development and validation of uv-spectroscopic method

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
Kalyankar et al.
World Journal of Pharmacy and Pharmaceutical Sciences
SJIF Impact Factor 2.786
Volume 3, Issue 8, 1742-1751.
Research Article
ISSN 2278 – 4357
DEVELOPMENT AND VALIDATION OF UV-SPECTROSCOPIC
METHOD FOR THE ESTIMATION OF DIDANOSINE IN TABLET
Mohammad Zameeruddin, Kalyankar Swapnil S*., Solanke S. B., Jadhav S. B.,
Kadam V.S. and Bharkad V. B.
Department of Quality Assurance, SSS Indira College of Pharmacy Vishnupuri, Nanded
431606
ABSTRACT
Article Received on
05 June 2014,
A simple, sensitive, rapid and accurate UV-Spectroscopic method has
Revised on 30 June 2014,
Accepted on 25 July 2014
been developed for the estimation of Didanosine in bulk drug and
tablet dosage form. In order to increase value of absorbtivity and
subsequently LOD the drug was dissolved in water and acetonitrile
*Correspondence for Author
Kalyankar Swapnil S
(90:10) and absorbance was measured at 249.60 nm. The linearity of
method was found to be between 1-14 µg/ml. The method was
Department of Quality
Assurance, SSS Indira College
validated based on ICH guidelines. Hence useful for the routine
of Pharmacy Vishnupuri,
analysis of Didanosine.
Nanded 431606
KEY WORDS: Didanosine, Acetonitrile, UV-Spectrophotometric
Method, ICH Guidelines.
INTRODUCTION
Antiretroviral (ARV) therapy is potent, convenient and usually well tolerated, capable of
reducing HIV blood concentration to undectable values within a few weeks from treatment
initiation and of inducing a robust and sustained cluster of differentiation antigen (CD4 Tcell) gain.
[1]
Didanosine is chemically, 9-[(2R, 5S)-5-(hydroxymethyl) oxolan-2-yl]-6.9-
dihydro-3H-purin-6-one. It is white, not hygroscopic crystalline powder having melting point
160–163 °C, water soluble i.e., (27.3 mg/ml at 25 °C and pH 6.2); soluble in
dimethylsulfoxide; slightly soluble in ethanol and methanol; insoluble in chloroform. It is
sensitive to acidic pH, but stable at neutral or slightly alkaline pH. At pH less than 3,
complete hydrolysis to hypoxanthine and 2′, 3′-dideoxyribose occurs in less than 2 min at
27°C. [2]Mechanism of Action: Didanosine is a synthetic nucleoside analogue of the naturally
occurring nucleoside deoxyadenosine in which the 3'-hydroxyl group is replaced by
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hydrogen. Intracellularly, didanosine is converted by cellular enzymes to the active
metabolite, dideoxyadenosine 5'-triphosphate. Dideoxyadenosine 5'-triphosphate inhibits the
activity of HIV-1 reverse transcriptase both by competing with the natural substrate,
deoxyadenosine 5'-triphosphate (DATP), and by its incorporation into viral DNA causing
termination of viral DNA chain elongation. At therapeutic concentrations, DATP inhibits
HIV replication by inhibiting HIV transcriptase enzyme.[3] Chemical structure is given in
Figure No. 1
O
N
HN
N
N
HO
CH
2
O
Didanosine
Figure No. 1 Chemical structure of Didanosine
Several methods have been reported for their determination in bulk, pharmaceutical dosage
forms. These methods include UV-Spectrophotometric
[4-8]
, HPLC
[9-15]
method. These
techniques were associated lack of sensitivity, time-consuming (HPLC). Spectrophotometry
is considered more convenient alternative technique because of its inherent simplicity,
adequate sensitivity and availability in all quality control laboratories. Therefore, the aim of
the present study was directed to the development of new simple spectrophotometric method
in solid dosage form.
Strategy for Validation of Methods
The validity of a specific method should be demonstrated in laboratory experiments using
samples or standards that are similar to the unknown samples analysed in the routine. The
preparation and execution should follow a validation protocol, preferably written in a stepby-step instruction format. [16]
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MATERIALS AND METHODS
Instrument used
A Shimadzu UV –Visible spectrophotometer model 1800 with 1cm matched quartz cells
were used for measuring the absorbance.
Chemicals and reagents
Didanosine pure drug was obtained as a gift sample for Cipla, pvt Ltd, Kurkumbh. Marketed
preparation was procured from the local market under the commercially available brand name
Dinex EC. All the chemicals used were of analytical grade.
