Entecavir

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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6H-Purin-6-one, 2-amino-1,9-dihydro-9-[(1S,3R,4S)-4-hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl]-, monohydrate;

9-[(1S,3R,4S)-4-Hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl]guanine monohydrate CAS RN®: 209216-23-9; UNII: 5968Y6H45M.

Anhydrous 277.28

1 DEFINITION

Entecavir is a monohydrate and contains NLT 98% and NMT 102% of entecavir (C₁₂H₁₅N₅O₃), calculated on the anhydrous basis.

2 IDENTIFICATION

Change to read:

A.SPECTROSCOPIC IDENTIFICATION TESTA (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

PROCEDURE

Solution A: Acetonitrile and water (3:97)

Solution B: Acetonitrile

Mobile phase: See Table 1. (Note-The gradient elution times are established on an HPLC system with a dwell volume of approximately 1.0 mL]

System suitability stock solution: 1.0 mg/ml, of USP Entecavir System Suitability Mixture RS in methanol

System suitability solution: 0.2 mg/mL of USP Entecavir System Suitability Mixture RS in Solution A from System suitability stock solution

Standard stock solution: 1.0 mg/mL of USP Entecavir Monohydrate RS in methanol. Sonicate as needed.

Standard solution: 0.2 mg/mL of USP Entecavir Monohydrate RS in Solution A from the Standard stock solution

Sample stock solution: 1.0 mg/mL of Entecavir in methanol. Sonicate as needed.

Sample solution: 0.2 mg/ml. of Entecavir in Solution A from Sample stock solution

Chromatographic system

(See Chromatography (621) System Suitability)

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm x 25-cm, 5-um packing L1

Flow rate: 1 mL/min

Injection volume: 10 μL

System suitability

Samples: System suitability solution and Standard solution

[Note-See Table 2 for the relative retention times of the components in the System suitability solution.]

Suitability requirements

Resolution: NLT 3.5 between entecavir 1-epimer and entecavir, NLT 2.0 between entecavir and 8-hydroxy entecavir, System suitability solution

Tailing factor: 0.8-1.5 for entecavir, System suitability solution

Relative standard deviation: NMT 1.59%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of entecavir (C₁₂H₁₅N₅O₃) in the portion of Entecavir taken:

Result = (r_U/(r_S) × (C_S /C_U) x 100

r_U = peak response of entecavir from the Sample solution

r_S = peak response of entecavir from the Standard solution

C_S = concentration of USP Entecavir Monohydrate RS in the Standard solution (mg/ml)

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

Acceptance criteria: 98%-102% on the anhydrous basis

4 IMPURITIES

ORGANIC INPURITIES

Solution A, Solution B, Mobile phase, System suitability stock solution, System suitability solution, Sample stock solution, Sample

solution, and Chromatographic system: Proceed as directed in the Assay.

Standard stock solution: Use the Standard solution from the Assay.

Standard solution: 0.2 µg/ml. of USP Entecavir Monohydrate RS in Solution A from the Standard stock solution.

System suitability

Samples: System suitability solution and Standard solution

[Note-See Table 2 for relative retention times of the components in the System suitability solution]

Suitability requirements

Resolution: NLT 3.5 between entecavir 1-epimer and erntecavir, NLT 2.0 between entecavir and 8-hydroxy entecavir, System suitability solution

Tailing factor: 0.8-1.5 for entecavir, System suitability solution

Relative standard deviation: NMT 10.0%, Standard solution

Analysis

Samples: Sample solution and Standard solution

Calculate the percentage of each impurity in the portion of Entecavir taken:

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

r_U = peak response of each impurity from the Sample solution

r_S = peak response of entecavir from the Standard solution

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

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

F = relative response factor (see Table 2)

Acceptance criteria: See Table 2. Disregard any peak less than 0.05%

^a 9-[(3aS,4S,6S,6aR)-3a,6-Dihydroxyhexahydro-1H-cyclopenta[c]furan-4-yl]guanine.

^b 9-[(1R,3R,4S)-4-Hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl]guanine.

^c 9-[(1S,3S,4S)-4-Hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl]guanine.

^d 8-Hydroxy-9-[(1S,3R,4S)-4-hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl]guanine.

^e 9-[(1S,3R,4R)-4-Hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl]guanine.

^f 8-Methoxy-9-[(1S,3R,4S)-4-hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl]guanine.

^g 9-[(1S,3R,4S)-4-Hydroxydimethylsilyl-3-(hydroxymethyl)-2-methylenecyclopentyl]guanine.

^h For information only; quantitated in the test for Limit of Entecavir Related Compound A.

ⁱ Includes the sum of all the impurities found in the tests for Limit of Entecavir Related Compound A and Organic Impurities.

LIMIT OF ENTECAVIR RELATED COMPOUND A

Solution A: 0.1% (v/v) trifluoroacetic acid in water

Solution B: 0.1% (v/v) trifluoroacetic acid in acetonitrile

Mobile phase: See Table 3. Nor-The gradient elution times are established on an HPLC system with a dwell volume of approximately 1.0 mL]

Standard solution: 2 μg/mL of USP Entecavir Related Compound A RS in methanol

Sample solution: 1.0 mg/mL of Entecavir in methanol. Sonicate as needed.

Chromatographic system

Mode: LC

Detector: UV 254 nm

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

Temperatures

Autosampler: 4°

Column: 30°

Flow rate: 2 mL/min

Injection volume: 10 μL

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: 0.8–1.5

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of entecavir related compound A in the portion of Entecavir taken:

Result = (r_U/(r_S) × (C_S /C_U) x 100

r_U = peak response of entecavir related compound A from the Sample solution

r_S = peak response of entecavir related compound A from the Standard solution

C_S = concentration of USP Entecavir Related Compound A.RS in the Standard solution (mg/mL)

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

Acceptance criteria: NMT 0.1%

5 SPECIFIC TESTS

WATER DETERMINATION (921), Method I. Method Ic: 5.5%-7.0%

OPTICAL ROTATION (7815), Procedures. Specific Rotation

Sample solution: 10 mg/mL of Entecavir in a mixture of dimethylformamide and methanol (50:50)

Acceptance criteria: +24° to +30°

6 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in well-closed containers, protected from light. Store at room temperature.