Acyclovir

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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(C₈H₁₁N₅O₃) 225.21 (IRA 1-Jun-2024)

6H-Purin-6-one, 2-amino-1,9-dihydro-9-[(2-hydroxyethoxy)methyl]-;

9-[(2-Hydroxyethoxy)methyl]guanine CAS RN®: 59277-89-3; UNII: X4HES1O11F.

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1 DEFINITION

Acyclovir contains NLT 98.0% and NMT 102.0% (IRA 1-Jun-2024) of acyclovir (C₈H₁₁N₅O₃), calculated on the anhydrous basis.

2 IDENTIFICATION

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K or 197A

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

3.1 Procedure

Mobile phase: Glacial acetic acid in water (1 in 1000)

Standard stock solution: 0.5 mg/mL of USP Acyclovir RS prepared as follows. Dissolve about 25 mg of USP Acyclovir RS in 5 mL of 0.1 N sodium hydroxide in a 50-mL volumetric flask, and dilute with water to volume.

Standard solution: 0.1 mg/mL of USP Acyclovir RS from Standard stock solution in 0.01 N sodium hydroxide

Sample stock solution: 0.5 mg/mL of Acyclovir prepared as follows. Dissolve about 100 mg of Acyclovir in 20 mL of 0.1 N sodium hydroxide in a 200-mL volumetric flask, and dilute with water to volume.

Sample solution: 0.1 mg/mL of Acyclovir from Sample stock solution in 0.01 N sodium hydroxide

3.2 Chromatographic system

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; 10-μm packing L1

Flow rate: 3 mL/min

Injection volume: 20 μL

3.3 System suitability

Sample: Standard solution

3.4 Suitability requirements

Tailing factor: NMT 2

Relative standard deviation: NMT 0.73% (IRA 1-Jun-2024)

3.5 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of acyclovir ((C₈H₁₁N₅O₃)) in the portion of Acyclovir taken:

Result = (r_u/r_s) × (C_s/C_u) × 100

r_u = peak response of acyclovir from the Sample solution

r_s = peak response of acyclovir from the Standard solution

C_s = concentration of USP Acyclovir RS in the Standard solution (mg/mL)

C_u = concentration of Acyclovir in the Sample solution (mg/mL)

Acceptance criteria: 98.0%–102.0% (IRA 1-Jun-2024) on the anhydrous basis

4 IMPURITIES

4.1 Organic Impurities

Buffer A: 3.48 g/L of dibasic potassium phosphate anhydrous (IRA 1-Jun-2024) in water. Adjust with phosphoric acid to a pH of 3.1.

Buffer B: 3.48 g/L of dibasic potassium phosphate anhydrous (IRA 1-Jun-2024) in water. Adjust with phosphoric acid to a pH of 2.5.

Solution A: Acetonitrile and Buffer A (1:99)

Solution B: Acetonitrile and Buffer B (50:50)

Mobile phase: See Table 1.

Table 1

Diluent: Dimethyl sulfoxide and water (20:80 v/v)

System suitability solution: 0.01 mg/mL of USP Acyclovir RS and 0.5 μg/mL each of USP Acyclovir Related Compound A RS and USP Acyclovir Related Compound F RS prepared as follows. Dissolve a suitable amount of USP Acyclovir RS, USP Acyclovir Related Compound A RS, and USP Acyclovir Related Compound F RS in 20% of the (IRA 1-Jun-2024) flask volume of Dimethyl sulfoxide and dilute with water to volume.

Standard stock solution: 0.1 mg/mL of USP Acyclovir RS prepared as follows. Dissolve a suitable amount of USP Acyclovir RS in 20% of the (IRA 1-Jun-2024) flask volume of dimethyl sulfoxide and dilute with water to volume.

Sensitivity solution: 0.3 μg/mL of USP Acyclovir RS from Standard stock solution in Diluent

Standard solution: 0.001 mg/mL (IRA 1-Jun-2024) of USP Acyclovir RS from Standard stock solution in Diluent

Sample solution: 1 mg/mL of Acyclovir prepared as follows. Dissolve a suitable amount of Acyclovir in 20% of the (IRA 1-Jun-2024) flask volume of dimethyl sulfoxide and dilute with water to volume.

4.2 Chromatographic system

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

4.3 System suitability

Samples: System suitability solution, Sensitivity solution, and Standard solution

[Note-The relative retention times in Table 2 are provided as information that could aid in peak assignment.]

Table 2 (IRA 1-Jun-2024)

^a 2-Amino-1,7-dihydro-6H-purin-6-one. (IRA 1-Jun-2024)

^b 2-Amino-9-(2-hydroxyethyl)-1,9-dihydro-6H-purin-6-one. (IRA 1-Jun-2024)

^c 2-Amino-7-[(2-hydroxyethoxy)methyl]-1,7-dihydro-6H-purin-6-one.

^d Unknown structure.

^e N-(6-Oxo-6,9-dihydro-1H-purin-2-yl)acetamide. (IRA 1-Jun-2024)

^f 2-Amino-9-({2-[(2-amino-6-oxo-1,6-dihydro-7H-purin-7-yl)methoxy]ethoxy}methyl)- 1,9-dihydro-6H-purin-6-one. (IRA 1-Jun-2024)

^g 1,2-Bis[(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methoxy]ethane.

^h N-(9-Acetyl-6-oxo-6,9-dihydro-1H-purin-2-yl)acetamide. (IRA 1-Jun-2024)

ⁱ Bis({9-[(2-hydroxyethoxy)methyl]guanine}-N²-yl)methane.

^j 2-{[2-(Acetylamino)-6-oxo-1,6-dihydro-9H-purin-9-yl]methoxy}ethyl acetate.

4.4 Suitability requirements

Resolution: NLT 1.0 between acyclovir related compound F and acyclovir related compound A, System suitability solution

Relative standard deviation: NMT 5.0%, Standard solution

Signal-to-noise ratio: NLT 10, Sensitivity solution

4.5 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of any 

(IRA 1-Jun-2024) impurity in the portion of Acyclovir taken:

Result = (r_u/r_s) × (C_s/C_u) × (1/F) × 100

r_u = peak response of each (IRA 1-Jun-2024) impurity from the Sample solution

r_s = peak response of acyclovir from the Standard solution

C_s = concentration of USP Acyclovir RS in the Standard solution (mg/mL)

C_u = concentration of Acyclovir in the Sample solution (mg/mL)

F = relative response factor (See Table 3 (IRA 1-Jun-2024) )

Acceptance criteria: See Table 3.(IRA 1-Jun-2024) Reporting threshold is 0.03%.

Table 3 (IRA 1-Jun-2024)

5 SPECIFIC TESTS

Water Determination 〈921〉, Method I: NMT 6.0%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers. Store at room temperature. Protect from light and moisture.

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USP Reference Standards 〈11〉

USP Acyclovir RS

USP Acyclovir Related Compound A RS

2-[(2-Amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methoxy]ethyl acetate.

C₁₀H₁₃N₅O₄ 267.25 (IRA 1-Jun-2024)

USP Acyclovir Related Compound F RS

N-[9-[(2-Hydroxyethoxy)methyl]-6-oxo-6,9-dihydro-1H-purin-2-yl]acetamide.

C₁₀H₁₃N₅O₄ 267.25 (IRA 1-Jun-2024)