Method
Preparation of stock solution
Standard stock solution of Didanosine was prepared by dissolving accurately weighed 10mg
of Didanosine in water and acetonitrile (90:10) in 100ml volumetric flask to give a
concentration of 100µg/ml. From the above stock solution 1ml was pipette out into 10ml
volumetric flask and dilution was made with mixture of water and acetonitrile (90:10) to
obtain concentration 10µg/ml. The samples was then scanned in UV spectrophotometer from
a range of 200-400nm against water and acetonitrile (9:1) as blank and the wavelength
corresponding to maximum absorbance in water and acetonitrile was found at 249.60nm.
Figure No.1: UV Spectrum of Didanosine
Preparation of standard calibration curve
For the preparation of standard calibration curve, concentration of 1-9µg were prepared by
pipetting out 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.10, 0.11, 0.12, 0.13 and 0.14 ml from
the Stock solution in to a 10ml volumetric flask and made up the volume with water and
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acetonitrile (90:10). The absorbance of each solution was measured at 249.60nm against
water and acetonitrile (90:10) as blank. Calibration curve of the drug was then plotted by
taking the absorbance obtained on y-axis and the concentration of the solution on x-axis
(Figure2). The curve showed linearity in the range of 1-14µg/ml with correlation coefficient
0.999.
Figure No. 2: Calibration curve Didanosine
Table No.01: Optical and Regression Characteristics
Sr. No
1
2
3
4
Parameters
Slope
Intercept
Correlation coefficient
Linearity range (µg/ml)
Didanosine
0.064
0.003
0.999
1-14
Application of proposed method for estimation of didanosine in Bulk drug.
Pipette out 0.4ml from the 100µg/ml solution in to a 10ml volumetric flask and made up the
volume with water and acetonitrile (90:10). The absorbance of each solution was measured at
249.60nm against water and acetonitrile (90:10) as blank.
Table No.02: Analysis of Didanosine laboratory mixture
Sr. No.
1
2
3
Taken Concentration (µg/ml)
4
4
4
Absorbance Obtained Concentration (µg/ml)
0.259
4
0.258
3.98
0.259
4
Mean*
SD
%RSD
* Indicates average of three determinations
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% Estimation
100.00
99.50
100.00
99.83
0.2886
0.2891
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Application of proposed method for estimation of Didanosine in Tablet
Twenty Tablets were weighed and ground to fine powder. An accurately weighed quantity
equivalent to 100 mg of Didanosine was transferred to 100 ml volumetric flask, sonicated for
10 min. and volume was made up to the mark with the distilled water and Acetonitrile
(90:10) and filtered through whatmann filter paper (no.41). Aliquot portion (0.4 ml) was
transferred to 10 ml volumetric flask and volume was adjusted to mark with Distilled water
and Acetonitrile (90:10) the absorbance was recorded.
Table No. 03: Analysis of Didanosine laboratory formulation mixture
Sr. No.
1
2
3
Concentration (µg/ml)
4
4
4
Absorbance
0.257
0.258
0.256
Mean*
SD
%RSD
% Label Claim
99.00
99.50
98.75
99.08
0.3818
0.3854
* Indicates average of three determinations
VALIDATION
Linearity
The linearity of the analytical method was its ability to elicit test results which are directly
proportional to analyze concentration in samples within a given range. The drug showed
linearity in the range of 1-9µg/ml with correlation coefficient 0.999. Linearity data are shown
in Table.
Table No.04: Linearity table of Didanosine
Concentration(µg/ml)* Absorbance
1
0.070 ± 0.0047
2
0.139±0.0045
3
0.198±0.0085
4
0.259±0.0020
5
0.327±0.0045
6
0.393±0.0060
7
0.462±0.0055
8
0.518±0.0061
9
0.589±0.0050
10
0.649±0.0040
11
0.709±0.0060
12
0.785±0.0080
13
0.849±0.0090
14
0.909±0.0070
* Indicates average of three determinations
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Precision
Precision studies were carried out to ascertain the reproducibility of the proposed method.
Repeatability was determined by preparing three replicates of same concentration of the
sample and the absorbance was measured. Intraday precision study was carried out by
preparing drug solution of same concentration and analyzing it at three different times in a
day. The same procedure was followed for three different days to determine interday
precision. The results were reported as %RSD. The precision result showed a good
reproducibility with percent relative standard deviation less than 2. The results of intraday
and interday precision studies are shown in Table.
Table No.05: Intra-day precision data
Sr. No.
I
II
III
Interval of Time
Intra-day
Concentration* (µg/ml)
4
4
4
Absorbance
0.259±0.004
0.260±0.007
0.259±0.009
Mean*
SD
%RSD
% Estimation
100.00
100.25
100.00
100.08
0.1443
0.1442
* Indicates average of three determinations
Table No.06: Intre-day precision data
Sr. No.
