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UV-SPECTROPHOTOMETRIC ESTIMATION OF ACYCLOVIR IN BULK AND

PHARMACEUTICAL DOSAGE FORMS

Article · August 2013

DOI: 10.7897/2277-4572.02451

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 Padala Narayana Raju et al: Estimation of Acyclovir in bulk and pharmaceutical dosage forms

Journal of Pharmaceutical and Scientific Innovation

www.jpsionline.com
Research Article

UV-SPECTROPHOTOMETRIC ESTIMATION OF ACYCLOVIR IN BULK AND PHARMACEUTICAL

DOSAGE FORMS
Narayana Raju Padala1, Dey Baishakhi2, Fathi H Assaleh3, Prakash Katakam3,4* and Babu Rao Chandu3
1
KLR College of Pharmacy, Khammam, India
2
School of Medical Science and Technology, IIT Kharaghpur, India
3
Faculty of Pharmacy, University of Zawia, Zawia, Libya
4
Nirmala College of Pharmacy, Guntur, India
*Corresponding Author Email: pkatakam9@gmail.com
DOI: 10.7897/2277-4572.02451
Published by Moksha Publishing House. Website www.mokshaph.com
All rights reserved.

Received on: 12/07/13 Revised on: 20/08/13 Accepted on: 25/08/13

ABSTRACT
Analytical method development being a vital part of pre formulation-formulation research and development obviates the need to develop reliable, effective,
eco friendly and cost effective methodologies for routine analysis of active pharmaceutical ingredients. UV spectroscopy is one of the earliest, yet of wide
applications in drug analysis in different stages of formulations and quality control; despite the availabilities of sophisticated chromatographic techniques and
other hyphenated techniques. Current research attempts to develop simple, sensitive, accurate, precise and economical UV spectrophotometric methods for the
routine analysis of acyclovir in bulk and pharmaceutical dosage forms using two separate alkaline media, 0.1N NaOH (method A) and 0.1N KOH (method B)
and validate them as per ICH guidelines. In both the methods maximum absorbance was observed at 264 nm. Beer’s law was obeyed in the concentration of
2.5-40 µg / mL in method A and 2.5-30 µg / mL in method B with correlation coefficient of 0.999. The % recovery carried out by adding known amount of
standard drug to pre-analyzed tablet solutions was 98.75 ± 0.52 % to 99.78 ± 0.69 % (method A) and 98.55 ± 0.31 % to 99.78 ± 0.22 % (method B). Intra and
interday precision expressed in % RSD were 0.38 ± 0.01 and 0.27 ± 0.02 - 0.44 ± 0.01 respectively and the percent purity was 99.85 ± 0.05 %. The methods
were validated statistically as per ICH guidelines and the results obtained were within the acceptance criteria for the parameters relating to linearity, accuracy,
precision.
Keywords: Acyclovir, dosage forms, UV spectrophotometric, alkaline medium, validation.

