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Validation of UV Spectrophotometric Method

for Quantitative Determination of Entacapone
in Tablets Using Experimental Design of
PlackettBurman for Robustness Evaluation
and Comparison with HPLC
a

C. S. Paim , H. Gonalves , A. Lange , D. Miron & M. Steppe

Programa de PsGraduao em Cincias Farmacuticas, Faculdade de

Farmcia, Universidade Federal do Rio Grande do Sul., Brazil
Published online: 31 Mar 2008.

To cite this article: C. S. Paim , H. Gonalves , A. Lange , D. Miron & M. Steppe (2008): Validation of UV
Spectrophotometric Method for Quantitative Determination of Entacapone in Tablets Using Experimental
Design of PlackettBurman for Robustness Evaluation and Comparison with HPLC, Analytical Letters, 41:4,
571-581
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Analytical Letters, 41: 571581, 2008

Copyright # Taylor & Francis Group, LLC
ISSN 0003-2719 print/1532-236X online
DOI: 10.1080/00032710801912730

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PHARMACEUTICAL ANALYSIS

Validation of UV Spectrophotometric
Method for Quantitative Determination of
Entacapone in Tablets Using Experimental
Design of Plackett-Burman for Robustness
Evaluation and Comparison with HPLC
C. S. Paim, H. Goncalves, A. Lange, D. Miron, and M. Steppe
Programa de Pos-Graduacao em Ciencias Farmaceuticas, Faculdade de
Farmacia, Universidade Federal do Rio Grande do Sul., Brazil

Abstract: Validation of UV spectrophotometric method for quantitative determination

of entacapone in tablets using acetonitrile as solvent. The validation of analytic method
was realized through the study of the following analytic parameters: specify linearity,
precision, accuracy, and robustness. The excipients of the formulation did not interfere
at 305 nm, demonstrating the specificity of the method. The method was linear
(r 0.99996) at concentrations ranging from 3.0 to 20.0 mg ml21, precise (repeatability and intermediated precision), exact (method of standard addition), and robust.
The results confirmed that the method is valid and useful to the routine quality
control of entacapone in coated tablets. The method was compared to a high-performance liquid chromatography (HPLC) method, which was previously developed and
validated to the same drug. There was not a significant difference between the
methods for entacapone quantitation.
Keywords: Entacapone, validation, experimental design, spectrophotometric method,
stability

Received 5 October 2007; accepted 9 December 2007

Address correspondence to C. S. Paim, Programa de Pos-Graduacao em Ciencias
Farmaceuticas, Faculdade de Farmacia, Universidade Federal do Rio Grande do
Sul., Av. Ipiranga, 2752 Lab. 402, Porto Alegre-RS CEP 90610-000, Brazil. E-mail:
csoldatelli30@hotmail.com
571

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C. S. Paim et al.

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INTRODUCTION
Parkinsons disease (PD) is a neurodegenerative, slowly progressive disorder
characterized by bradykinesia, resting tremor, rigidity, and postural reflex
impairment with associated characteristic eosinophilic cytoplasmatic
inclusions (Bernheimer et al. 1973).
The standard treatment for PD consists of intake of levodopa, a dopamine
precursor, with carbidopa or benserazide, peripheral aromatic amino acid descarboxylase inhibitor, to prevent the breakdown of levodopa to dopamine
outside the brain and thereby reducing peripheral unwanted effects, in
affixed combination (association). The response to association is generally
stable during the initial years of treatment. However, due to the progressive
degeneration of the dopamine system, the neuronal buffer capacity is
believed to be reduced. At the stage, the patient may switch within seconds
from a state of relatively good mobility to one of severe parkinsonism,
giving rise to the term on-off phenomenon (Rajput et al. 2002).
The fixed combination improved the brain bioavailability of levodopa,
through peripheral dopa-decarboxylase inhibition. However, following dopadecarboxylase inhibition, more levodopa is metabolized by the enzyme
catechol-O-methyl transferase (COMT) in the gastrointestinal tract, liver, and
kidney, resulting in high circulating levels of 3-O-methyldopa. Thus, concomitant inhibition of COMT will reduce the metabolism of levodopa, will increase
the amount of levodopa available for conversion into dopamine in the brain, and
should improve the efficacy of the association (Myllila et al. 2006).
Entacapone
(E)-2-cyano-N,N-diethyl-3-(3,4-dihidroxy-5-nitrophenil)acrylamide (The Merck 2001; Fig. 1) is an orally active, nitrocatechol derivative with a selective and reversible inhibitory effect on COMT (Holm and
Spencer 1999).
Although there are many works describing the determination of entacapone in biological fluids (Wikberg et al. 1992; Keski-Hynnila et al. 1998;
Keski-Hynnila et al. 2000, Keski-Hynnila et al. 2001; Ramakrishna et al.
2005), there are only two studies of quantitative determination of this drug
in tablets (Rajeswari et al. 2006; Paim et al. 2007).
The work developed by Paim and collaborators (2007) described the
development and validation of a stability-indicate method by HPLC.
Rajeswari and collaborators (2006) described the quantitation of entacapone

Figure 1. Chemical structure of entacapone.

