Clozapine

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

Clozapine contains NLT 98.0% and NMT 102.0% of clozapine (C₁₈H₁₉CIN₄), calculated on the dried basis.

2 IDENTIFICATION

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

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

Mobile phase: Methanol and water (80:20). To each liter add 0.75 mL of triethylamine.

Diluent: Methanol and water (80:20)

System suitability stock solution: Transfer 10 mg of USP Clozapine RS to a suitable container, add 5 mL of 0.1 N hydrochloric acid VS and heat for 2 h at 90°. Transfer this solution to a 100-mL volumetric flask, add 15 mL of water, and dilute with methanol to volume.

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

System suitability solution: System suitability stock solution and Standard solution (50:50)

Sample solution: 0.1 mg/mL of Clozapine in Diluent

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 257 nm

Column: 4.6-mm × 25-cm; 5-µm packing LZ

Flow rate: 1 mL/min

Injection volume: 10 µL

Run time: NLT 3 times the retention time of clozapine

System suitability

Samples: Standard solution and System suitability solution

Suitability requirements

Resolution: NLT 1.5 between clozapine and any other peak, System suitability solution

Tailing factor: NMT 1.5, Standard solution

Relative standard deviation: NMT 0.73%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of clozapine (C₁₈H₁₉CIN₄) in the portion of Clozapine taken:

Result = (r_u/r_s) x (C_s/C_u) × 100

r_u = peak response of clozapine from the Sample solution

r_s = peak response of clozapine from the Standard solution

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

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

Acceptance criteria: 98.0%-102.0% on the dried basis

4 IMPURITIES

4.1 RESIDUE ON IGNITION (281): NMT 0.1%

Change to read:

4.2 ORGANIC IMPURITIES

Buffer: 2.0 g/L of monobasic potassium phosphate in water. Adjust with phosphoric acid to a pH of 2.4. [NOTE-The pH of this solution must not be below 2.4.]

Solution A: Acetonitrile, methanol, and Buffer (10:10:80)

Solution B: Acetonitrile, methanol, and Buffer (40:40:20)

Mobile phase: See Table 1.

Table 1

Diluent: Methanol and water (80:20)

System suitability solution: Dissolve 4 mg of USP Clozapine Resolution Mixture RS in 4 mL of methanol, add 1 mL of water, and dilute with Diluent to 10 mL.

Standard solution: 0.75 µg/mL of USP Clozapine RS in Diluent

Sample solution: 750 µg/mL of Clozapine prepared as follows. Transfer a suitable quantity of Clozapine to a suitable volumetric flask.

Dissolve in 80% of the flask volume of methanol, sonicate for about 3 min, and dilute with water to volume.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 257 nm

Column: 4.6-mm x 15-cm; 5-µm packing 11

Flow rate: 1.2 mL/min

Injection volume: 20 µL

System suitability

Samples: System suitability solution and Standard solution

[NOTE- See Table 2 for the relative retention times.]

Suitability requirements

Resolution: NLT 2.5 between demethyl clozapine and clozapine, System suitability solution

Relative standard deviation: NMT 5.0% for clozapine, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each related compound and any unknown impurity in the portion of Clozapine taken:

Result = (r_u/r_s) x (C_s/C_u) × (1/F) × 100

r_u= peak response of any impurity from the Sample solution

r_s = peak response of clozapine from the Standard solution

C_s = concentration of USP Clozapine RS from the Standard solution (µg/mL)

C_u = concentration of Clozapine from the Sample solution (µg/mL)

F = relative response factor of the impurity (see Table 2)

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

Table 2

a 8-Chloro-11-(piperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine.

b1-[2-[(2-Amino-4-chlorophenyl)amino]benzoyl]-4-methylpiperazine.

C8-Chloro-5,10-dihydro-11H-dibenzo[b,e] [1,4]diazepin-11-one.

d 1,4-Bis(8-chloro-5H-dibenzo[b,e] [1,4]diazepin-11-yl)piperazine.

5 SPECIFIC TESTS

5.1 LOSS ON DRYING (731)

Sample: Dry at 105° for 4h.

Acceptance criteria: NMT 0.5%

6 ADDITIONAL REQUIREMENTS

6.1 PACKAGING AND STORAGE: Preserve in well-closed containers.

6.2 USP REFERENCE STANDARDS (11).

USP Clozapine RS

USP Clozapine Resolution Mixture RS

Contains a mixture of the following 5 compounds:

Clozapine.

Chlorodibenzodiazepinone;

8-Chloro-5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one.

C₁₃H₉CIN₂O     244.68

Didiazepinyl piperazine;

1,4-Bis(8-chloro-5H-dibenzo[b,e] [1,4]diazepin-11-yl)piperazine.

C₃₀H₂₄CI₂N₆  539.46

Demethyl clozapine;

8-Chloro-11-(piperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine.

C₁₇H₁₇CIN₄    312.80

Benzoyl methylpiperazine analog;

1-[2-[(2-Amino-4-chlorophenyl)amino]benzoyl]-4-methylpiperazine.

C₁₈H₂₁CIN₄O    344.84

[NOTE-The contents have previously been referred to as clozapine, Impurity A, Impurity B, Impurity C, and Impurity D, respectively.]