Available online at www.ijntps.org | ISSN: 2277 – 2782
INTERNATIONAL JOURNAL OF NOVEL TRENDS IN PHARMACEUTICAL SCIENCES
RESEARCH ARTICLE
Analytical Method Development and Validation of Caffeine
in Tablet Dosage Form By Using UV- Spectroscopy
1
2
S.Sethuraman , K.Radhakrishnan , T.Arul Solomon
2
1
Department of chemistry, SCSVMV University, Kanchipuram
Department of pharmaceutical analysis, Jagans college of pharmacy, Nellore.
2
Article Info
Article history
Received 25 Sep 2013
Revised 8 Oct 2013
Accepted 17 Oct 2013
Available online 30 Oct 2013
Keywords
Caffeine,
validation.
UV-spectroscopy,
Abstract
Analytical method development and validation plays important role in the
discovery, development and manufacture of pharmaceuticals. A simple, rapid and
reproducibe UV- spectrophotometric method for the quantitative determination of
caffeine in tablet formulation was developed and validated in the present work.
The parameters lineartiy, specificity, precision, accuracy, robustness and
ruggedness was studied according to ICH guidelines. The wavelength 270nm was
selected for the estimation of drug using distilled water as a solvent. The drug
obeyed Beer-lamber’ts law over the concentration range 10-50µg/ml. The accuracy
of the method was assessed by recovery studies and was found between 99.46100.67%. The method was successfully applied for routine analysis of this drug in
formulations.
INTRODUCTION
Caffeine is a central nervous system and metabolic
stimulant, and is used both recreationally and
medically to reduce physical fatigue and to restore
alertness when drowsiness occurs [1-3]. It produces
increased wakefulness, faster and clearer flow of
thought, increased focus, and better general body
coordination. According to literature survey, it
revealed that caffeine was quantitatively assayed in
Tea, coffee and food beverages using liquid
chromatography, UV-Visible spectroscopy How-ever
no UV-Spectrophotometry method was proposed
for the estimation of caffeine by using of water as a
solvent in Tablet dosage forms [4-7]. In the present
study to develop a simple, accurate and precise UV
spectroscopic method for estimation of caffeine in
tablet dosage form. The validation carried out as per
ICH guidelines [8-10].
All the chemicals used were of analytical
grade. A gift sample of caffeine obtained from
Microlabs, Hosur, was used as working standard. The
formulation of caffeine tablet was purchased from
online shopping.
Solubility test
Solubility test for the drug Caffeine was
performed by using various solvents. The solvents
include Water, Methanol, Ethanol, Acetonitrile,
Hydrochloric Acid (HCl), Sodium Hydroxide, Nitric
acid, Sulphuric acid and Chloroform.
DETERMINATION OF λ max
Preparation of stock solution
Standard stock solution of Caffeine was
prepared by dissolving 100mg of Caffeine in 100ml
of distilled water which gives 1000 µg/ml.
EXPERIMENTAL WORK
Apparatus
A Systronics double beam UV visible
spectrophotometer model 2202, band width of 2nm
wavelength accuracy ±0.5nm and two matched
quartz cells with 1cm pathlength was used for all
spectral measurements.
Preparation of working solution
From the above stock solution 10ml was
transferred into 100ml volumetric flask and volume
was made up to the mark with distilled water to
make 100µg/ml. Then the sample was scanned with
Materials
S.Sethuraman
Email: su_sethuraman@yahoo.com
To whom correspondence should be addressed:
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S.Sethuraman et al., Analytical Method Development And Validation Of Caffeine In Tablet Dosage Form By Using
UV- Spectroscopy
UV-Vis Spectrophotometer in the range 200-400nm
against distilled water as blank and the wavelength
corresponding to maximum absorbance was noted
which is its λ-max i.e. at 270nm.
Fig 1. λ-max of Caffeine
Preparation of calibration curve
One ml of this 100µg/ml solution was
further diluted and the volume was made up to 10ml
by using method to produce 10µg/ml solution. 2ml,
3ml, 4ml and 5ml of 50µg/ml solution were diluted
and the volume was made up to 10ml using
methanol to produce 20µg/ml, 30µg/ml, 40µg/ml,
50µg/ml
solutions
respectively.
Then
the
construction of calibration curve was done by taking
the above prepared solutions of different
concentration ranging from 10-50µg/ml. Then taking
the absorbance calibration curve was plotted taking
concentration on x-axis and absorbance on y-axis
which showed a straight line. This straight line
obeyed linearity in the concentration range of
10-50µg/ml. The correlation coefficient was found to
be 0.999
Fig 2. Linearity curve of Caffeine
Assay of Caffeine Tablet (200mg)
A quantity of powder equivalent to 100mg
of Caffeine was taken in a 100ml volumetric flask
and it was dissolved and diluted up to the mark with
distilled water. The resultant solution was
ultrasonicated for 5 minutes. The solution was then
filtered using Whatmann filter paper No. 40. From
the filtrate, appropriate dilutions were made with
distilled water to obtain the desired concentration
(50 µg/ml). This solution was then analysed in UV
and the result was indicated by % recovery given in
table 4.