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Validation of UV Spectrophotometric Method

573

for spectrophotometry in the region of visible at 689 nm, based on the reaction
of entacapone with ferric chloride and potassium ferrocyanide.
The method validation described in this work was fulfilled through the
evaluation of the analytic parameters of specificity, linearity, precision
(repeatability and intermediate precision), and accuracy (ICH 2005; USP 30
2007). Experimental design was used to evaluate method robustness
(Snyder et al. 1997; Vander Heyden et al. 2001).
The objective of this work was to develop and to validate a simple, fast,
and low-cost method for UV spectrophotometry to quantify entacapone in
coated tablets. In addition, the comparison of these results with those
obtained from the HPLC analysis (Paim et al. 2007) was presented.
EXPERIMENTAL
Samples
Entacapone substance reference (98.0%) was characterized by differential
scanning calorimetry (DSC), infrared spectroscopy (IR), nuclear magnetic
resonance (NMR) spectroscopy, and nonaqueous titration of weak acids
(Paim et al. 2007).
Comtan 200 mg of entacapone was purchased in the market. The tablets
are manufactured by Orion of Finland and for commercialization in Brazil are
imported, packed, and distributed by Novartis Biociencias S. A.
Instrumentation and Conditions
Spectral and absorbance measurements were performed with a UV-Vis
Shimadzu model UV 160A using 10 mm quartz cells and detection at 305 nm.
Standard Solutions Preparation
Entacapone substance reference was accurately weighed (10 mg) and
dissolved in a 100 ml volumetric flask with acetonitrile to generate a concentration of 0.1 mg ml21 of analyte. An aliquot of 2 ml of this solution was
diluted in a 20 ml volumetric flask (0.1 mg ml21) and the volume was
completed with the same solvent.
Sample Solutions Preparation
Twenty tablets were weighed and finely powdered. An amount equivalent to
100 mg of entacapone was transferred to a 100 ml volumetric flask with 60 ml
of acetonitrile. This volumetric flask was kept in an ultrasonic bath for 15
minutes and shaken by a mechanical shaker for 15 minutes. The volume

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C. S. Paim et al.

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was completed with the same solvent. An aliquot of 2 ml of this solution was
diluted in a 20 ml volumetric flask (0.1 mg ml21) and the volume was
completed with acetonitrile. In according to the previous, a new dilution
was realized, obtaining a final concentration of 10 mg ml21. All determinations were conducted in triplicate.

Method Validation
The evaluation of the specificity of the method was performed by preparing a
placebo containing the same excipients of the commercial product.
The linearity was evaluated through the construction of three calibration
curves, prepared in the same day, with six concentrations (3.0, 4.0, 5.0, 8.0,
10.0, and 20 mg ml21). The calculation of linear regression was employed
by the method of least squares. The curves were validated by means of the
analysis of variance.
The determination of precision was realized through seven tablet samples,
at the same concentration (20.0 mg ml21), under the same experimental conditions in the same day for intraday precision (repeatability) and on three
different days for interday precision (intermediate precision). The relative
standard deviation (RSD) was determined.
The accuracy was calculated in relation of the percentage of recovery by
the assay of the known added amount of entacapone substance reference in
the samples solutions using three concentrations levels covering the specified
range (12.0, 14.0, and 20.0 mg ml21) and three replicates of each concentration.
The robustness testing was performed in order to evaluate the susceptibility of measurements due to deliberate variations in analytic conditions
(ICH 2005; USP 30 2007). The study was accomplished through experimental
design of Plackett Burman, which allows the execution of a minimum number
of experiments for study of the selected factors (Vander Heyden et al. 2001).
The factors and levels studied for the robustness test are presented in Table 1.
After determination of the number of real factors to be examined, the
remaining columns in the design were defined as dummy factors. A dummy
factor is an imaginary factor for which the change from one level to the
other has no physical meaning. The experiments and their specified levels
are represented in Table 2.

Table 1.

Factors and levels investigated in the robustness test

Factors
Mechanically shaken (time)
Shaken in an ultrasonic bath (time)
Wavelength (nm)

Nominal

Level (21)

Level (1)

15
15
305

12
12
302

18
18
308

Validation of UV Spectrophotometric Method

575

Table 2. The selected Plackett-Burman design for the verification of the robustness of
analytic method

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Exp Ultrasonic bath

1
2
3
4
5
6
7
8

1
21
21
1
21
1
1
21

l
1
1
21
21
1
21
1
21

Dummy Dummy Dummy Mechanical shaken Dummy

1
1
1
21
21
1
21
21

21
1
1
1
21
21
1
21

1
21
1
1
1
21
21
21

21
1
21
1
1
1
21
21

21
21
1
21
1
1
1
21

Note: Ultrasonic bath shaken in an ultrasonic bath; l wavelength.

