INTRODUCTION:

Acyclovir, also known as acyclovir, is an antiviral medication. Acyclovir is White, crystalline powder, soluble in diluted hydrochloric acid; slightly soluble in water; insoluble in alcohol.

Acyclovir is primarily used for the treatment of herpes simplex virus infections, chickenpox, and shingles. It treats cold sores around the mouth (caused by herpes simplex), shingles (caused by herpes zoster), and chickenpox.^[1]

Figure 1: Chemical Structure of Acyclovir

The ability to increase the aqueous solubility can be a valuable aid for increasing the efficacy and/or reducing adverse effects for certain drugs. Following approaches can be employed to enhance the aqueous solubility of poorly soluble drugs. Complexation, Use of co-solvents, Alteration of pH, Use of surfactants, Supercritical fluid re-crystallization, Micronization, Solid dispersion, Eutectic mixture, Hydrotropic solubalisation, Mixed hydrotropic solubalisation, Mixed solvency.^[3-7]

The above mentioned methods have been used widely in various fields of pharmacy. However, applications of ‘Mixed Hydrotropic Solubilization’ and ‘Mixed Solvency’ have not been explored to appreciable extent in various fields of pharmacy.^[8-11]

The significance of study is to improve the aqueous solubility of Acyclovir and enhance its efficacy and reduces its side effects by using hydrotropic agents.

It is new, simple, cost effective, safe accurate, precise and environmental friendly method for the analysis (Titrimetric and Spectrophotometric) of poorly water soluble drugs. By use of hydrotropic agent we can avoids the problem of residual toxicity, error due to Volatility, pollution, cost etc.

MATERIAL AND METHODS:

Materials:

1) Urea.

2) Sodium Benzoate.

3) Acyclovir.

Materials was purchased from Jinedra Scientifics, Jalgaon

Instrumentation:

1. UV spectrophotometric method was performed on double beam UV – visible spectrophotometer (Shimadzu, model 1800) having two matched quartz cells with 1 cm light path.

Figure 2 UV Spectrophotometer

2. Ultra Sonicator (Model No-CD4820)

Methods:

Preparation of Hydrotropic solution:

Accurately 2.0 gm sodium benzoate and 2.0 gm urea 10gm was weighed and transferred in 100ml volumetric flask. Distilled Water was slowly added to dissolves both hydrotropes then volume was make up to the mark and prepared 2.0% solution of each.

Preliminary Solubility Study:

Accurately 10 mg drug was weighed and transferred in 10 ml volumetric flask. 0.5 ml of mixed blend of hydrotropes was added up to total drug soluble. When saturated solution was started to formed stop the addition of hydrotropic solution and make up the volume up to the mark with distilled water then the solubility of drug in hydrotropic solution was determined. The clear solution of Acyclovir was obtained of 1000µg/ml.

Preparation of Stock and working standards Solutions:

Preparation of standard stock solution:

10 mg of standard drug sample was weighed accurately and transferred to 10 ml volumetric flask. 5 ml of blend solution (mixed hydrotropic solution) was added and dissolved the drug completely by vigorous shaking. When no particles remain in solution, make up the volume up to the mark with distilled water. This is stock solution (1000 µg/ml). Diluted 0.5 ml of this solution up to 10 ml with distilled water to get concentration of 50 µg/ml solution (SS_5).

Preparation of its reagent blank:

5 ml of the mixed hydrotropic solution was diluted up to 10 ml with distilled water (reagent stock solution--RS). Pipette out 0.5 ml of this solution (RS) and diluted it up to 10 ml with distilled water to gave reagent blank (RB_5). For blank correction distilled water was used on UV. The absorbance of SS_5 against RB_5 at 339 nm (Acyclovir) was determined. In the same way, SS_10 was prepared and note absorbance against RB_10. For 20, 30, and 40 mcg/ml procedure was repeated. Calibration curve was plotted.

(Mixed hydrotropic solution contains 2%w/v sodium benzoate, 2%w/v urea,).

