Efavirenz

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

Efavirenz contains NLT 98.0% and NMT 102.0% of C₁₄H₉ClF₃NO₂, calculated on the anhydrous, solvent-free basis.

2 IDENTIFICATION

Change to read:

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K (CN 1-May-2020)

Sample: Dry at 105° for 30 min, and cool in dessicator.

Change to read:

B. Spectroscopic Identification Tests 〈197〉, Ultraviolet-Visible Spectroscopy: 197U (CN 1-May-2020)

Solvent: Methanol

Standard solution: 10 μg/mL of USP Efavirenz RS in Solvent

Sample solution: 10 μg/mL of Efavirenz in Solvent

Acceptance criteria: Meets the requirements

3 ASSAY

Procedure

[Note—Protect solutions of efavirenz from light. Use of polypropylene HPLC vials is recomended to avoid possible degradation from certain types of glass vials.]

Diluent: Acetonitrile and water (1:1)

Solution A: Methanol, trifluoroacetic acid, and water (1:0.005:9). [Note—Use only freshly-opened trifluoroacetic acid, ≤6 months.]

Solution B: Methanol, trifluoroacetic acid, and water (9:0.005:1). [Note—Use only freshly-opened trifluoroacetic acid, ≤6 months.]

Mobile phase: See the gradient table below.

Standard solution: 250 μg/mL of USP Efavirenz RS and 1.0 μg/mL of USP Efavirenz Related Compound B RS in Diluent. [Note—Dissolve in about 65% of the flask volume in Diluent and shake for 30 min or until dissolved before diluting with Diluent to volume.]

Sample solution: 250 μg/mL of Efavirenz in Diluent. [Note—Dissolve in about 65% of the flask volume of Diluent, and shake for 30 min or until dissolved before diluting with Diluent to volume.]

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 250 nm

Column: 4.6-mm × 15-cm; 5-μm packing L10

Column temperature: 40°

Flow rate: 1.5 mL/min

Injection size: 35 μL

System suitability

Sample: Standard solution

Suitability requirements

Resolution: NLT 1.2 between efavirenz related compound B and efavirenz

Relative standard deviation: NMT 1.0% for efavirenz

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of C₁₄H₉ClF₃NO₂ in the portion of Efavirenz taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U= peak response for efavirenz from the Sample solution

r_S = peak response for efavirenz from the Standard solution

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

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

Acceptance criteria: 98.0%–102.0% on the anhydrous, solvent-free basis

4 IMPURITIES

Inorganic Impurities

Residue on Ignition 〈281〉: NMT 0.2%, use a platinum crucible

Organic Impurities

Procedure 1

Diluent, Solution A, Solution B, Sample solution, and Chromatographic system: Proceed as directed in the Assay.

System suitability solution: Use the Standard solution prepared as directed in the Assay.

Standard solution: 1.25 μg/mL of USP Efavirenz RS in Diluent, prepared from the System suitability solution

System suitability

Samples: System suitability solution and Standard solution

Suitability requirements

Resolution: NLT 1.2 between efavirenz related compound B and efavirenz, System suitability solution

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

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of any individual impurity in the portion of Efavirenz taken:

Result = (r_U/r_S) × (C_S/C_U) × (1/F) × 100

r_U = peak response for any individual impurity from the Sample solution

r_S= peak response for efavirenz from the Standard solution

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

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

F = relative response factor (see Impurity Table 1)

Acceptance criteria

Individual impurities: See Impurity Table 1. [Note—Disregard any peak less than 0.05%.]

Total impurities: NMT 1.0%

Impurity Table 1

^a (S)-2-(2-Amino-5-chlorophenyl)-4-cyclopropyl-1,1,1-trifluorobut-3-yn-2-ol.

^b (S,E)-6-Chloro-4-(2-cyclopropylvinyl)-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one.

^c (S,E)-6-Chloro-4-(pent-3-en-1-ynyl)-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one.

^d (S,Z)-6-Chloro-4-(pent-3-en-1-ynyl)-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one.

^e (S)-6-Chloro-4-(3-methylbut-3-en-1-ynyl)-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one.

^f (S)-6-Chloro-4-(pent-1-ynyl)-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one.

^g (S)-6-Chloro-4-{[(2RS,2RS)-2-methylcyclopropyl]ethynyl}-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one.

^h (S)-Methyl 4-chloro-2-(4-cyclopropyl-1,1,1-trifluoro-2-hydroxybut-3-yn-2-yl)phenylcarbamate.

ⁱ (S)-6-Chloro-4-(cyclopropylethynyl)-1-(4-methoxybenzyl)-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one.

^j (S)-N-(4-Chloro-2-(4-cyclopropyl-1,1,1-trifluoro-2-hydroxybut-3-yn-2-yl)phenyl)-4-methoxybenzamide.

^k 6-Chloro-2-cyclopropyl-4-(trifluoromethyl)quinoline.

