Bendamustine Hydrochloride for Injection

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

DOWNLOAD PDF HERE

1 DEFINITION

Bendamustine Hydrochloride for Injection is a sterile, lyophilized mixture of Bendamustine Hydrochloride and Mannitol. It contains NLT 90.0% and NMT 110.0% of the labeled amount of bendamustine hydrochloride (C₁₆H₂₁CI₂N₃O₂ . HCI).

2 IDENTIFICATION

A. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

Change to read:

B. ASPECTROSCOPIC IDENTIFICATION TESTS (197), Ultraviolet-Visible Spectroscopy: 1970 (CN 1-MAY-2020)

Sample solution: Nominally equivalent to 5 µg/mL of bendamustine hydrochloride, from Bendamustine Hydrochloride for Injection, in water

Acceptance criteria: Meets the requirements

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: Nominally equivalent to 4.2 mg/mL of bendamustine hydrochloride in Diluent, from Bendamustine Hydrochloride for

Injection

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

Sample: Standard solution

Suitability requirements

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

Tailing factor: NMT 2.0

Relative standard deviation: NMT 1.0%

Analysis

Samples: Standard solution and Sample solution

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

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution 

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

C_U = nominal concentration of bendamustine hydrochloride in the Sample solution (mg/mL)

Acceptance criteria: 90.0%-110.0%

4 PERFORMANCE TESTS

UNIFORMITY OF DOSAGE UNITS (905): Meets the requirements

5 IMPURITIES

5.1 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 B RS, USP Bendamustine Related Compound C RS, USP Bendamustine Related Compound D RS, USP Bendamustine Related Compound E RS, USP Bendamustine Related Compound F 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 for Injection taken:

Result = (r_U/{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 0.1%.

Name

Bendamustine related compound A

Relative Retention Time

Table 2

^a It is a free base of USP Bendamustine Related Compound B RS: 4-(1-Methyl-5-morpholino-1H-benzimidazol-2-yl)butanoic acid.

^b This process impurity is controlled in the drug substance monograph. It is included in the table for identification only, and it is not to be reported in the total impurities.

6 SPECIFIC TESTS

BACTERIAL ENDOTOXINS TEST (85): Meets the requirements

STERILITY TESTS (71): Meets the requirements

PARTICULATE MATTER IN INJECTIONS (788): Meets the requirements for small-volume injections

PH (791): 2.5-3.5 in a constituted solution prepared as directed in the labeling

OTHER REQUIREMENTS: Meets the requirements in Injections and Implanted Drug Products (1)

7 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve as described in Packaging and Storage Requirements (659), Injection Packaging. Store at controlled room temperature.

LABELING (7): Meets the requirements

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 B RS

4-(1-Methyl-5-morpholino-1H-benzimidazol-2-yl)butanoic acid hydrochloride.

C₁₆H₂₁N₃O₃ . xHCI

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 F RS

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

C₂₂H₃₃CI₂N₃O₇ 522.42

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