Method validation
Validation is a process of establishing
documented evidence, which provides a high degree
of assurance that a specific activity will consistently
produce a desired result or product meeting its
predetermined
specifications
and
quality
characteristics. The validation for UV method
development was performed using parameters like
Linearity, Accuracy, Precision, Robustness, and
Ruggedness.
Linearity
Various aliquots were prepared from the
secondary stock solution (100 µg/ml) ranging from
10-50 µg/ml. The samples were scanned in UV-Vis
Spectrophotometer against distilled water as blank.
It was found that the selected drug shows linearity
between the ranges of 10-50 µg/ml.(Table no.3)
Accuracy
Solutions were prepared in triplicate at
levels 80%, 100% and 120% of test concentration
using Caffeine working Standard as per the test
method and taken absorbance of each solution in
triplicate. The recovery results showed that the
proposed method has an acceptable level of
accuracy for Caffeine which is from 80% - 120% of
test concentration is from 99.51 % - 100.01 %(Table
4).
Precision
Precision of the method was demonstrated
by intraday and interday variation studies. In
intraday variation study nine different solutions of
same concentration 50µg/ml were analyzed three
times in a day i.e. from morning, afternoon and
evening and the absorbance is note. In the interday
variation studies, solution of same concentration
50µg/ml were analyzed three times for the three
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S.Sethuraman et al., Analytical Method Development And Validation Of Caffeine In Tablet Dosage Form By Using
UV- Spectroscopy
consecutive days and the absorbance result mean,
standard deviation and %RSD was calculated and
summarised results given in table 4.
Specificity
Got spectrum in the range of 200nm to
400nm for appropriate concentration of sample,
Blank, and placebo.
Fig 3. Spectrum for blank
Fig 4. Spectrum for caffeine tablet excipients
Fig 5. UV- Spectrum of standard Caffeine
VOLUME 3 | NUMBER 4 | OCT | 2013
Robustness
Robustness of the method was determined
by carrying out the analysis under different λ-max
i.e. at 268nm and at 272nm. The respective
absorbances of 50µg/ml were noted and the result
was indicated as %RSD and given in table no.1
Ruggedness
Ruggedness of the method was determined
by carrying out the analysis by different analyst and
the respective absorbance of 50 µg/ml was noted.
The result was indicated as %RSD and given in table
no.2
RESULTS AND DISCUSSION
The drug was analyzed at 270nm in distilled
water using UV-Visible spectrophotometer. Optical
characteristics such as Beer’s law limits, intercept and
slope has been calculated using regression equation,
which has been presented in Table 3.
Precision was determined by analyzing the
drug at particular concentration for five times on the
same day. Inter day precision was determined
similarly, analyzing the samples daily, for three
consecutive days. The results are summarised in
Table 4.
To ensure the accuracy method, recovery
studies were performed by standard addition
method at 80%,100% and 120% levels of drug
concentration, to the pre-analyzed samples and
percent recovery values were calculated. Recovery
experiment indicated the absence of interferences
from the commonly encountered pharmaceutical
additives and excipients.
The linearity studies were performed by
plotting different concentration of standard solution
against their respective absorbances. Caffeine were
found to be linear in the concentration range of 1050µg/ml. Correlation co-efficient value were found
to be 0.999, calibration curve shows that it obeys
Beer’s law limit within the concentration range.
The proposed method was found to be
simple, accurate, precise, simple, sensitive, robust
and cost effective. The results of the validation tests
were found to be satisfactory and therefore this
method can be applied successfully for the
estimation of Caffeine in Tablet dosage form.
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S.Sethuraman et al., Analytical Method Development And Validation Of Caffeine In Tablet Dosage Form By Using
Uv- Spectroscopy
Table 1. Robustness
Concentration(µg/ml)
At 268nm
Absorbance
50
50
50
50
50
50
Statistical
analysis
Mean=0.5353
0.535
0.535
0.536
0.537
0.535
0.534
Concentration(µg/ml)
At 272nm
Absorbance
50
50
50
50
50
50
SD=0.00103
%RSD=0.19
Statistical
analysis
Mean=0.5348
0.535
0.536
0.535
0.534
0.533
0.536
SD=0.00116
%RSD=0.21
Table 2. Ruggedness
Concentration(µg/ml)
Analyst 1
Absorbance
50
50
50
50
50
50
0.535
0.536
0.535
0.535
0.537
0.535
Statistical
analysis
Mean=0.5355
Concentration(µg/ml)
50
50
50
50
50
50
SD=0.000837
%RSD=0.15
Analyst 2
Absorbance
0.534
0.535
0.535
0.534
0.536
0.535
Statistical
analysis
Mean=0.5348
SD=0.000753
%RSD=0.14
Table 3. Optical characteristics of Caffeine
Beer’s law limit(µg/ml)
10-50µg/ml
Correlation coefficient
0.999
Regression equation(Y*)
0.009x + 0.042
Slope
0.009
Intercept
0.042
Table 4. Summary of validation
Parameter
Result
Linearity indicated by correlation coefficient
0.999
Precision indicated by % RSD
0.26%
Accuracy indicated by % recovery
Specificity
99.9044%
No interferences of impurity
Range
10µg-50µg/ml
Linear regression equation
0.009x + 0.042
Robustness indicated by %RSD
0.19%
Assay indicated by % recovery
99.89%
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UV- Spectroscopy
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