Lamivudine
(la miv' ue deen).
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C8H11N3O3S 229.26
2(1H)-Pyrimidinone, 4-amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-, (2R-cis)-;    
(–)-1-[(2R,5S)-2-(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine     [134678-17-4].
DEFINITION
Lamivudine contains NLT 98.0% and NMT 102.0% of C8H11N3O3S, calculated on the anhydrous and solvent-free basis.
IDENTIFICATION
•  B. The retention time of the major peak of the Sample solution corresponds to that of the System suitability solution, as obtained in the test for Lamivudine Enantiomer.
ASSAY
•  Procedure
Buffer:  1.9 g/L of ammonium acetate in water. Adjust with acetic acid to a pH of 3.8 ± 0.2 before final dilution.
Mobile phase:  Methanol and Buffer (5:95)
System suitability solution:  0.25 mg/mL of USP Lamivudine Resolution Mixture B RS in Mobile phase
Standard solution:  0.25 mg/mL of USP Lamivudine RS in Mobile phase
Sample solution:  0.25 mg/mL of Lamivudine in Mobile phase
Chromatographic system 
Mode:  LC
Detector:  UV 277 nm
Column:  4.6-mm × 25-cm; packing L1
Column temperature:  35
Flow rate:  1 mL/min
Injection size:  10 µL
System suitability 
Samples:  System suitability solution and Standard solution
[Note—The relative retention times for lamivudine diastereomer and lamivudine are 0.9 and 1.0, respectively. ]
Suitability requirements 
Resolution:  NLT 1.5 between lamivudine and lamivudine diastereomer, System suitability solution
Relative standard deviation:  NMT 2.0% for lamivudine, Standard solution
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of lamivudine (C8H11N3O3S) in the portion of Lamivudine taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak response from the Sample solution
rS== peak response from the Standard solution
CS== concentration of USP Lamivudine RS in the Standard solution (mg/mL)
CU== concentration of Lamivudine in the Sample solution (mg/mL)
Acceptance criteria:  98.0%–102.0% on the anhydrous, solvent-free basis
IMPURITIES
•  Organic Impurities, Lamivudine Enantiomer
Buffer:  7.7 g/L of ammonium acetate in water
Mobile phase:  Methanol and Buffer (5:95)
System suitability solution:  0.25 mg/mL of USP Lamivudine Resolution Mixture A RS in water
Sample solution:  0.25 mg/mL of Lamivudine in water
Chromatographic system 
Mode:  LC
Detector:  UV 270 nm
Column:  4.6-mm × 25-cm; packing L45
Column temperature:  15–30 (constant temperature)
Flow rate:  1 mL/min
Injection size:  10 µL
System suitability 
Sample:  System suitability solution
[Note—The relative retention times for lamivudine and lamivudine enantiomer are 1.0 and 1.2, respectively. ]
Suitability requirements 
Resolution:  NLT 1.5 between lamivudine and lamivudine enantiomer
Analysis 
Sample:  Sample solution
Calculate the percentage of the lamivudine enantiomer in the portion of Lamivudine taken:
Result = [rU/(rU + rS)] × 100
rU== peak response of the lamivudine enantiomer
rS== peak response of lamivudine
Acceptance criteria:  NMT 0.3%
•  Organic Impurities, Other Related Compounds
Buffer, Mobile phase, Standard solution (Lamivudine), System suitability solution, Suitability requirements, and Chromatographic system:  Proceed as directed in the Assay.
Standard solution:  0.625 µg/mL of salicylic acid in Mobile phase
Sample solution:  0.25 mg/mL of Lamivudine in Mobile phase
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of salicylic acid in the portion of Lamivudine taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak response of salicylic acid from the Sample solution
rS== peak response of salicylic acid from the Standard solution
CS== concentration of salicylic acid in the Standard solution (mg/mL)
CU== concentration of Lamivudine in the Sample solution (mg/mL)
Calculate the percentage of other individual impurities in the portion of Lamivudine taken:
Result = (rU/rT) × 100
rU== peak response of each impurity other than salicylic acid from the Sample solution
rT== sum of the responses of all the peaks
Acceptance criteria:  See Table 1.
Table 1
Name Relative
Retention
Time
Acceptance
Criteria,
NMT (%)
Specified impurity 1 0.4 0.3
Specified impurity 2 0.9 0.2
Salicylic acid 1.0 0.1
Any other individual impurity 0.1
Total impurities 0.6
•  Organic Impurities, Residual Solvents
Internal standard solution:  Dilute 1 mL of 2-pentanone with dimethyl sulfoxide and water (1:1) to 100.0 mL.
Standard solution:  Transfer 10 mL of Internal standard solution to a 100-mL volumetric flask. Add 100 µL of each of the following: dehydrated alcohol, isopropyl acetate, methanol, and triethylamine. Dilute with dimethyl sulfoxide and water (1:1) to volume.
Sample solution:  Transfer 5 g of Lamivudine to a 100-mL volumetric flask, add 10 mL of Internal standard solution, and dilute with dimethyl sulfoxide and water (1:1) to volume.
Chromatographic system 
Mode:  GC
Detector:  Flame ionization
Column:  0.53-mm × 50-m column, coated with a 5-µm film of phase G1
Temperature 
Injector:  150
Detector:  250
Column:  See Table 2.
Table 2
Initial
Temperature
()
Temperature
Ramp
(/min)
Final
Temperature
()
Hold Time at Final
Temperature
(min)
70 70 3
70 30 200 6.5
Injection size:  0.5 µL
Injection type:  Split flow rate, 320 mL/min
Carrier gas:  Hydrogen (at pressure 5 psig)
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of each residual solvent in the portion of Lamivudine taken:
Result = (RU/RS) × (CS/CU) × 100
RU== peak response ratios of respective analyte to the internal standard from the Sample solution
RS== peak response ratios of respective analyte to the internal standard from the Standard solution
CS== concentration of the respective analyte in the Standard solution (mg/mL)
CU== concentration of the Sample solution (mg/mL)
Acceptance criteria:  See Table 3.
Table 3
Name Acceptance
Criteria,
NMT (%)
Alcohol 0.2
Isopropyl acetate 0.2
Methanol 0.1
Triethylamine 0.1
Total residual solvents 0.3
SPECIFIC TESTS
•  Light Absorption
Mode:  UV-Vis
Sample solution:  50 mg/mL in water
Analytical wavelength:  440 nm
Cell:  4 cm
Acceptance criteria:  Absorptivity NMT 0.0015
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in well-closed, light-resistant containers. Store at room temperature.
•  USP Reference Standards 11
USP Lamivudine RS Click to View Structure
USP Lamivudine Resolution Mixture A RS Click to View Structure
USP Lamivudine Resolution Mixture B RS Click to View Structure
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 3632
Pharmacopeial Forum: Volume No. 32(2) Page 346