1. INTRODUCTION:

Chemical formula of Acyclovir is [2-amino-9(2-hydroxyethoxy) methyl)-1, 9 dihydro-6Hpurine-6-one]. Acyclovir is an antiviral agent. Acyclovir is a white crystalline powder. Acyclovir is sparingly soluble in water freely soluble in dimethyl sulfoxide (DMSO) and very slightly soluble in alcohol^1-4.

Figure 1: Structure of Acyclovir

Acyclovir is used to control the symptoms of infection involving herpes simplex virus (HSV) type-1 and type-2 which causes herpes simplex, varicella zoster virus (VZV) causes shingles and chickenpox. Acyclovir also known as acycloguanosine, antiviral drug which is used to control the symptoms of infection involving herpes simplex virus (HSV) type-1 and type-2 which causes herpes simplex, or varicella zoster virus (VZV) causes shingles and chickenpox^6-8.

2. MATERIAL AND METHOD:

2.1 Chemicals and reagents:

The drug Acyclovir was obtained as gift sample from Aadhar Life Sciences. HPLC grade Methanol and water (Merck) Mumbai, India. 0.45µm Millipore syringe filters (Ultipor^®N₆₆^®Nylon 6, 6 Membrane) were from Pall Life Sciences, India.

2.2 Instruments:

Analytical balance (Aczet CY224C), HPLC (Agilent 1260 Infinity II autosampler), Vortex machine (Remi CM 101 plus), Sonicator (Labman).

2.3 Chromatographic equipment and conditions:

The analysis was carried out on HPLC (Agilent 1260 Infinity II) equipped with auto sampling and diode array detector (DAD). The separation was carried out on Luna C18 (150*4.6mm,5µm) using Methanol and water in the ratio of 50:50 respectively, with injection volume 10µl and flow rate of 1ml/min with column temperature of 30º C±0.8ºC at 248nm.

2.4 Preparation of standard stock solution:

10mg of Acyclovir API was transferred into 10ml of volumetric flask containing 5ml of Methanol. The solution was ultrasonicated for 15 minutes. The volume was made upto the mark with Methanoland vortex for 5min.

2.5 Preparation of Gel stock solution:

5mg of AcyclovirTablet was weighed and transferred into a 10ml volumetric flask. It was dissolved in sufficient amount of methanol and sonicated for 10 minutes. Volume was made up to the mark with methanol and vortexed for 5 minutes.

3. Validation of Analytical Method^9-16:

a. Specificity:

The analytical parameter specificity is the ability to detect the analyte in presence of other components example impurities, degradants, matrix etc. From the Tablet stock solution 0.1ml solution was spiked out from the above stock and volume was made up to the mark using water. The solution was vortexed for 5 minutes. The solution was filtered using 0.45µm Millipore syringe filter. This solution was assayed at 248nm using mobile phase Methanol: water (50:50).

Figure 2: Chromatogram of Acyclovir

b. Linearity and Range:

Linearity of an analytical procedure is its ability to obtain test results which is directly proportional to the concentration (amount) of analyte present in the sample.

Range of an analytical procedure is the differential interval between the upper and lower concentration (amount) of analyte present in the sample.

The standard stock solution was further diluted with mobile phase to obtain solutions having the following concentrations i.e. 40, 45, 50, 55 and 60 having range of 40–60µg/ml respectively.

Figure 3: Linearity overlay of Acyclovir

c. Precision:

Precision of an analytical parameter is the closeness of test results to that of the true value obtained from multiple sampling of a homogenous sample under the prescribed experimental conditions. Precision was evaluated for reproducibility, inter-day and intra-day analysis.

1ml of sample was spiked out from the standard stock solution into a 10ml volumetric flask; volume was made upto mark using water. This solution was vortexed for 5 minutes filtered using 0.45µ Millipore syringe filter and filled into HPLC vials.

d. Accuracy:

The accuracy of an analytical parameter is the closeness of the test results to true value. Accuracy of the proposed method was done by standard addition method or recovery study by spiking out the standard stock solution.

e. LOD and LOQ:

The limit of detection (LOD) gives information about the lowest amount of analyte present in the sample which can be easily detected but not necessarily quantified.