INTRODUCTION specctrofluorometry and UV-spectrophotometric techniques

Development of a properly validated, stability indicating, for the analytical estimation of ACV1,6-9. Some of the
specific analytical method for the routine analysis of drugs is spectrophotometric methods are based on oxidative-coupling
of imperative necessity since the design of the drug delivery reactions using mostly ceric ammonium sulphate or
system is related to it. Analytical method development is an potassium persulfate as oxidizing reagents followed by
integral part of pre formulation and formulation development coupling with 3-methylbenzothiazoline 2-one hydrazone
research1. Sophisticated chromatographic methods with (MBTH reagent) to form colored chromogens detectable at
HPLC, HPTLC which are being employed for analysis are 630 nm. Another method reported oxidative-coupling
relatively expensive; many methods necessitate analyte reaction with MBTH in presence of FeCl3 and the deep green
extraction from respective sample matrices and hence colored species detectable at 616 nm3,10-12. One method also
complicated sample preparation steps, use of internal mentioned about the reaction of ACV with metals like Cu (II)
standards for analysis thus increasing the time required and and Co (II) using borax or NaOH buffers in nonaqueous
error in recovery2. Hence there is always a need to develop a media and the absorbance’s of the formed complexes
simple, sensitive, cost effective and less time consuming measured at 290 nm3. The objective of the current study was
method for the selected drug to aid during various steps of to develop a simple, accurate UV-method in two separate
formulation design. UV-vis spectrophotometric method is alkaline media for the routine estimation of ACV with
one of the earliest, yet easy, sensitive, relatively cost effective minimum processing steps.
method applied for drug estimations in both small and large
scale RandDs1-3. Acyclovir (ACV), chemically known as 9- MATERIALS AND METHODS
[(2-hydroxyethoxy) methyl] guanine is a purine nucleoside Chemicals
analog active against Herpes Simplex Virus (HSV) both type Acyclovir was a kind gift sample from Torrent Pharma Ltd.,
1 and type 2 and marketed under trade names like Zovirax, Ahmedabad. NaOH and KOH (AR grades) and acyclovir
Zovir (GSK), Acivir, Axovir, Herpex etc. ACV is official in tablets were purchased from local market. All reagents were
USP and BP, very selective in action and low in cytotoxicity prepared a fresh with double distilled water.
(Figure 1)1-3. ACV interacts with HSV thymidine kinase and
DNA polymerase and inhibits viral DNA synthesis. Cellular Instrumentation
enzymes convert ACV to ACV-triphosphate which All the spectral and absorbance measurements were made on
competitively inhibits viral DNA polymerase and acts as a an ELICO SL-59, UV-VIS spectrophotometer by using 1 cm
chain terminator when incorporated into viral DNA. ACV match quartz cells.
causes suicide inactivation when the terminated DNA
template containing ACV binds the viral DNA polymerase Method Development
leading to its irreversible inactivation3-5. Literature surveys An accurately weighed amount of acyclovir (pure and tablet
have reported some HPLC and immunoassay techniques, powder) equivalent to 100 mg was dissolved 100 mL of 0.1N

JPSI 2 (4), Jul–Aug 2013, 40-43

 Padala Narayana Raju et al: Estimation of Acyclovir in bulk and pharmaceutical dosage forms

NaOH (method A) and 100 mL of 0.1 N KOH (method B) inter day study solutions of same concentration (5 µg / mL)
and further dilutions were made with 0.1 N NaOH and 0.1 N were prepared, analyzed and results presented as % RSD.
KOH in method A and method B respectively. A series of
standard solutions containing 2.5-40 μg / mL of acyclovir Drug Content Estimation in Formulations
were prepared in 0.1 N NaOH and a series of standard ACV content in the marketed formulations was estimated by
solutions containing 2.5-30 μg / mL of acyclovir were this method. Twenty tablets were weighed; the mean weight
prepared in 0.1 N KOH and absorbances were measured at was determined and finely powdered. An accurately weighed
264 nm against reagent blank. Recovery studies were carried tablet powder equivalent to its labeled amount (200, 400 or
out by adding a known quantity of pure drug to the pre- 800 mg) of ACV was transferred into 100 mL volumetric
analyzed formulation and following the same methodology. flask containing either 0.1 N KOH or NaOH and sonicated
Percentage recovery was calculated from the amount of drug for 10 minutes. After achieving complete solubility of the
found. drugs, the volume was made up to the mark using the alkali
solution. The resulting solution was filtered through 0.45 μm
Method Validation membrane filter. From the filtrate, a 5 mL of solution was
Method validation is the process of establishing documented transferred into 10 mL volumetric flask and volume was
evidence to provide a high degree of assurance that a specific made up to mark with alkali solution to obtain the
activity will consistently produce a desired result or product concentration of 25 μg mL-1 ACV which were then subjected
meeting its predetermined specifications and quality to proposed methods and the amount of ACV was determined
characteristics. The analytical method development of ACV using calibration curves using the two developed methods.
in bulk and pharmaceutical dosage forms was validated in
terms of accuracy, intra and inter day precision, linearity and Recovery Studies
percent recovery13,14. To further validate the accuracy of the method developed,
analytical recovery studies were performed by adding known
Linearity amount of pure drug to pre-analyzed samples of the tablet
Appropriate concentrations of stock solutions were assayed formulations with 2, 10 and 20 µg / mL concentration range.
as per the developed methods. Beer-Lambert’s concentration Percent analytical recovery values were obtained by
ranges were found to be 2.5-40 μg / mL in (method A) and comparison between concentrations obtained from spiked
2.5-30 in (method B). samples against actual added concentrations (2, 10 and 20 µg
/ mL) covering the specified range.
Accuracy
Accuracy was determined by performing recovery studies on RESULTS
marketed formulations (tablets) and for prepared solutions Calculation of percent purity
containing known amount of drug by standard addition A 1 250 50 50
method in which standard drug were added at three different % purity = %1cm    
A 100 wt .taken 5 5
concentration levels (80 %, 100 %, 120 %) to pre analyzed
amount present
samples as per ICH guidelines. The recovery studies were % purity =  100
carried out in triplicates at each level. dose of tablet

