Lamivudine

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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C₈H₁₁N₃O₃S 229.26

C₈H₁₁N₃O₃S · 0.2 CH₃OH 235.66

C₈H₁₁N₃O₃S · 0.2 H₂O 232.86

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 CAS RN®: 134678-17-4; UNII: 2T8Q726O95.

1 DEFINITION

Lamivudine contains NLT 98.0% and NMT 102.0% of lamivudine (C₈H₁₁N₃O₃S ), calculated on the anhydrous and solvent-free basis. If labeled as a methanol solvate, it contains NLT 98.0% and NMT 102.0% of lamivudine (C₈H₁₁N₃O₃S ), calculated on the anhydrous, methanol-free, and solvent-free basis.

2 IDENTIFICATION

Change to read:

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

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 Limit of Lamivudine Enantiomer.

3 ASSAY

Procedure

Buffer: Transfer about 1.9 g of ammonium acetate to a 1000-mL volumetric flask, dissolve in about 900 mL of water, adjust with acetic acid to a pH of 3.8 ± 0.2, and dilute with water to volume.

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

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 277 nm

Column: 4.6-mm × 25-cm; packing L1

Column temperature: 35°

Flow rate: 1 mL/min

Injection volume: 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 = (r_U/r_S)x(C_S/C_U) × 100

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution

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

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

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

If labeled as a methanol solvate: 98.0%–102.0% on the anhydrous, methanol-free, and solvent-free basis

OTHER COMPONENTS

Content of Methanol (if labeled as lamivudine methanol solvate)

Diluent: N,N-Dimethylformamide and t-butanol (500:1)

Standard solution: 0.625 mg/mL of methanol in diluent. Transfer 2.0 mL of this solution into a headspace vial, and immediately seal the vial.

Sample solution: Transfer 50 mg of Lamivudine to a headspace vial, add 2.0 mL of Diluent, and immediately seal the vial.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: GC headspace

Detector: Flame ionization

Column: 0.53-mm × 75-m, coated with a 3-μm lm of phase G43

Temperatures

Injector: 180°

Detector: 250°

Column: See Table 1.

Table 1

Injection volume: 1.0 mL

Injection type: Split ratio, 15:1

Carrier gas: Helium

Flow rate: 6 mL/min

Headspace samplers

Oven: 95°

Loop: 175°

Transfer line: 175°

Equilibrium time: 10 min

System suitability

Sample: Standard solution

[Note—The relative retention times for methanol and t-butanol are 1.0 and 1.9, respectively.]

Suitability requirements

Tailing factor: NMT 2.0 for methanol

Column efficiency: NLT 25,000 for methanol

Relative standard deviation: NMT 5.0% for methanol

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of methanol in the portion of lamivudine methanol solvate taken:

Result = (R_U/R_S)x(C_S/C_U) × 100

R_U = peak response ratio of methanol to t-butanol from the Sample solution

R_S = peak response ratio of methanol to t-butanol from the Standard solution

C_S = concentration of methanol in the Standard solution (mg/mL)

C_U = concentration of Lamivudine (as methanol solvate) in the Sample solution (mg/mL)

Acceptance criteria: 2.0%–3.0%

4 IMPURITIES

4.1 Limit of 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

(See Chromatography 〈621〉, System Suitability.)

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 volume: 10 μL

System suitability

Sample: System suitability solution

[Note—The relative retention times for lamivudine and the lamivudine enantiomer are 1.0 and 1.2, respectively.]

Suitability requirements

Resolution: NLT 1.5 between lamivudine and the lamivudine enantiomer

Analysis

Sample: Sample solution

Calculate the percentage of the lamivudine enantiomer in the portion of Lamivudine taken:

Result = [r_U/(r_U + r_S)] × 100

r_U = peak response of the lamivudine enantiomer

r_S = peak response of lamivudine

Acceptance criteria: NMT 0.3%

Other Related Compounds Buffer, Mobile phase, System suitability solution, Standard solution, Chromatographic system, and System suitability: Proceed as directed in the Assay.

Salicylic acid standard solution: 0.625 μg/mL of USP Salicylic Acid RS in Mobile phase

Sample solution: 0.25 mg/mL of Lamivudine in Mobile phase

Analysis

Samples: Salicylic acid standard solution and Sample solution

Calculate the percentage of salicylic acid in the portion of Lamivudine taken:

Result = (r_U/r_S)x(C_S/C_U) × 100

r_U = peak response of salicylic acid from the Sample solution

r_S = peak response of USP Salicylic Acid RS from the Salicylic acid standard solution

C_S = concentration of salicylic acid in the Salicylic acid standard solution (mg/mL)

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

Calculate the percentage of other individual impurities in the portion of Lamivudine taken:

Result = (r_U/r_T) × 100

r_U = peak response of each impurity other than salicylic acid from the Sample solution

r_T = sum of the responses of all the peaks

Acceptance criteria: See Table 2.

Table 2

^a (2RS,5SR)-5-(Cytosine-1-yl)-1,3-oxathiolane-2-carboxylic acid.

^b 1–[(2RS,5RS)-2-(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine.

4.2 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 the Internal standard solution to a 100-mL volumetric flask. Add 100 μL 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 the Internal standard solution, and dilute with dimethyl sulfoxide and water (1:1) to volume.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: GC

Detector: Flame ionization

Column: 0.53-mm × 50-m, coated with a 5-μm lm of phase G1

Temperatures

Injector: 150°

Detector: 250°

Column: See Table 3.

Table 3

Injection volume: 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 = (R_U/R_S)x(C_S/C_U) × 100

R_U = peak response ratio of the respective analyte to the internal standard from the Sample solution

R_S = peak response ratio of the respective analyte to the internal standard from the Standard solution

C_S = concentration of the respective analyte in the Standard solution (mg/mL)

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

Acceptance criteria: See Table 4.

Table 4

5 SPECIFIC TESTS

Water Determination, Method Ic〈921〉: NMT 2.0%

Light Absorption

(See Ultraviolet-Visible Spectroscopy 〈857〉.)

Mode: Vis

Sample solution: 50 mg/mL in water

Analytical wavelength: 440 nm

Cell: 4 cm

Acceptance criteria: Absorptivity NMT 0.0015

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in well-closed, light-resistant containers. Store at room temperature.

Labeling: Where it is a methanol solvate form, the label so indicates.

USP Reference Standards 〈11〉

USP Lamivudine RS

USP Lamivudine Resolution Mixture A RS

USP Lamivudine Resolution Mixture B RS

USP Salicylic Acid RS