitrile and 0.1 mL of triethylamine. Sonicate for 5 min and add 15 μL of (R)-(+)-alpha-methylbenzyl isocyanate. Heat the mixture at 70° for 20 min.

System suitability solution: To 4 mg of USP Ethambutol Hydrochloride RS, add 4 mL of acetonitrile and 0.1 mL of triethylamine. Sonicate for 5 min and add 15 μL of (R)-(+)-alpha-methylbenzyl isocyanate. Heat the mixture at 70° for 20 min. Add 40 μL each of Impurity stock solution A and Impurity stock solution B.

Standard stock solution: To 4 mg of USP Ethambutol Hydrochloride RS, add 4 mL of acetonitrile and 0.1 mL of triethylamine. Sonicate for 5 min and add 15 μL of (R)-(+)-alpha-methylbenzyl isocyanate. Heat the mixture at 70° for 20 min.

Standard solution: Dilute 0.5 mL of Standard stock solution with acetonitrile to 100.0 mL.

Sample solution: To 4 mg of Ethambutol Hydrochloride, add 4 mL of acetonitrile and 0.1 mL of triethylamine. Sonicate for 5 min and add 15μL of (R)-(+)-alpha-methylbenzyl isocyanate. Heat the mixture at 70° for 20 min.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 215 nm

Column: 4.6-mm × 10-cm; 3-μm packing L1

Temperatures

Autosampler: 10°

Column: 40°

Flow rate: 1 mL/min

Injection volume: 10 μL

System suitability

Samples: System suitability solution and Standard solution

[Note—The relative retention times for ethambutol related compound B, ethambutol, and ethambutol related compound A are 0.91, 1.0, and 1.3, respectively.]

Suitability requirements

Resolution: NLT 1.2 between ethambutol related compound B and ethambutol; NLT 3.0 between ethambutol and ethambutol related compound A, System suitability solution

Relative standard deviation: NMT 5.0%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of any individual impurity in the portion of Ethambutol Hydrochloride taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

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

r_S = peak response of ethambutol from the Standard solution

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

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

Acceptance criteria: See Table 2. The reporting threshold is 0.05%.

Table 2

^a Calculate any individual impurity and total impurities from the relative retention times of 0.75 to 1.5 with reference to ethambutol.

Change to read:

Limit of Aminobutanol

Solution A: 12.4 mg/mL of boric acid in water. Adjust with 5 N sodium hydroxide to a pH of 9.0 prior to final dilution.

Fluorescamine solution: 0.1 mg/mL of fluorescamine in acetone

Standard solution: 5 μg/mL of USP Aminobutanol RS

Sample solution: 0.5 mg/mL of Ethambutol Hydrochloride

Instrumental conditions

(See Fluorescence Spectroscopy 〈853〉.)

Mode: Fluorometry

Analytical wavelength: 485 nm, with the excitation wavelength at about 385 nm

Cell: 1 cm

Analysis

Samples: Standard solution and Sample solution

Pipet a 10-mL portion of the Sample solution into a glass-stoppered, 100-mL conical flask, and add 10 mL of water and 20 mL of Solution A. To another 100-mL flask, add 10.0 mL of the Sample solution, 10.0 mL of the Standard solution, and 20 mL of Solution A. Place the flasks on a magnetic stirrer, and while the contents are being stirred rapidly, add 10 mL of Fluorescamine solution rapidly. Insert the stoppers in the flasks, invert, and shake briefly. After 1 min, accurately timed, determine the relative fluorescence intensities of both solutions in a suitable fluorometer.

Acceptance criteria: The fluorescence intensity of the solution from the Sample solution is NMT (ERR 1-Aug-2020) the difference between the intensities of the two solutions (NMT 1.0%). (ERR 1-Aug-2020)

5 SPECIFIC TESTS

Optical Rotation 〈781S〉, Procedures, Specific Rotation

Sample solution: 100 mg/mL in water

Acceptance criteria: +6.0° to +6.7°

Loss on Drying 〈731〉

Analysis: Dry at 105° for 2 h.

Acceptance criteria: NMT 0.5%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in well-closed containers.

USP Reference Standards 〈11〉

USP Aminobutanol RS C H NO 89.14

USP Ethambutol Hydrochloride RS

USP Ethambutol Related Compound A RS

((2R,2′S)-2,2′-[Ethane-1,2-diylbis(azanediyl)]dibutan-1-ol)dihydrochloride.

C₁₀H₂₄N₂O₂ · 2HCl 277.23

USP Ethambutol Related Compound B RS

((2R,2′R)-2,2′-[Ethane-1,2-diylbis(azanediyl)]dibutan-1-ol)dihydrochloride.

C₁₀H₂₄N₂O₂ · 2HCl 277.23