Analysis of Marketed Formulation by proposed method:

20 Tablets (Ocuvir 400DT) was accurately weighed, and reduced to fine powder. An accurately weighed powder sample equivalent to 10 mg of Acyclovir was transferred to 10 ml volumetric flask and hydrotropic blend was added and sonicated it for 20 min. The solution was filtered through Whatmann filter paper no. 41. The filtrate was further diluted with distilled water to get final concentration (1000µg/ml). From this solution 30µg/ml was prepared. The absorbance of sample solution was measured at 339 nm and the results are shown in Table 1 and Table 6.

Analytical method validation:

The proposed method has been developed and validated for the determination of Acyclovir in pharmaceutical dosage forms. The method was validated according to the validation of analytical procedures provided in the ICH guidelines and draft guidance for the industry: analytical procedures and methods validation.

Analytical methods were validated to ensure consistent, reliable, and accurate results. The Analytical methods were validated in terms of linearity, accuracy, precision; LOD and LOQ. Method validation of the drugs were performed over a 3-day period.

Evaluation of Linearity:

Standard solutions were evaluated for the linearity within the concentration range of 10- 50 µg/mL for Acyclovir. The absorbance was plotted against drug concentration and the linearity was thus calculated by the linear regression equation y= mx + c, where y represents the peak area and x represents drug concentration in µg/mL. A correlation coefficient of approximately 0.999 or more was considered as desirable for all calibration curves.

Determination of accuracy:

Accuracy was determined by performing recovery studies by spiking different concentrations of pure drug in the pre-analyzed samples within the analytical concentration range of the proposed method at three different set at level of 80%, 100%, and 120%. The amount of Acyclovir was calculated at each level and % recoveries were computed. The percentage recovery study was carried out for 80%, 100%, and 120%.

Determination of limit of detection (LOD) and limit of quantification (LOQ):

The limit of detection (LOD) was determined by lower concentrations of Acyclovir. The limit of

Quantification (LOQ), which is the lowest quantifiable concentration, was also determined from range of concentrations analyzed for the LOD determination.

Determination of Precision:

Inter-day validation was conducted with three sets of three samples of different concentrations of drug (10, 20, 30 µg/mL). As for the intra-day validation, three sets of three different drug samples were assayed and evaluated with reference to one calibration curve on the same run. The precision of the methods were determined for both intra-day and inter-day variations using multiple analysis of different concentrations of samples on three different days.

Repeatability:

Repeatability was carried out using a minimum of six determinations at one of the test concentrations.

RESULT AND DISCUSSION:

Method development and optimization of the spectrophotometric method:

Proper wave length selection of the methods depends upon the nature of the sample and its solubility. To develop a rugged and suitable spectrophotometric method for the quantitative determination of Acyclovir, the analytical conditions were selected.

Study of Spectra and Selection of Wavelength:

The aliquot portion of standard stock solutions of Acyclovir was diluted appropriately with distilled water to obtain concentration of 10 mg/ml of drug. The solution of drug was scanned in the range of 400 – 200 nm. The UV absorbance spectrum of Acyclovir is shown in Figure 2.

All solutions were filtered prior to the use through whatmann filter paper.

Figure 2 UV Spectrum of standard for Selection of wavelength

From the spectrum 339 nm wavelength was selected for estimation of drug.

Assay:

The average absorbance of the drug was computed from the spectrophotometer and the amount of the drug present in the tablet dosage form was calculated by using the regression equation obtained for the pure drug. The relevant results are furnished in Table no.1

Table no. 1 Assay

Sr. No.	Conc.	Amt taken	Abs	Amt Found	% Label Claim
1	30	24.87	0.402	31.00	103.33
2	30	24.87	0.403	31.08	103.61
3	30	24.87	0.407	31.42	104.72
4	30	24.87	0.407	31.42	104.72
5	30	24.87	0.402	31.00	103.33
6	30	24.87	0.402	31.00	103.33
		Mean	0.40	31.15	103.84
		SD	0.00	0.21	0.05
		%RSD	0.61	0.66	0.05

METHOD VALIDATION:

The method was validated according to the validation of analytical procedures provided in the ICH guidelines and draft guidance for the industry: analytical procedures and methods validation.

