Entecavir Monohydrate CAS 209216-23-9 Assay 98.0%~102.0% API USP EP Standard Antiviral Hepatitis B Infection

Entecavir

C12H15N5O3·H2O 295.29
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 [209216-23-9]; UNII: 5968Y6H45M.
Anhydrous 277.28
DEFINITION
Entecavir is a monohydrate and contains NLT 98% and NMT 102% of Entecavir (C12H15N5O3), calculated on the anhydrous basis.
IDENTIFICATION
Change to read:
• A. SPECTROSCOPIC IDENTIFICATION TESTS <197>, Infrared Spectroscopy: 197A or 197K (CN 1-May-2020)
• B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.
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.]
Table 1 Time (min) Solution A (%) Solution B (%)
0 100 0
8 100 0
50 77 23
75 17 83
90 100 0
100 100 0
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 × 25-cm; 5-µm 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 1.5%, Standard solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of entecavir (C12H15N5O3) in the portion of Entecavir taken:
Result = (ru /rs ) × (Cs /Cu ) × 100
ru = peak response of entecavir from the Sample solution
rs = peak response of entecavir from the Standard solution
Cs = concentration of USP Entecavir Monohydrate RS in the Standard solution (mg/mL)
Cu = concentration of Entecavir in the Sample solution (mg/mL)
Acceptance criteria: 98%-102% on the anhydrous basis
IMPURITIES
• ORGANIC IMPURITIES
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 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 10.0%, Standard solution
Analysis
Samples: Sample solution and Standard solution
Calculate the percentage of each impurity in the portion of Entecavir taken:
Result = (ru /rs ) × (Cs /Cu ) × (1/F) × 100
ru = peak response of each impurity from the Sample solution
rs = peak response of entecavir from the Standard solution
Cs = concentration of USP Entecavir Monohydrate RS in the Standard solution (mg/mL)
Cu = 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%.
Table 2
Name Relative Retention Time Relative Response Factor Acceptance Criteria, NMT (%)
Furoentecavir a 0.73 1.0 0.1
Entecavir 1-epimer b 0.93 1.0 0.1
Entecavir 3-epimer c 0.96 1.0 0.1
Entecavir 1.0 - -
8-Hydroxy entecavir d 1.03 0.67 0.1
Entecavir 4-epimer e 1.08 1.0 0.1
8-Methoxy entecavir f 1.27 0.67 0.1
4-Dimethylsilyl entecavir g 1.84 1.0 0.1
Entecavir related compound A 3.41 - -
Any unspecified impurity - 1.0 0.1
Total impurities i - - 0.3
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. [ NOTE- The gradient elution times are established on an HPLC system with a dwell volume of
approximately 1.0 mL.]
Table 3
Time (min) Solution A (%) Solution B (%)
0 65 35
8 53 47
8.1 65 35
11 65 35
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
(See Chromatography <621>, System Suitability.)
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 = (ru /rs ) × (Cs /Cu ) × 100
ru = peak response of entecavir related compound A from the Sample solution
rs = peak response of entecavir related compound A from the Standard solution
Cs = concentration of USP Entecavir Related Compound A RS in the Standard solution (mg/mL)
Cu = concentration of Entecavir in the Sample solution (mg/mL)
Acceptance criteria: NMT 0.1%
SPECIFIC TESTS
• WATER DETERMINATION <921>, Method I, Method Ic: 5.5%-7.0%
• OPTICAL ROTATION <781S>, Procedures, Specific Rotation
Sample solution: 10 mg/mL of Entecavir in a mixture of dimethylformamide and methanol (50:50)
Acceptance criteria: +24° to +30°
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in well-closed containers, protected from light. Store at room temperature.
• USP REFERENCE STANDARDS <11>
USP Entecavir Monohydrate RS
USP Entecavir Related Compound A RS
3-Benzyl-4-silyl entecavir;
9-[(1S,3R,4S)-4-Dimethylphenylsilyl-3-(benzyloxymethyl)-2-methylenecyclopentyl]guanine.
C27H31N5O2Si 485.65
USP Entecavir System Suitability Mixture RS
The mixture contains entecavir monohydrate and the following impurities (other impurities may also be present):
Entecavir 1-epimer.
8-Hydroxy entecavir.
8-Methoxy entecavir.