I
II
III
Interval of Time
Inter-day
Concentration(µg/ml) *
4
4
4
Absorbance
0.259±0.007
0.260±0.005
0.260±0.008
Mean*
SD
%RSD
% Estimation
100.00
100.25
100.25
100.16
0.1443
0.4140
* Indicates average of three determinations
Accuracy
Accuracy of the proposed method was determined using recovery studies. The recovery
studies were carried out by adding different amounts (80%, 100% and 120%) of the pure drug
to the pre-analysed formulation. The solutions were prepared in triplicates and the %
recovery was calculated. The results are shown in Table.
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Table No.19: Recovery study data
Level of Recovery
Added concentration (µm/ml)
3.2
3.2
3.2
4.0
4.0
4.0
4.8
4.8
4.8
80%
100%
120%
Amount recovered (µm/ml)
3.15
3.21
3.18
3.98
4.01
3.96
4.79
4.76
4.73
% Recovery
98.44
100.31
99.37
99.50
100.25
99.00
99.77
98.15
98.50
Table No. 19: Statistical validation of recovery study data
Level of recovery
% Mean recovery*
80%
99.37333
100%
99.58333
120%
98.80667
* Indicates average of three determinations
SD
0.935004
0.629153
0.852428
% RSD
0.940901
0.631785
0.862723
Ruggedness
Ruggedness was determined by carrying out analysis by two different analysts and the
respective absorbance was noted and the results were indicated as % RSD. The results are
shown in Table.
Table no.21: Ruggedness data (Analyst 1st)
Sr. No.
I
II
III
Analyst
1st
Concentration (µg/ml)
4
4
4
Absorbance
0.258
0.260
0.259
Mean*
SD
%RSD
% Estimation
99.50
100.25
100.00
99.91
0.3818
0.3822
* Indicates average of three determinations
Table no.21: Ruggedness data (Analyst 2nd)
Sr. No.
I
II
III
Analyst
2nd
Concentration(µg/ml)
4
4
4
Absorbance
0.258
0.259
0.259
Mean*
SD
%RSD
% Estimation
99.50
100.00
100.00
99.83
0.2886
0. 2891
* Indicates average of three determinations
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Robustness
Analysis was carried out at two different ratio concentrations, acetonitrile: water; 10:90v/v
and ratio 9:91v/v to determine the robustness of the method and the respective absorbance
was measured. The results were indicated as %RSD.
Table No.24: Robustness data (Ace: water; 10:90)
Sr. No.
I
II
III
Ratio of acetonitrile:water
Concentration(µg/ml)
4
4
4
10:90
Absorbance
0.260
0.259
0.259
Mean*
SD
%RSD
%Estimation
100.25
100.00
100.00
100.08
0.1443
0.1442
* Indicates average of three determinations
Table No.24: Robustness data (Ace:water; 9:91)
Sr. No.
I
II
III
Ratio of acetonitrile:water Concentration (µg/ml)
4
9:91
4
4
Absorbance
0.258
0.259
0.259
Mean*
SD
%RSD
% Estimation
99.50
100.00
100.00
99.83
0.2886
0. 2891
* Indicates average of three determinations ±
LOQ and LOD
Limit of detection (LOD) is the lowest amount of analyte in the sample that can be detected.
Limit of quantification (LOQ) is the lowest amount of analyte in the sample that can be
quantitatively determined by suitable precision and accuracy. LOQ and LOD was determined
using the following equation LOQ-10s/m, LOD-3.3s/m where s is the standard deviation of
the response and m is the slope of the related calibration curve.
Table No.27: LOD & LOQ
Name of the drug
Didanosine
LODµg/ml LOQµg/ml
0.1073
0.3252
RESULTS AND DISCUSSION
A simple UV spectroscopic method was developed and validated for the estimation of
Didanosine using the solvent acetonitrile: water in ratio of 10:90.
Linearity range for
Didanosine was obtained as 1-14 µg/ml and its wavelength of detection was 249.60 nm. The
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regression analysis using the method of least squares was made for the slope 0.064, intercept
0.003 and correlation (r2) 0.999 obtained from different concentrations. The proposed
method was applied for pharmaceutical formulation and % estimation Didanosine was found
to be 99.08.
The % recovery for Didanosine was found to be in the range 98.80 – 99.58 and % RSD
values less than 2 indicative of accuracy of the method. The method was found to be precise
as indicated by the inter-day, intra-day and repeatability analysis; showing % RSD less than
2. The result did not show any statistical difference between operators suggesting that method
developed was rugged the method was validated based on ICH guidelines. Useful for the
routine analysis of Didanosine.
CONCLUSION
The developed method can be concluded to be simple, accurate, reliable and economical. The
proposed method is specific without any interference of excipients and hence can be used for
the routine analysis of Didanosine in bulk and in pharmaceutical formulation.
ACKNOWLEDGEMENT
The author is thankful to SSS’s Indira college of Pharmacy for providing facilities to utilize
library, internet and instrument in the college.
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