Bendamustine 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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C₁₆H₂₁CI₂N₃O₂ . HCI  394.72

1H-Benzimidazole-2-butanoic acid, 5-[bis(2-chloroethyl)amino]-1-methyl-, monohydrochloride;

4-(5-[Bis(2-chloroethyl)amino]-1-methyl-1H-benzimidazole-2-yl)butanoic acid monohydrochloride CAS RN^®: 3543-75-7.

Bendamustine (free base)

C₁₆H₂₁CI₂N₃O₂  358.26 CAS RN^®: 16506-27-7.

Monohydrate

C₁₆H₂₁CI₂N₃O₂ . HCI . H₂O  412.74 CAS RN^®: 1374784-02-7.

1 DEFINITION

Bendamustine Hydrochloride is anhydrous or contains one molecule of hydration. The anhydrous form contains NLT 98.0% and NMT 102.0% of bendamustine hydrochloride (C₁₆H₂₁CI₂N₃O₂ . HCI), calculated on the as-is basis. The monohydrate form contains NLT 98.0% and NMT 102.0% of bendamustine hydrochloride (C₁₆H₂₁CI₂N₃O₂ . HCI), calculated on the anhydrous and solvent-free basis.

2 IDENTIFICATION

Change to read:

A. ASPECTROSCOPIC IDENTIFICATION TESTS (197), Infrared Spectroscopy: 197A or 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.

C. IDENTIFICATION TESTS-GENERAL (191), Chemical Identification Tests, Chloride

3 ASSAY

3.1 PROCEDURE

Solution A: 0.1% (v/v) trifluoroacetic acid in water

Solution B: 0.1% (v/v) trifluoroacetic acid in acetonitrile

Mobile phase: See Table 1.

Table 1

Diluent: 1-Methyl-2-pyrrolidone and Solution A (1:1)

Standard solution: 4.2 mg/mL of USP Bendamustine Hydrochloride RS in Diluent

Sample solution: 4.2 mg/mL of Bendamustine Hydrochloride in Diluent

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 15-cm; 5-µm packing L60

Temperatures

Autosampler: 2°-8°

Column: 30°

Flow rate: 1 mL/min

Injection volume: 2 µL

Analysis time: 25 min

System suitability

[NOTE-The slower syringe draw rate and higher detector sampling rate can be applied in order to improve the precision.]

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 2.0

Relative standard deviation: NMT 1.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of bendamustine hydrochloride (C₁₆H₂₁CI₂N₃O₂ . HCI) in the portion of Bendamustine Hydrochloride taken:

Result = (r_U/r_S) x (C_S/C_U) × 100

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution s

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

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

Acceptance criteria: 98.0%-102.0% on the as-is basis for the anhydrous form; 98.0%-102.0% on the anhydrous and solvent-free basis for the monohydrate form

4 IMPURITIES

4.1 RESIDUE ON IGNITION (281): NMT 0.1%

Change to read:

4.2 ORGANIC IMPURITIES

Mobile phase, Diluent, Standard solution, Sample solution, and Chromatographic system: Proceed as directed in the Assay.

System suitability solution: 4.2 mg/mL of USP Bendamustine Hydrochloride RS, and 0.02 mg/mL each of USP Bendamustine Related Compound A RS, USP Bendamustine Related Compound C RS, USP Bendamustine Related Compound D RS, USP Bendamustine Related Compound E RS, USP Bendamustine Related Compound G RS, USP Bendamustine Related Compound H RS, and USP Bendamustine Related Compound I RS in Diluent

Sensitivity solution: 2 µg/mL of USP Bendamustine Hydrochloride RS in Diluent, from the Standard solution

System suitability

Samples: System suitability solution and Sensitivity solution

Suitability requirements

Resolution: NLT 5 between the bendamustine related compound G and bendamustine peaks; NLT 4 between the bendamustine related compound H and bendamustine related compound I peaks, System suitability solution

Signal-to-noise ratio: NLT 10, Sensitivity solution

Analysis

Sample: Sample solution

Calculate the percentage of each impurity in the portion of Bendamustine Hydrochloride taken:

Result = (r /{Σ[r_U × (1/F)] + r_S}) × (1/F) × 100

r_U = peak area of each impurity from the Sample solution

F = relative response factor for each impurity (see Table 2)

r_S = peak area of bendamustine from the Sample solution 

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

Table 2

5 SPECIFIC TESTS

WATER DETERMINATION (921), Method I, Method la: NMT 1.0% for the anhydrous form; 3.0%-5.5% for the monohydrate form

BACTERIAL ENDOTOXINS TEST (85): Meets the requirements

MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECIFIED MICROORGANISMS (62): The total aerobic microbial count is NMT 103 cfu/g. The total

combined molds and yeasts count is NMT 102 cfu/g.

6 ADDITIONAL REQUIREMENTS

6.1 PACKAGING AND STORAGE

Preserve in well-closed containers. Store at room temperature.

6.2 USP REFERENCE STANDARDS (11)

USP Bendamustine Hydrochloride RS

USP Bendamustine Related Compound A RS

4-(5-[Bis(2-hydroxyethyl)amino]-1-methyl-1H-benzimidazol-2-yl)butanoic acid.

C₁₆H₂₃N₃O₄  321.38

USP Bendamustine Related Compound C RS

Ethyl 4-(5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzimidazol-2-yl)butanoate.

C₁₈H₂₇N₃O₄  349.43

USP Bendamustine Related Compound D RS

4-(5-[(2-Chloroethyl)amino]-1-methyl-1H-benzimidazol-2-yl)butanoic acid.

C₁₄H₁₈CIN₃O₂  295.77

USP Bendamustine Related Compound E RS

4-{5-[(2-Chloroethyl) (2-hydroxyethyl)amino]-1-methyl-1H-benzimidazol-2-yl}butanoic acid.

C₁₆H₂₂CIN₃O₃  339.82

USP Bendamustine Related Compound G RS

4-[6-(2-Chloroethyl)-3,6,7,8-tetrahydro-3-methylimidazo [4,5-h] [1,4]benzothiazin-2-yl]butanoic acid.

C₁₆H₂₀CIN₃O₂S  353.86

USP Bendamustine Related Compound H RS

4-[5-((2-[(4-(5-[Bis(2-chloroethyl)amino]-1-methyl-1H-benzimidazol-2-yl}butanoyl)oxy]ethyl)(2-chloroethyl)amino)-1-methyl-1H-benzimidazol-2-yl]butanoic acid.

C₃₂H₄₁CI₃N₆O₄  680.07

USP Bendamustine Related Compound I RS

Ethyl 4-(5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzimidazol-2-yl)butanoate.

C₁₈H₂₅CI₂N₃O₂  386.32