Linearity and Range:

A linear relationship was obtained between the absorbance for the drug and corresponding concentration. The mean standard calibration curves are presented in Fig.3. The calibration curves in Fig.3 exhibit linearity over the concentration range of 10-50 μg/mL for Acyclovir with regression coefficient 0.991. The methods (R² =0.991) provided a good correlation between absorbance and drug concentration.

Table no.2 Linearity

Sr. No.	Concentration (μg/mL)	Absorbance at 339nm
1	0	0
2	10	0.169
3	20	0.288
4	30	0.407
5	40	0.511
6	50	0.615

Figure 3 Linearity

Limit of detection (LOD) and limit of quantification (LOQ):

The limit of detection (LOD) was evaluated by determining the minimum level of concentration for Acyclovir that could be detected using this analytical method. The limit of quantification (LOQ) was studied by estimating the minimum concentration that could be quantified with acceptable accuracy and precision. The LOD for Acyclovir was determined to be 1.21μg/mL and LOQ was found to be 3.66μg/mL.

Table no.3 LOD and LOQ

Sr No.	Conc	Abs I	II	III	Mean	SD	% RSD
2	10	0.169	0.17	0.165	0.17	0.003	1.57
3	20	0.288	0.29	0.286	0.29	0.002	0.69
4	30	0.407	0.405	0.412	0.41	0.004	0.88
5	40	0.511	0.522	0.512	0.52	0.006	1.18
6	50	0.615	0.603	0.615	0.61	0.007	1.13
					Avg SD	0.0044

Precision:

The intra-day and inter-day precision of the assay method were studied by analyzing replicates at 3 different concentration levels: 10, 20, 30 μg/mL for Acyclovir. The results are shown in Table 4 and 5. The precision of this method reflected by relative standard deviation (%RSD) of replicates was not more than 2% for interday and intraday precision study. (Table-4 and 5).

Accuracy:

The amount of Acyclovir was calculated at each level and % recoveries were computed. The percentage recovery study was carried out for 80%, 100%, and 120%. The results are shown in table no. 6.

Table no.6 Accuracy

80%
Sr no.	µg/ml	Amt added	Abs	Amt found	Amt rcvd	% rcvd
1	30	24	0.325	24.58	24.81	103.36
2	30	24	0.324	24.50	24.72	103.01
3	30	24	0.325	24.58	24.81	103.36
			Mean	24.56	24.78	103.24
			SD	0.05	0.05	0.20
			%RSD	0.20	0.19	0.19

100%
Sr no.	µg/ml	Amt added	Abs	Amt found	Amt recvd	% Recovered
1	30	30	0.407	31.42	31.64	105.46
2	30	30	0.408	31.50	31.72	105.74
3	30	30	0.405	31.25	31.47	104.91
			Mean	31.39	31.61	105.37
			SD	0.13	0.13	0.42
			%RSD	0.41	0.40	0.40

120
Sr no.	µg/ml	Amt added	Abs	Amt found	Amt rcvd	% Recovered
1	30	36	0.488	38.17	38.39	106.64
2	30	36	0.49	38.33	38.56	107.10
3	30	36	0.488	38.17	38.39	106.64
			Mean	38.22	38.44	106.79
			SD	0.10	0.10	0.27
			%RSD	0.25	0.25	0.25

Repeatability:

The results of repeatability are shown in Table no.7 which predicted that the relative standard deviation (%RSD) was found to be not more than 2%.

Table no.7 Repeatability

Sr. No.	Conc	Abs	Amt Found	%Amt Found
1	30	0.407	31.42	104.72
2	30	0.406	31.33	104.44
3	30	0.407	31.42	104.72
4	30	0.407	31.42	104.72
5	30	0.408	31.50	105.00
6	30	0.408	31.50	105.00
		Mean	31.43	104.77
		SD	0.06	0.21
		%RSD	0.20	0.20

SUMMARY:

Table no. 8 Summary

Sr. No.	Validation parameters		Result
1	UV detection wavelength (nm)		339
2	Linearity range (µg/ml)		10-50
3	Standard regression equation		y=0.012x+0.030
4	Correlation coefficient (R²)		R²= 0.991
5	Precision (% RSD) Interday Intraday		0.8566 0.5300
6	Repeatability (n=6)		0.20
7	% Recovery (Accuracy, n=9)	80	103.24
		100	105.37
		120	106.79
8	LOD (µg/ml)		1.21
9	LOQ (µg/ml)		3.66
10	Assay (% Label claim)		101.48

CONCLUSION:

The solubility of acyclovir in distilled water was found to be 0.525mg/ml, where as in this solvent mixture was found to be 2 mg/ml. Hence the solubility was increased as compared with distilled water. In the given study the aqueous solubility of acyclovir was found to be 2 mg/ml that give the satisfactory result with regression coefficient of about 0.991. The proposed method was new, simple, cost effective, accurate, sensitive, free from pollution and precise and can be adopted for routine analysis of Acyclovir in tablet dosage form. Presence of hydrotropic agent do not shows any significant interference in the spectrophotometric assay thus further confirming the applicability and reproducibility of the developed method.

ACKNOWLEDGMENT:

All authors are very thank full to Dr. R. K. Maheshwari for his guidance in hydrotrophy and also thanks to institute to provide facilities required for research.

REFERENCES:

1. https://pubchem.ncbi.nlm.nih.gov/compound/acyclovir

2. B. A. Kate, Phulzalke S. B., Deshmukh M. T. Asian Journal of Biomedical and Pharmacutical Science, 2016.

3. V.M. Dharashive, Garud S ChanaleAjit and SR. Devne Effect of Hydrotropes on Solubility of Drugs Int Journal of Pharmaceutical and Chemical Sciencs Volume 5 (1) jan- march 2016.

4. V.Sampath Kumar, C. Raja, C. Jayakumar A Review on Solubility Enhancement Using Hydrotropic Phenomena Int. Journal of Pharmacy and Pharmaceutical Science Volume 6, Issue 6, 2014.

5. Momin Shiraz Mohammad Shafi and Ravindra Bhanudas Saudagar a Review On: Novel Solubility Enhancement Technique Hydrotropy World Journal of Pharmaceutical Research Volume 4, Issue 2, 2015 324-332.

6. Md. Ali Sajid, Vicky Choudhary Solubility Enhancement methods with importantnce of Hydrotropy Journal of Drug Delivery and Therapeutics; 2012 ,2(6). Page no.96-101

7. V. Gurumurthy, R Deveswaran, S. Bharath B.V. Basvaraj and V. Madhavan: Application of Mixed Cosolvency Concept in Spectrophotometric estimation of acyclovir tablets Pelagia Research Library, Der Chemica Sinica ,2011,2(6):198-203.

8. S.V. Kadam, D.M. Shinkar, R.B. saudagar, Review on Solubilty Enhancement Techniques, Department of Pharmaceutics, KCTS RGS College of Pharmacy, IJPBS, Volume 3.

9. Gawai Mamata N., Aher Smita S., Saudagar Ravindra B: Mix Hydrotropy Technique for Solubility Enhancement-A Review, International Journal of Institutional Pharmacy And Life Sciences,5(5): Sepember-october2015.

10. R. K. Maheshwari, Arpna Indurkhya, Novel application of Mixed Hydrotropic Solubiliza Technique in the Formulation and Evaluation of Hydropic Solid Dispersion of Aceclofenac, Depaetment of Pharmacy, Shri G.S. Institute of Technology and Science, Indore 452 003, India

11. Yong Cui; Research Article Hydrotropic Solubilization By Urea Derivatives: A Molecular Dynamics Simulation Study, Journal of Pharmaceutics Volume 2013.

Received on 17.12.2018 Accepted on 19.01.2019

Asian J. Pharm. Ana. 2019; 9(1): 25-29.

DOI: 10.5958/2231-5675.2019.00007.3

Sr. No.	Conc	Abs	II	III	Mean	Amt Found	% Amt Fnd	RSD
1	10	0.179	0.175	0.177	0.18	12.25	122.50	1.13
2	20	0.298	0.297	0.296	0.30	22.25	111.25	0.34
3	30	0.462	0.463	0.462	0.46	36.03	120.09	0.12