Efavirenz
C14H9ClF3NO2 315.67
1H-Benzo[d][1,3]oxazin-2(4H)-one, 6-chloro-4-cyclopropylethynyl-4-trifluoromethyl;
(S)-6-Chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one
[154598-52-4].
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C14H9ClF3NO2 315.67
1H-Benzo[d][1,3]oxazin-2(4H)-one, 6-chloro-4-cyclopropylethynyl-4-trifluoromethyl;
(S)-6-Chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one
[154598-52-4].DEFINITION
Efavirenz contains NLT 98.0% and NMT 102.0% of C14H9ClF3NO2, calculated on the anhydrous, solvent-free basis.
IDENTIFICATION
• A. Infrared Absorption 
197K
197K
Sample:
Dry at 105
for 30 min, and cool in dessicator.
for 30 min, and cool in dessicator.
• B. Ultraviolet Absorption 197U
197U
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
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. ]
6 months. ]
Solution B:
Methanol, trifluoroacetic acid, and water (9:0.005:1). [Note—Use only freshly-opened trifluoroacetic acid, 6 months. ]
6 months. ]
Mobile phase:
See the gradient table below.
| Time (min) |
Solution A (%) |
Solution B (%) |
|---|---|---|
| 0 | 60 | 40 |
| 16 | 50 | 50 |
| 23 | 35 | 65 |
| 28 | 30 | 70 |
| 29 | 20 | 80 |
| 31 | 20 | 80 |
| 32 | 60 | 40 |
| 40 | 60 | 40 |
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
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 = (rU/rS) × (CS/CU) × 100
| rU | = | = peak response for efavirenz from the Sample solution |
| rS | = | = peak response for efavirenz from the Standard solution |
| CS | = | = concentration of USP Efavirenz RS in the Standard solution (mg/mL) |
| CU | = | = concentration of efavirenz in the Sample solution (mg/mL) |
Acceptance criteria:
98.0%–102.0% on the anhydrous, solvent-free basis
IMPURITIES
Inorganic Impurities
• Residue on Ignition 281:
NMT 0.2%, use a platinum crucible
281:
NMT 0.2%, use a platinum crucible
• Heavy Metals, Method II 231:
NMT 20 ppm
231:
NMT 20 ppm
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 = (rU/rS) × (CS/CU) × (1/F) × 100
| rU | = | = peak response for any individual impurity from the Sample solution |
| rS | = | = peak response for efavirenz from the Standard solution |
| CS | = | = concentration of USP Efavirenz RS in the Standard solution (mg/mL) |
| CU | = | = 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
| Name | Relative Retention Time |
Relative Response Factor |
Acceptance Criteria, NMT (%) |
|---|---|---|---|
| Efavirenz aminoalcohola | 0.48 | 0.26 | 0.15 |
| Efavirenz ethene analogb (efavirenz related compound B) | 0.93 | 0.91 | 0.40 |
| Efavirenz pent-3-ene-1-yne (cis)c | 1.16 | 1.0 | 0.10q |
| Efavirenz pent-3-ene-1-yne (trans)d | 1.16 | 1.0 | 0.10q |
| Efavirenz penteneynee | 1.16 | 1.0 | 0.10q |
| Efavirenz pentyne analogf | 1.2 | 1.0 | 0.15 |
| Methyl efavirenzg | 1.28 | 1.0 | 0.10 |
| Efavirenz amino alcohol methyl carbamateh | 1.33 | 0.83 | 0.10 |
| N-Benzylefavirenzi | 1.8 | 0.71 | 0.25 |
| Efavirenz benzoylaminoalcoholj | 1.9 | 0.56 | 0.15 |
| Quinoline analogk | 1.45 | 2.0 | 0.10 |
| Efavirenz amino alcohol ethyl carbamatel | 1.53 | 0.83 | 0.10 |
| Unidentified impuritym | 1.60 | 1.0 | 0.10 |
| Efavirenz amino alcohol bis(ethoxycarbonyl)n | 1.63 | 0.34 | 0.10 |
| Unidentified impurityo | 2.1 | 1.0 | 0.10 |
| Cyclobutenylindole analogp | 2.18 | 0.48 | 0.10 |
| Any other unknown individual impurity |
— | 1.0 | 0.10 |
|
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 trifluoroacetic acid, 6 months. ]
6 months. ]
Solution B:
Acetonitrile, trifluoroacetic acid, and water (8:0.005:2). [Note—Use only freshly-opened trifluoroacetic acid, 6 months. ]
6 months. ]
Mobile phase:
See the gradient table below.
| Time (min) |
Solution A (%) |
Solution B (%) |
|---|---|---|
| 0 | 100 | 0 |
| 40 | 0 | 100 |
| 45 | 0 | 100 |
| 45.1 | 100 | 0 |
| 50 | 100 | 0 |
Standard solution:
1.25 µg/mL of USP Efavirenz RS in Diluent
Sample solution:
250 µg/mL of Efavirenz in Diluent
Chromatographic system
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 = (rU/rS) × (CS/CU) × (1/F) × 100
| rU | = | = peak response for each of the three specified impurities from the Sample solution |
| rS | = | = peak response for efavirenz from the Standard solution |
| CS | = | = concentration of USP Efavirenz RS in the Standard solution (µg/mL) |
| CU | = | = 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
| Name | Relative Retention Time |
Relative Response Factor |
Acceptance Criteria, NMT (%) |
|---|---|---|---|
| Efavirenz | 1.0 | 1.0 | — |
| Efavirenz pent-3-ene-1-yne (cis)a | 1.10 | 1.1 | 0.10 |
| Efavirenz pent-3-ene-1-yne (trans)b | 1.13 | 1.1 | 0.10 |
| Efavirenz penteneynec | 1.14 | 1.0 | 0.10 |
|
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.
|
|||
SPECIFIC TESTS
• Completeness of Solution 641:
Meets the requirements
641:
Meets the requirements
Solvent:
Methanol
Sample solution:
50 mg/mL of Efavirenz in Solvent
• Water Determination, Method Ic 921
921
Sample:
100 mg/mL of Efavirenz in methanol
Acceptance criteria:
NMT 0.5%
• Enantiomeric Purity
Mobile phase:
Hexane and ethanol (97:3)
Retention time solution:
1 g/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
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 × [rR/(rR + rS)]
| rR | = | = peak response of (R)-efavirenz enantiomer from the Sample solution |
| rS | = | = peak response of (S)-efavirenz enantiomer from the Sample solution |
Acceptance criteria:
NMT 0.5% of (R)-efavirenz enantiomer
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in well-closed containers, protected from light, and store at controlled room temperature.
• USP Reference Standards 11
11
USP Efavirenz RS 
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USP Efavirenz Related Compound B RS
(S,E)-6-Chloro-4-(2-cyclopropylvinyl)-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one.
C14H11ClF3NO2 317.69
(S,E)-6-Chloro-4-(2-cyclopropylvinyl)-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one.
C14H11ClF3NO2 317.69
USP Efavirenz Racemic RS
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Behnam Davani, Ph.D., M.B.A.
Senior Scientific Liaison 1-301-816-8394 |
(SM12010) Monographs - Small Molecules 1 |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 3010
Pharmacopeial Forum: Volume No. 36(3) Page 661