Precision (intraday and inter day) The percent purity of the drug was found to be 99.85 ± 0.05
Precision was demonstrated by intraday and inter day %. Data for optical characteristics and linearity of ACV are
variation studies. Intraday precision was determined by presented in Table 1 and the calibration curve of ACV for
taking different solutions of same concentration (5 µg / mL) method A and method B in Figure 2. .
and analyzed thrice a day, results indicated by % RSD. In the

Table 1: Optical Characteristics and Linearity of Acyclovir

Parameters Method A Method B

Maximum absorbance (λmax , nm) 264 264
Beer’s law limits (μg / mL) 2.5-40 μg / mL 2.5-30 μg / mL
Sandell’s sensitivity (μg / cm2 / 0.001 absorbance unit) 0.01930 0.01780
Molar extinction coefficient (L mole-1 cm-1) 1.156 x 10 4 1.257 x 10 4
% Range of error
0.05 confidence limits 0.24750 0.26422
0.01 confidence limits 0.37594 0.39091
Correlation coefficient (r) 0.9998 0.9996
Regression equation (Y*)
Slope (a) 0.0518 0.5657
Intercept (b) -0.00114 -0.00242
Y* = b + aC, where C is concentration in μg / mL and Y is absorbance unit.

Table 2: Estimation of Acyclovir from its Pharmaceutical Formulations

Sample Label Amount Amount obtained (mg) % Recovery by proposed method

(mg) Method A Method B Method A Method B
Tablet1 200 197.5 ± 0.9 197.1 ± 0.5 98.75 ± 0.52 98.55 ± 0.31
Tablet 2 400 393.0 ± 1.2 396.2 ± 1.7 98.25 ± 0.35 99.05 ± 0.19
Tablet 3 800 798.3 ± 1.6 796.9 ± 1.8 99.78 ± 0.69 99.78 ± 0.22

JPSI 2 (4), Jul–Aug 2013, 40-43

 Padala Narayana Raju et al: Estimation of Acyclovir in bulk and pharmaceutical dosage forms

Table 3: Table for Accuracy Data of Acyclovir

Sample No. (%) Conc (µg / mL) Pure drug % Recovery Statistical mean Standard deviation (± SD) % Recovery SD
S1:80 10 6 0.0681 0.0681 1.0 × 10-4 0.15
S2:80 10 6 0.0682
S3:80 10 6 0.0680
S4:100 10 8 0.0821 0.0820 1.5 × 10-4 0.19
S5:100 10 8 0.0822
S6:100 10 8 0.0819
S7:120 10 10 0.0979 0.0977 1.5 × 10-4 0.16
S8:120 10 10 0.0978
S9:120 10 10 0.0976