^l (S)-Ethyl 4-chloro-2-(4-cyclopropyl-1,1,1-trifluoro-2-hydroxybut-3-yn-2-yl)phenylcarbamate.

^m Relative retention time of 1.60.

ⁿ (S)-Ethyl 4-chloro-2-[4-cyclopropyl-2-(ethoxycarbonyloxy)-1,1,1-trifluorobut-3-yn-2-yl]phenylcarbamate.

^o Relative retention time of 2.1.

^p Ethyl 5-chloro-2-cyclobutenyl-3-(trifluoromethyl)-1H-indole-1-carboxylate.

^q [Note—If results exceed 0.10%, perform Procedure 2 to separate the three coeluting impurities and ensure that each impurity meets the limit.]

Procedure 2

[Note—Perform Procedure 2 in addition to Procedure 1 if the result for the total of the three impurities at a relative retention time of 1.16 in

Procedure 1 exceeds 0.10%.]

Diluent: Acetonitrile, trifluoroacetic acid, and water (55:0.05:45)

Solution A: Acetonitrile, trifluoroacetic acid, and water (4:0.005:6). [Note—Use only freshly-opened triuoroacetic acid, ≤6 months.]

Solution B: Acetonitrile, trifluoroacetic acid, and water (8:0.005:2). [Note—Use only freshly-opened triuoroacetic acid, ≤6 months.]

Mobile phase: See the gradient table below.

Standard solution: 1.25 μg/mL of USP Efavirenz RS in Diluent

Sample solution: 250 μg/mL of Efavirenz in Diluent

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 250 nm

Column: 4.6-mm × 25-cm; 5–μm packing L1

Column temperature: 35°

Flow rate: 1.5 mL/min

Injection size: 20 μL

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 1.5 for efavirenz

Relative standard deviation: NMT 5.0% for efavirenz

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each of the three specified impurities in the portion of Efavirenz taken:

Result = ((r_U/r_S) × (C_S/C_U) × (1/F) × 100

r_U = peak response for each of the three specified impurities from the Sample solution

r_S= peak response for efavirenz from the Standard solution

C_S = concentration of USP Efavirenz RS in the Standard solution (μg/mL)

C_U = concentration of Efavirenz in the Sample solution (μg/mL)

F = relative response factor (see Impurity Table 2)

Acceptance criteria

Individual impurities: See Impurity Table 2. [Note—Disregard any peak less than 0.05%.]

Impurity Table 2

^a (S,E)-6-Chloro-4-(pent-3-en-1-ynyl)-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one.

^b (S,Z)-6-Chloro-4-(pent-3-en-1-ynyl)-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one.

^c (S)-6-Chloro-4-(3-methylbut-3-en-1-ynyl)-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one.

5 SPECIFIC TESTS

Completeness of Solution 〈641〉: Meets the requirements

Solvent: Methanol

Sample solution: 50 mg/mL of Efavirenz in Solvent

Water Determination, Method Ic〈921〉

Sample: 100 mg/mL of Efavirenz in methanol

Acceptance criteria: NMT 0.5%

Enantiomeric Purity

Mobile phase: Hexane and absolute alcohol (97:3)

Retention time solution: 1 mg/mL of USP Efavirenz RS in Mobile phase

Standard solution: 10 μg/mL of USP Efavirenz Racemic RS in Mobile phase

Sample solution: 1 mg/mL of Efavirenz in Mobile phase

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 250 nm

Column: 4.6-mm × 25-cm column; 5-μm packing L10 connected in-line and before 4.6-mm × 25-cm column; 10-μm packing L40

Column temperature: 35°

Flow rate: 1.0 mL/min

Injection size: 20 μL

System suitability

Samples: Standard solution and Retention time solution

Suitability requirements

Efavirenz identification: Identify the (S)-efavirenz enantiomer peak, Retention time solution

Resolution: NLT 3.0 between (R)-efavirenz enantiomer and (S)-efavirenz enantiomer, Standard solution

Relative standard deviation: NMT 5.0% for (R)-efavirenz enantiomer

Analysis

Samples: Retention time solution, Standard solution, and Sample solution

[Note—Verify the identification of the efavirenz peak based on the chromatogram of the Retention time solution. The relative retention times for (R)-efavirenz enantiomer and (S)-efavirenz enantiomer are 0.88 and 1.00, respectively.]

Calculate the percentage of (R)-efavirenz enantiomer in the portion of Efavirenz taken:

Result = 100 × [r_R/(r_R+ r_S)]

r_R = peak response of (R)-efavirenz enantiomer from the Sample solution

r_S= peak response of (S)-efavirenz enantiomer from the Sample solution

Acceptance criteria: NMT 0.5% of (R)-efavirenz enantiomer

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in well-closed containers, protected from light, and store at controlled room temperature.

USP Reference Standards 〈11〉

USP Efavirenz RS

USP Efavirenz Related Compound B RS

(S,E)-6-Chloro-4-(2-cyclopropylvinyl)-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one.

C₁₄H₁₁ClF₃NO₂ 317.69