Dacarbazine

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

Dacarbazine contains NLT 97.0% and NMT 102.0% of dacarbazine (C₆H₁₀N₆O).

[CAUTION-Great care should be taken in handling Dacarbazine, as it is a potent cytotoxic agent.]

2 IDENTIFICATION

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

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

3 ASSAY

3.1 PROCEDURE

Protect solutions containing dacarbazine from light and store at 2°-8°.

Solution A: 4.1 g/L of sodium acetate in water. Adjust with phosphoric acid to a pH of 7.0.

Solution B: 4.1 g/L of sodium acetate in water. Adjust with phosphoric acid to a pH of 5.5. Mix acetonitrile and this solution (25:75).

Mobile phase: See Table 1. Return to original conditions, and equilibrate the system for 10 min.

Table 1

Diluent: 0.1 mg/mL of citric acid in water

Standard solution: 0.1 mg/mL of USP Dacarbazine RS in Diluent

Sample solution: 0.1 mg/mL of Dacarbazine in Diluent

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 250 nm

Column: 4.6-mm x 25-cm; 5-µm packing 11

Autosampler temperature: 2"-8"

Flow rate: 1.0 mL/min

Injection volume: 20 µL

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 1.5

Relative standard deviation: NMT 0.73%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of dacarbazine (C₆H₁₀N₆O) in the portion of Dacarbazine 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 Dacarbazine RS in the Standard solution (mg/mL)

C_u = concentration of Dacarbazine in the Sample solution (mg/mL)

Acceptance criteria: 97.0%-102.0%

3.2 IMPURITIES

3.3 RESIDUE ON IGNITION (281): NMT 0.1%

Change to read:

ORGANIC IMPURITIES

Protect solutions containing dacarbazine and its related compounds from light and store at 2°-8°.

Solution A, Solution B, Mobile phase, and Chromatographic system: Proceed as directed in the Assay.

Diluent: 4.0 mg/mL of citric acid in water

Standard stock solution: Use the Standard solution, prepared as directed in the Assay.

Standard solution: 4.0 µg/mL of USP Dacarbazine RS prepared as follows. Transfer a suitable amount of Standard stock solution into a volumetric flask and dilute with water.

Dacarbazine related compound A standard solution: 0.04 mg/mL of USP Dacarbazine Related Compound A RS in water. Sonicate to ensure complete dissolution.

Dacarbazine related compound B standard solution: 0.04 mg/mL of USP Dacarbazine Related Compound B RS in water. Sonicate to ensure complete dissolution.

Sensitivity solution: 2.0 µg/mL of USP Dacarbazine RS in water from Standard solution

Sample solution: 4.0 mg/mL of Dacarbazine in Diluent. Sonicate to ensure complete dissolution.

System suitability

Samples: Dacarbazine related compound A standard solution, Dacarbazine related compound B standard solution, and Sensitivity solution

Suitability requirements

Relative standard deviation 1: NMT 5.0%, Dacarbazine related compound A standard solution

Relative standard deviation 2: NMT 5.0%, Dacarbazine related compound B standard solution

Signal-to-noise ratio: NLT (ERR 1-Dec-2021) 10, Sensitivity solution

Analysis

Samples: Standard solution, Dacarbazine related compound A standard solution, Dacarbazine related compound B standard solution, and Sample solution

Calculate the percentage of 5-aminoimidazole-4-carboxamide (free base of USP Dacarbazine Related Compound A RS) in the portion of Dacarbazine taken:

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

r_u = peak response of 5-aminoimidazole-4-carboxamide from the Sample solution

r_s = peak response of dacarbazine related compound A from the Dacarbazine related compound A standard solution S

C_s = concentration of USP Dacarbazine Related Compound A RS in the Dacarbazine related compound A standard solution (mg/mL)

C_u = concentration of Dacarbazine in the Sample solution (mg/mL)

M_r1 = molecular weight of 5-aminoimidazole-4-carboxamide, 126.12

M_r2 = molecular weight of USP Dacarbazine Related Compound A RS, 162.58

Calculate the percentage of 2-azahypoxanthine (anhydrous) (anhydrous form of USP Dacarbazine Related Compound B RS) in the portion of Dacarbazine taken:

Result = (r_u/r_s) x (C_s/C_u) × 100

r_u = peak response of 2-azahypoxanthine (anhydrous) from the Sample solution

r_s = peak response of dacarbazine related compound B from the Dacarbazine related compound B standard solution

C_s = concentration of USP Dacarbazine Related Compound B RS in the Dacarbazine related compound B standard solution (mg/mL)

C_u = concentration of Dacarbazine in the Sample solution (mg/mL)

Calculate the percentage of each individual unspecified impurity in the portion of Dacarbazine taken:

Result = (r_u/r_s) x (C_s/C_u) × 100

r_u = peak response of each unspecified impurity from the Sample solution

r_s = peak response of dacarbazine from the Standard solution S

C_s = concentration of USP Dacarbazine RS in the Standard solution (mg/mL)

C_u = concentration of Dacarbazine in the Sample solution (mg/mL)

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

Table 2

a It is the free base of USP Dacarbazine Related Compound A RS (C₄H₆N₄O).

b It is the anhydrous form of USP Dacarbazine Related Compound B RS (C₄H₃N₅O).

3.4 LIMIT OF DIMETHYLAMINE

System suitability stock solution A: 20 mg/mL of dimethylamine in water prepared as follows. Transfer USP Dimethylamine Solution RS into a suitable volumetric flask and dilute with water to volume.

System suitability stock solution B: 20 mg/mL of triethylamine in water

System suitability solution: System suitability stock solution A and System suitability stock solution B (1:1). Transfer 5 µL of this solution into a 20-mL headspace vial by syringe through the cap septum.

Standard solution: 10 mg/mL of dimethylamine in water prepared as follows. Transfer USP Dimethylamine Solution RS into a suitable volumetric flask and dilute with water to volume.

Standard: Transfer 5 µL of Standard solution into a 20-mL headspace vial by syringe through the cap septum (0.05 mg).

Sample: Transfer 100 mg of Dacarbazine into a 20-mL headspace vial, cap and seal. Add 5 µL of water by syringe through the cap septum.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: GC

Detector:

Flame ionization

Column: 30-m x 0.53-mm 1.0-µm coating of phase G50

Temperatures

Injection port: 210°

Detector: 300°

Column: See Table 3.

Table 3

Headspace operating parameters: [NOTE-Headspace operating parameters can be modified in order to optimize the performance.]

Temperatures

Oven: 100°

Loop: 110°

Transfer line: 120"

Times

Vial equilibration: 10 min

Pressurization: 0.5 min

Loop fill: 0.25 min

Loop equilibration: 0.05 min

Injection: 0.5 min

Loop size: 1.0 mL

Carrier gas: Helium

Flow rate: 2 mL/min

Injection type: Split, split ratio 25:1

System suitability

Samples: Standard and System suitability solution

Suitability requirements

Resolution: NLT 1.0 between the dimethylamine and triethylamine peaks, System suitability solution

Tailing factor: NMT 2.0, Standard

Relative standard deviation: NMT 15.0%, Standard

Analysis

Samples: Standard and Sample

Calculate the percentage of dimethylamine in the portion of Dacarbazine taken:

Result = (r_u/r_s) x (W_s/W_u) x 100

r_u = peak response of dimethylamine from the Sample

r_s = peak response of dimethylamine from the Standard

W_s = weight of dimethylamine in the Standard (mg) 

W_u = weight of Dacarbazine in the Sample (mg)

Acceptance criteria: NMT 0.05%

4 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight, light-resistant containers, in a refrigerator.

USP REFERENCE STANDARDS (11)

USP Dacarbazine RS

USP Dacarbazine Related Compound A. RS

5-Aminoimidazole-4-carboxamide hydrochloride.

C₄H₆N₄O . HCI     162.58

USP Dacarbazine Related Compound B RS

2-Azahypoxanthine monohydrate.

C₄H₃N₅O . H₂O      155.12

USP Dimethylamine Solution RS

40% of dimethylamine in water

(CH₃)₂NH      45.09