Limit of quantitaion (LOQ) of an analytical parameter gives information about the lowest amount of analyte present in the sample which can be easily detected, and quantified with suitable precision and accuracy.

LOD and LOQ were determined using the following equations;

LOD= 3.3*σ/S

LOQ= 10*σ/S

Where, σ = Standard deviation, and S= slope of the regression coefficient.

4. RESULTS AND DISCUSSION:

4.1 Method Development:

The proposed HPLC method was developed and optimized for a series of trails in the terms of mobile phase selection, composition, wavelength, choice of stationary phase of column, flow rate and column temperature. Acyclovir showed the absorbance maxima 248nm. Hence this wavelength was chosen as a working wavelength for the proposed HPLC method.

4.2 Method Validation:

The optimum ratio of mobile phase for Methanol and water was finalized that 50:50 respectively, at the flow rate of 1ml/min. The separation was achieved using Luna C18 column at the column temperature of 30°C±0.8°C.

Sample and standard samples were injected in the volume of 20µl via autosampler. The retention time of Acyclovir in this proposed method was found to be 2.718minutes.

Table 1: Specificity results

Sample	Weight (mg)	Area	Assay
WS	5.1	3231466	-
Acyclovir	87.3	2257396	99.23

Table 2: Linearity results

% Concentration	Concentration µg/ml	Area
80	40	2495622
90	45	2787611
100	50	3115408
110	55	3427482
120	60	3730868

Figure 4: Calibration curve of Acyclovir

Table 3: Precision results

Sample	Area	TP	ASY	Resolutions
Reps1	3223463	10485	1.09	2.67
Reps2	3231957	10490	1.09	2.67
Reps3	3234896	10574	1.03	2.67
Reps4	3195524	10274	0.94	2.74
Reps5	3226924	10566	1.05	2.67
Average	3222553	10477.8	1.04	2.684
STDEV	14080.38
%RSD	0.436

Table 4: Accuracy results

	Wt. of API (mg)	Conc.		STD Area
	5.1	508.47		3115408
% Conc.	Spike Amount	Area	Amount recovery	% Recovery	Average	STDEV	%RSD
80	40.6776	2495622	40.731	100.3	100.03	0.254781541	0.2
80	40.6776	2495622	40.731	100.3	100.03	0.254781541	0.2
100	50.847	3115408	50.847	100	100.01	0.146207527	0.1
100	50.807	3115409	50.849	100.02	100.01	0.146207527	0.1
120	61.0164	3730868	60.892	99.80	99.79	0.006212843	0.03
120	61.0164	3730867	60.890	99.78	99.79	0.006212843	0.03

Table 5: LOD and LOQ results

Parameter	µg/ml
LOD	3.10
LOQ	9.38

5. DISCUSSION:

1. Specificity:

The sample solution was assayed at 248nm and the results were found to be 99.70% w/w which fits within the range of 99.5-100.5% w/w. Hence the method was specific.

2. Linearity:

Linearity of sample was performed for range of 40-60µg/ml, and the regression coefficient was found to be 1.0. Hence the method was linear within given range.

3. Precision:

As per ICH guidelines the limit for precision is NMT 2% RSD, the above developed method shows the precision of 0.436 %RSD, which complies with the ICH guidelines. Hence the method was precise.

4. Accuracy:

Accuracy was performed for 80µg/ml, 100µg/ml, 120µg/ml and the % recovery was found to be 100.3, 100.01, and 99.79% respectively. The developed method was accurate.

5. LOD and LOQ:

LOD was found to be 3.10µg/ml, LOQ was found to be 9.38µg/ml.

Hence the developed method was validated for all the above parameters.

