Ethambutol Hydrochloride
(eth am' bue tol hye'' droe klor' ide).
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C10H24N2O2·2HCl 277.23
1-Butanol, 2,2¢-(1,2-ethanediyldiimino)bis-, dihydrochloride, [S-(R*,R*)]-;    
(+)-2,2¢-(Ethylenediimino)-di-1-butanol dihydrochloride     [1070-11-7].
Ethambutol Hydrochloride contains NLT 98.0% and NMT 100.5% of C10H24N2O2·2HCl, calculated on the dried basis.
•  B. Identification Tests—General, Chloride 191: Meets the requirements
Sample:  100 mg/mL in water
•  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 mL of 0.1 N perchloric acid is equivalent to 13.86 mg of C10H24N2O2·2HCl.
Acceptance criteria:  98.0%–100.5% on the dried basis
Inorganic Impurities 
•  Heavy Metals, Method II 231: NMT 20 ppm
Organic Impurities 
•  Procedure 1: Ordinary Impurities 466
Sample solution:  Methanol
Standard solution:  Methanol
Eluant:  Methanol and ammonium hydroxide (18:1)
Visualization:  16
•  Procedure 2: Limit of Aminobutanol
Solution A:  1.24 g of boric acid to a 100-mL volumetric flask. Dissolve in 90 mL of water, adjust with 5 N sodium hydroxide to a pH of 9.0, and dilute with water to volume.
Fluorescamine solution:  0.1 mg/mL of fluorescamine in acetone
Standard solution:  5.0 µg/mL of USP Aminobutanol RS
Sample solution:  0.5 mg/mL of Ethambutol Hydrochloride
Spectrometric conditions 
Mode:  Fluorometry
Analytical wavelength:  485 nm, with the excitation wavelength at about 385 nm
Cell:  1 cm
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 the difference between the intensities of the two solutions (NMT 1.0%).
•  Limit of Total Stereoisomers
Solution A:  60 mg/mL of (R)-(+)-alpha-methylbenzyl isocyanate in acetonitrile
Solution B:  Acetonitrile and water (1:1)
Mobile phase:  Methanol and water (13:7)
Standard solution 1:  Transfer 13 mg of USP Ethambutol Hydrochloride RS to a 10-mL volumetric flask. Add 2.0 mL acetonitrile and 260 µL of triethylamine, and mix for 1 min. Add 650 µL of Solution A, and mix for another min. Dilute with Solution B to volume.
Standard solution 2:  Prepare as directed for Standard solution 1 using USP Ethambutol Related Compound A RS and USP Ethambutol Related Compound B RS.
System suitability solution:  Mix equal volumes of Standard solution 1 and Standard solution 2.
Sample solution:  Prepare as directed for Standard solution 1 using Ethambutol Hydrochloride.
Chromatographic system 
Mode:  LC
Detector:  UV 254 nm
Column:  3.9-mm × 15-cm; packing L1
Flow rate:  1.7 mL/min
Injection size:  20 µL
Run time:  2.3 times the retention time of ethambutol
System suitability 
Samples:  Standard solution 1 and System suitability solution
[Note—The relative retention times for ethambutol related compound B, ethambutol, and ethambutol related compound A are 0.85, 1.0, and 1.4, respectively. ]
Suitability requirements 
Resolution:  NLT 3.0 between ethambutol and ethambutol related compound A, System suitability solution
Relative standard deviation:  NMT 2.0%, Standard solution 1
Sample:  Sample solution
Calculate the percentage of stereoisomers in the portion of Sample taken:
Result = (rU/rT) × 100
rU== peak response of ethambutol related compound A or ethambutol related compound B from the Sample solution
rT== sum of all peak responses of ethambutol related compound A, ethambutol related compound B and ethambutol from the Sample solution
Calculate the percentage of total stereoisomers:
Result = % ethambutol related compound A + % ethambutol related compound B
Acceptance criteria 
Total impurities:  NMT 4.0%
•  Optical Rotation, Specific Rotation 781S: +6.0 to +6.7
Sample solution:  100 mg/mL in water
•  Loss on Drying 731: Dry a sample at 105 for 2 h: it loses NMT 0.5% of its weight.
•  Packaging and Storage: Preserve in well-closed containers.
•  USP Reference Standards 11
USP Aminobutanol RS Click to View Structure
    C4H11NO        89.14
USP Ethambutol Hydrochloride RS Click to View Structure
USP Ethambutol Related Compound A RS
    C10H24N2O2        204.31
USP Ethambutol Related Compound B RS
    C10H24N2O2        204.31
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Topic/Question Contact Expert Committee
Monograph Behnam Davani, Ph.D., M.B.A.
Senior Scientific Liaison
(SM12010) Monographs - Small Molecules 1
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USP35–NF30 Page 3123
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