Table 4: Table for Interday Precision of Acyclovir

Sample No. (5 µg / ml) Abs 1 Abs 2 Abs 3 Average % RSD

S1 0.0424 0.0425 0.0424 0.13
S2 0.0426 0.0425 0.0425 0.13
S3 0.0423 0.0423 0.0424 0.14
S4 0.0427 0.0426 0.0427 0.13
S5 0.0422 0.0423 0.0424 0.23
S6 0.0424 0.0424 0.0425 0.14
% RSD 0.44 0.28 0.27

Figure 1: Structure of Acyclovir

Figure 2: Calibration Curves of Acyclovir for Developed Methods A

and B

The values obtained for the determination of acyclovir in cm2 / 0.001 (method A) and 0.01780 μg / cm2 / 0.001 (method
tablet formulations by the proposed method were provided in B) absorbance units showed that the methods was quite
Table 2. To evaluate the validity and reproducibility of the sensitive. Method validations followed by statistical analysis
methods known amounts of pure drug was added to were done as per ICH guidelines13,14. The percentage
previously analyzed pharmaceutical preparations and the recovery studies carried out by adding known amount of
mixtures analyzed by proposed method and the percent standard drug to pre-analyzed tablet solutions were 98.75 ±
recoveries are given in Tables 2. Results of validation studies 0.52 % to 99.78 ± 0.69 % (method A) and 98.55 ± 0.31 % to
relating to accuracy, precision (intra- and inter day) are 99.78 ± 0.22 % (method B). Accuracy studies carried out
presented in Tables 3 and 4. For intraday precision studies with different concentrations levels (80, 100 and 120 %)
average of 8 samples were taken with mean value (0.0424 ± showed the percentage recovery SD values of 0.15-0.19 %.
0.02), SD values (1.6 × 10-4) and % RSD (0.38 ± 0.01). The Results of the intra- and interday precision studies expressed
relationship amongst different RSD values i.e. RSD amongst in % RSD were 0.38 ± 0.01 and 0.27 ± 0.02 - 0.44 ± 0.01;
samples and RSD amongst different absorbances are respectively10-14. Thus the developed methods were found
presented in Table 4. sensitive, accurate, precise and reproducible and could be
used for the routine estimations of acyclovir in bulk and
DISCUSSION pharmaceutical formulations. Moreover the methods are very
The purpose of the current research was to develop an simple, use of analytical grade lab reagents makes it more
economical, easy, time saving, reliable UV spectroscopic cost effective, doesn’t require any sample pre treatment or
method for the routine estimation of ACV in bulk and any complicated methods of sample preparation and expected
pharmaceutical formulations. As per the developed methods, to be beneficial in small and large analytical and formulation
Beer’s law was obeyed in the concentration range of 2.5-40 RandDs.
μg / mL in (method A) and 2.5-30.0 μg / mL in (method B)
with a correlation coefficient (r) of 0.9998 and 0.9996 for ACKNOWLEDGEMENTS
methods A and B respectively, indicating a good linearity of The authors are grateful to Torrent Pharmaceutical, Ahmedabad, India for
providing gift samples of acyclovir. The authors are also grateful to Nirmala
the developed methods. Sandell’s sensitivity of 0.01930 μg /

JPSI 2 (4), Jul–Aug 2013, 40-43

 Padala Narayana Raju et al: Estimation of Acyclovir in bulk and pharmaceutical dosage forms

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necessary facilities to carry out the research work. Biomed Life Sci 2002; 772(2): 291-97. http://dx.doi.org/10.1016/S1570-
0232(02)00116-2
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Source of support: Nil, Conflict of interest: None Declared

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How to cite this article:

Padala Narayana Raju, Dey Baishakhi, Katakam Prakash, Babu Rao Chandu and Adiki Shanta Kumari. UV-spectrophotometric estimation of acyclovir in bulk
and pharmaceutical dosage forms. J Pharm Sci Innov. 2013; 2(4): 40-43.

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