Active ingredients do not show a significant absorption because of their poor solubility and lesser ionizing capacity. The present method describes the development of a new dissolution method using pH 7.4 buffers as medium followed by determination of Acyclovir content in tablets by UV Spectrophotometer by considering absorption maximum at 251 nm.

MATERIAL AND METHODS:

Apparatus:

Schimadzu1700 Double beam UV Spectrophotometer with 1 cm quartz cell, Electrolab Dissolution testing apparatus (8 Vessels), pH Meter and calibrated volumetric Borosilicate glassware’s were used for experiment..

Reagent and Chemicals:

Acyclovir was gifted by Agio Biopharma, Pune, India. Other reagents like Hydrochloric acid and various phosphate buffers were prepared from AR grade materials. Water obtained from a Milli-ORQ water purification system.

Selection of Wavelength:

The wavelength required for analysis of Acyclovir (3 ppm) was selected from the UV spectrum and it was found at 251 nm.

Preparation of Phosphate Buffers:

Reagents like 0.1N Potassium Dihydrogen Phosphate, 0.2M Sodium Hydroxide was mixed in different proportions as per USP to get required pH of the buffer. Each buffer was tested for its accurate pH with a calibrated pH meter and their actual pH values were noted down.

Solubility Characterization:

The pH solubility profile of acyclovir at room temperature was determined. Excess raw material was suspended in 10 ml of series of buffers adjusted with different pH. The suspension was equilibrated by shaking in water bath for 12 hr. Aliquots were withdrawn and filtered through 0.45µm hydrophilic filters. The solubility of acyclovir in presence of excipient was also studied in various buffers using above procedure for their quantification by UV. It was revealed that solubility of acyclovir were high in a pH 7.4 phosphate buffer after comparing with other buffers.

Study of Dissolution Parameters:

Dissolution parameters such as media volume, rpm, time interval and USP apparatus have been evaluated. Four different method conditions were adapted to conduct using single and multiple vessel dissolution apparatus and from this Method 4 is selected as an optimized method for data analysis.

Table No. 1 Dissolution Parameters

Method	Volume of Media	RPM	Time Intervals (Min)	Apparatus
Method - 1	900 ml	50	5, 10, 30, 45	USP Apparatus II
Method - 2	900 ml	100	5, 10, 30, 45	USP Apparatus II
Method - 3	900 ml	50	5, 10, 30, 45	USP Apparatus I
Method - 4	900 ml	100	5,10,15,20,25,30,45	USP Apparatus II

Quantitative Determination of the Drug Using Developed Method:

Label claim -Each film coated tablet contains Acyclovir equivalent to 200, 400, 800 mg.

Brand Name -Acivir 200 mg, Acivir 400 mg, Acivir 800 mg.

Manufacturer -CIPLA Pharmaceuticals.

Procedure for Dissolution of Acyclovir Raw Material and Tablets:

Electro lab dissolution test apparatus was set for equilibrium after adjusting the dissolution parameter. A series of 10 mg of pure Acyclovir raw material were accurately weighed and placed in 8 vessels separately. Dissolution testing was run continuously for 45 minutes with intermittent sample withdrawn for successive time interval of 5, 10, 15, 20, 25, 30, 45 min.

RESULTS AND DISCUSSIONS:

The spectrophotometric determination of Acyclovir was studied using UV Spectrophotometer. This proposed method was optimized by measuring the absorbance of Acyclovir in pH 7.4 phosphate buffer at wavelength of 251 nm. The parameters were optimized and fixed by evaluating the effects of excipients and coating materials. Interference studies were also conducted in pH 7.4 phosphate buffer. The proposed method has been further validated for its linearity, precision and accuracy. Different aliquots of standard solutions between 1-10 mg/ml were prepared. Absorbance values were noted with solutions of Acyclovir in pH 7.4 phosphate buffer. A linear plot was established with concentration of Acyclovir on X-axis and absorbance (A) on Y-axis. The Beer’s Law limits were found to be 1-10 mg/ml. A linear regression analysis of the results gave the following equation, y= 0.095x - 0.001 (r=0.999, n=6), where C is the concentration of Acyclovir in mg/ml. Slope was found to be 0.0009. The accuracy of the method was evaluated by analyzing standard solutions of Acyclovir. The results were compared with those obtained from an official method. The reproducibility of the proposed method was checked with six replicates of 6 mg/ml of Acyclovir in pH 7.4 phosphate buffer solution. The relative standard deviation (RSD) value was found to be ±0.004. The results were highly reproducible. The proposed method has been further validated for its linearity, precision and accuracy [7-11].

Table No. 2 Validation Summary of Proposed method

PARAMETERS	ACYCLOVIR
Linearity - Range	1-10 mg/ml
Linearity - Slope	0.0009
Linearity - Correlation Coefficient	0.999
Linearity - Equation of Line	y= 0.095x - 0.001
Intraday Precision (n=3)	RSD = 0.006
Interday Precision (n=3)	RSD = 0.004
Repeatability Precision (n=6)	RSD = 0.009
Ruggedness - Analyst 1	99.10%
Ruggedness - Analyst 2	99.27%
Robustness - Wavelength (+2nm)	RSD = 0.0075
Robustness - Wavelength (-2nm)	RSD = 0.0027

Evaluation of Dissolution and Dissolution Profile of Acyclovir Tablet [12]:

Under fixed dissolution parameters, a series of Acyclovir with different strength were evaluated for the determination of Q value and dissolution profile at different time intervals using ph 7.4 phosphate buffer.