6. CONCLUSION:

The proposed method of validation for estimation of Acyclovir in Tablet formulation has proven to be very specific, linear, accurate, precise, sensitive, and economic. The method exhibits simplicity in terms of short analysis time, isocratic mode of elution of mobile phase, effective and clear resolution with low LOD and LOQ values. The proposed method was developed and validated as per the ICH guidelines.

7. ACKNOWLEDGEMENT:

Authors are thankful to the Principal College of Pharmacy, Solapur for providing required facilities for research work.

8. REFERENCES:

1. Yadav PK, Chandanam S, Rao ST, Akkamma HG, Bharadwajan RG, Deepa MS, New analytical method development and validation of acyclovir by RP-HPLC. Unique J Pharmaceutical and Biological Sciences.2016; 04(02):20-26

2. Williams DA, Lemke TL, Editors. Foye’s Principles of Medicinal Chemistry. 7th edition Philadelphia: Lippincott Williams and Wilkins, Baltimore: Wolters and Kluwer Business;2013:1283

3. Umesh SD, Satyam ZC, Shweta A, Jadhav and Vaibhav RP, Spectrophotometric determination and validation of acyclovir in tablet dosage form. International J Parma Tech Research.2012; 4(4):1840-1845.

4. Bhagyashri PP, Sugandha VM. UV spectrophotometric estimation of acyclovir in bulk and tablet dosage form using area under curve method. World J Pharmacy and Pharmaceutical Sciences.2014; 3(8):1644-1651

5. Somshubhra G, Anirban S, D. Sathis Kumar, Satyaprabha J. A. Harani, D. Tanuja et al. Method development and validation of acyclovir in tablet form by RP-HPLC. Journal of Pharmacy Research. 2012;5(3):1785-1786.

6. Hayam ML, Stability indicating methods for the determination of acyclovir in the presence of its degradation product. Analytical Chem an Indian J.2010; 9 (4): 398-407

7. Ravishankar P, A. Niharika, S. Sireesha, O. Sai Koushiks, V. Himaja Development and validation of RP-HPLC method for quantitative estimation of acyclovir in bulk drug and tablets. J Chemical and Pharmaceutical Sciences.2015;08(01):73-80

8. Vilas C. and Milind UA. Validated stability indicating method HPLC assay method for acyclovir in bulk drug. International J Analytical, Pharmaceutical and Biomedical Science.2012;01(02)

9. Suchita VG, Varsha M, Jadhav and Vilasrao JK High pressure liquid chromatographic method development and validation for estimation of acyclovir in bulk and marketed formulation. International J. of Pharmaceutical Sciences and Research 2016;7(5):2194-2199

10. Indian Pharmacopeia 2007. Vol. II: 376.

11. Williams DA, Lemke TL. Foye’s Principles of Medicinal Chemistry. 7^th edition Philadelphia: Lippincott Williams and Wilkins, Baltimore: Wolters and Kluwer Business;2013.

12. https://en.wikipedia.org/wiki/Dapsone#cite_note-47

13. Dey AA, Dey SA. Estimation of Dapsone in bulk and dosage form by UV spectroscopic method. Indo American J Pharm Res. 2014;4(1):312-319.

14. Sarsam SA. Spectrophotometric and high-performance liquid chromatographic methods for the determination of Dapsone in a pharmaceutical preparation. Rafidian J Sci. 2013; 24:128-145.

15. Lida BM, Latifeh NR, Afshar MN, an ecofriendly and stability indicating RP-HPLC method for determination of Dapsone: Application to pharmaceutical analysis. Chiang Mai J Sci. 2016;43(3):620-629.

16. Nahla SS, Mohammed RR, Safaa TR, Maha HA, Mohammed WP, Validated TLC and HPLC stability indicating methods for the quantitative determination of Dapsone. J Planar Chromatography Modern TLC. 2012;25(1):65-71.

Received on 21.09.2020 Revised on 10.10.2020

Asian J. Pharm. Ana. 2021; 11(1):41-44.

DOI: 10.5958/2231-5675.2021.00008.9