Determination the Content of Acyclovir by High Performance Liquid Chromatography

The analytical method introduced in this article, with reference to the 2020 edition of the Pharmacopoeia of the People's Republic of China in Acyclovir test method, by using Wayeal high performance liquid chromatograph LC3200 series with a DAD detector.

1. Instrument Configuration and Experiment Method

1.1 Instrument Configuration

1.2 Experiment Method

1.2.1 Reagents Preparation

Table 2 List of Reagents

1.2.1.1 Test solution: Take 40mg of sample into a 200mL measuring flask, add 2mL of 0.4% sodium hydroxide to dissolve it, then add 25mL of 0.1% (V/V) phosphoric acid solution and dilute it with water to the scale, shake well.

1.2.1.2 Reference solution: Take 1mL of the test solution into a 100mL measuring flask, add 5mL of 0.1% phosphoric acid solution, dilute with water to scale and shake well.

1.2.1.3 Guanine control storage solution: Take 10mg of guanine reference into a 50mL measuring flask, add 5mL of 0.4% sodium hydroxide solution to dissolve it, then add 5mL of 0.1% phosphoric acid solution, dilute it with water to the scale, shake well.

1.2.1.4 Guanine reference solution: Take 1mL of guanine reference storage solution into a 100mL flask, dilute with water and shake well.

1.2.1.5 System suitability solution: Take appropriate amount of each of the reference solution and guanine reference solution, mix in equal volume and shake well.

1.2.2 Chromatography Condition

Table 3 Chromatography Conditions

Table 4 Mobile Phase Ratio

2. Experiment Result

2.1 System Suitability Solution

Fig 1 Test Chromatogram of System Suitability Solution

Table 5 Test Data System Suitability Solution

Note: From the above graph and the data in the table, it can be seen that Acyclovir and Guanine have better peak shapes and high theoretical plate number. The degree of separation is more than 3.0, which meets the requirements in pharmacopoeia.

2.2 Repeatability

Fig 2 Repeatability Chromatogram of 6 Injections of System Suitability

Table 6 Repeatability Data of 6 Injections of System Suitability Solution Retention Time

Table 7 Repeatability Data of 6 Injections of System Suitability Solution Peak Area

Note: According to the data in the above table, the RSD of retention time of guanine and acyclovir in the system suitability solution is 0.048% and 0.074%, and the RSD of peak area is 0.475% and 0.452%, respectively. The reproducibility results are good and meet the experimental requirements.

2.3 Test Solution

Fig 3 Chromatogram of Test Sample

Table 8 Test Data of Test Sample

Note: According to the above chromatogram and data shown, there are no other peaks in the chromatogram of the test sample, and the peak shape is good, the theoretical plate number is high, which meets the requirements of the limit of the pharmacopoeia under the item of guanine and other substances in the test method of acyclovir.

2.4 Note 

Sodium hydroxide is highly corrosive, so be careful to take precautions when preparing solutions.

3. Conclusion

The analytical method introduced in this article, with reference to the 2020 edition of the Pharmacopoeia of the People's Republic of China in Acyclovir test method, by using Wayeal high performance liquid chromatograph LC3200 series with a DAD detector. The experimental results showed that acyclovir and guanine had better peak shapes and high theoretical plate number. The degree of separation is higher than 3.0, which meets the requirements in the Pharmacopoeia. The retention time RSDs for guanine and acyclovir in the system suitability solutions are 0.048% and 0.074%, and the peak area RSDs are 0.475% and 0.452%, respectively. Repeatability results are good and meet the experimental requirements. There is no other peaks in the chromatogram of the test sample and the peak shape is good and theoretical plate number is high, which meets the requirements of the Pharmacopoeia for the limit of Acyclovir test method under the item of guanine and other substances.