Table No. 3 Drug Release of Acyclovir Tablet by Dissolution

Time (min)	Abs	Con. In 5 ml (mcg)	Con. In 900 ml (mcg)	Drug Release	% Drug Released
5	0.026	1.362	245.283	245.283	24.528
10	0.036	1.886	337.735	336.373	33.637
15	0.080	4.192	750.524	748.637	74.863
20	0.088	4.612	825.576	821.383	82.138
25	0.097	5.083	910.010	905.398	90.539
30	0.100	5.241	938.155	933.071	93.307
45	0.107	5.607	1003.82	998.584	99.85

Table No. 4 Drug Release of Acyclovir Bulk Drug by Dissolution

Time (min)	Abs	Con. In 5 ml (mcg)	Con. In 900 ml (mcg)	Drug Release	% Drug Released
5	0.801	41.98	7556.60	7556.60	75.566
10	0.876	45.91	8218.23	8176.25	81.762
15	0.942	49.37	8837.42	8791.50	87.915
20	0.963	50.47	9034.43	8985.06	89.850
25	1.013	53.09	9503.51	9453.03	94.530
30	1.016	53.24	9531.65	9478.56	94.785
45	1.065	55.81	9991.35	9938.10	99.381

Table No. 5 Dissolution of commercial Acyclovir by proposed methods

Product Label Claim	% Found( ±%RSD)	Average %RSD
Acivir 200 mg	99.60±0.26	99.63
	99.72±0.14
	99.57±0.27
Acivir 400 mg	100.40±0.11	100.21
	100.10±0.16
	100.14±0.19
Acivir 800 mg	98.90±0.42	99.20
	99.71±0.25
	99.01±0.38

CONCLUSION:

The proposed dissolution method is more precise, accurate and rapid than most of the reported methods and characterized by instrumental simplicity, cost effective in the use of reagents and time. No interference of excipient and coating material was found in this method. All aliquot solutions prepared were stable for 12hrs. Hence this method can be employed for routine evaluation and validation of Acyclovir tablet during the small scale, pilot scale and large production scale batches.

ACKNOWLEDGMENT:

We would like to thanks Principal and Management of Shivnagar Vidya Prasarak Mandal’s College of Pharmacy for providing all the facilities to complete this work successfully.

REFERENCES:

1. http// www.cimsasia.com/Acyclovir, retrieved 22 January.

2. Sweetman, S.C., Eds., In; Martindale, 33rd Edn., The Pharmaceutical Press., Inc., London, 2002, 612.

3. Stulzer H. K., Tagliari M. P., Murakami S., Silva M. A., Laranjeira M. C., Development and validation of an RP-HPLC method to quantitate acyclovir in cross-linked chitosan microspheres produced by spray drying, J Chromatogr Sci., 46(6), 2008, 496-500.

4. Tzanavaras P. D, Themelis D. G., High-throughput HPLC assay of acyclovir and its major impurity guanine using a monolithic column and a flow gradient approach, Journal of Pharmaceutical and Biomedical Analysis, 43(4), 2007, 1526-1530.

5. Basavaiah K., Prameela H. C., Chandrashekar U., Simple high-performance liquid chromatographic method for the determination of acyclovir in pharmaceuticals, Farmaco, 58(12), 2003, 1301-1306.

6. Henning P. A., Leung D. T., Sacks S. L., Development and validation of a plasma assay for acyclovir using high-performance capillary electrophoresis with sample stacking, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 772(2), 2002, 291-297.

7. Baokar Shrikrishna, Pawar Vinod, Patil R. N., Jagatap Rashmi, Ekatpure, Netrali, Validation of Simple and Rapid UV-Spectrophotometric Method with Stress Degradation Study for Sildenafil Citrate, Research J. Pharm. and Tech., 5 (2), 2012, 212-218.

8. Fifield F. W., Kealey D., 5th Edition, Black Well Science Ltd. Principles and Practice of Analytical Chemistry, 2000, 270 – 276.

9. Hokanson C., A Life Cycle Approach to the Validation of Analytical Methods During Pharmaceutical Product Development, Part – II: Changes and the Need for Additional Validation, Pharm. Tech., 92 -100, 1994.

10. Baokar Shrikrishna, Pawar Vinod, Sonawane S. H., High Performance Liquid Chromatographic Method Development and Validation of Cholesterol Inhibitor Drug, Journal of Pharmacy Research, 4(7), 2011, 2313-2316.

11. Baokar Shrikrishna B., Erande R. S., Shaikh S. G., Analytical Method Development and Validation for Estimation of Ezetimibe from Tablet Dosage Form by Using RP-HPLC, Int. J. of Res. in Pharm. and Biomed. Sci., 2 (2), 2011, 833-841.

12. Lokesh B. V. S., Raghava Naidu S., New Dissolution Method for the Evaluation of Enalapril Maleate Tablets using pH 7.2 Phosphate Buffer In Vitro and Determination of its Content by Validated UV Spectrophotometric Method, JASA, 2, 2007, 34-37.

Received on 20.12.2012

Modified on 30.12.2012

Accepted on 09.01.2013

Research Journal of Pharmaceutical Dosage Forms and Technology. 5(1): January- February, 2013, 46-48