The effects of the factors on response were calculated according to the

following equation:
P
P
Y
Y



Ex
N=2
N=2
where
X

Ex effects of the factor on response

X
Y and
Y
sums of the responses

where x is at the extreme levels () and (2), respectively

N number of experiments of the design
An error estimate of experiment was obtained according to the following
equation:

sP
2
Edummy
Ee
nerror
where
Ee experimental variability within the design
X
2
sum of squares of the nerror dummy
Edummy
After calculating the effects of the factors (Ex) and of the error of the
experiment (Ee), the significance of the factors was determined in analysis
through the accomplishment of the t-test statistic according to the following
equation:
t

Ex
Ee

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C. S. Paim et al.

where
Ex effects of the factor on response

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Ee experimental variability within the design

The value t-calculated was compared with t theoretical bicaudal for
a 0.05 and 4 degrees of freedom (gl).
The stability of entacapone in acetonitrile was studied by HPLC and spectrophotometric methods. Sample solutions of entacapone (10.0 mg ml21) were
prepared, and the stability was studied by performing the experiments and
looking for the change in the chromatographic pattern (HPLC) and the
decrease of absorbance (spectrophotometric) compared with those of freshly
prepared solutions.

RESULTS AND DISCUSSION

The UV-Vis method is very useful in quality control of pharmaceutical
products due to the potential of the great majority of the drugs to absorb
energy in these wavelengths. The absorption of UV-visible radiation occurs
through the excitation of electrons within the molecular structure to a
higher energy state. Although the selectivity depends on the chromophore
of the drug, the method presents a series of applications: quantification of
drugs in formulations where there is no interference from excipients, pka
determination, release of drugs from formulations with time in dissolution
testing, monitoring of the reaction kinetics of drug degradation, and identification of drugs starting from UV spectrum (Watson 2005).
Considering the solubility of the entacapone substance reference, the
solvents used in the development and validation of the analytic method were
ethanol, 50 mM phosphate buffer (NaH2PO4) pH 7.4, methanol, and acetonitrile.
Studies of stability of the drug were accomplished in each solvent for the
development and validation of the method. The decrease of the absorbance of
the drug in this maximum wavelength as a function of time was evaluated.
The first solvent to be studied was 50 mM phosphate buffer pH 7.4, in
which an alteration in the coloration was observed as a function of time.
Three solutions (10 mg ml21) were prepared, and measurements of the absorbance at 378 nm were carried out, in determinates times, to verify if the color
change in the entacapone solution was due to the degradation of the drug. In
the execution of the assay, 100% of entacapone was admitted at time zero. The
results are presented in Fig. 2. The results demonstrated that entacapone is not
stable in 50 mM phosphate buffer (NaH2PO4) pH 7.4, due to the decrease of
the absorbance as a function of time. The degradation arrived at 1% in 10
minutes and at 5% in 150 minutes. The results also indicated that the
decrease of the concentration was not influenced by the temperature and
light, and because of this, the use of this solvent was discarded.

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Validation of UV Spectrophotometric Method

Figure 2.

577

Stability of entacapone in 50 mM phosphate buffer (NaH2PO4), pH 7.4.

The preliminary results with ethanol demonstrated that the drug presented
two wavelengths of maximum absorption (303 nm and 387 nm) and in both it
was specific. However, the method presented linearity in the range studied just
in 303 nm. The evaluation of the stability of the drug in ethanol demonstrated
that it did not decrease in 303 nm for 3 hours, confirming the stability of the
drug in these conditions. The repeatability and intermediate precision tests
indicated the precision of the method. However, the recovery test demonstrated that the method did not present accuracy. Alternatively, the calibration
curve was accomplished using methanol, but the results were similar.
After these preliminary tests, the use of acetonitrile was analyzed for
development of the analytic method, in which the drug was shown to be
stable during the studied period (6 hours).
The specificity test demonstrated that there was not interference in the
determination of this drug. The UV spectra obtained through the analysis of
the placebo solution did not present any interference in the maximum of
305 nm of entacapone (Fig. 3).
Linearity was observed over the concentrations range of 3.0 to 20.0
mg ml21, with significantly high value of correlation coefficient (r 0.99996)

Figure 3.
tablets.

UV spectrum obtained through the analysis of placebo solution of coated

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578

C. S. Paim et al.

and the linear regression equation y 0.065992x 0.000721. The validity of the
assay was verified by means of the ANOVA. According to it, there is linear
regression and there is not deviation from linearity (p 0.05).
The experimental values obtained for the determination of the precision
of the analytic method are presented in Table 3. The low relative standard
deviation (RSD) obtained for the repeatability (1.52; 1.21; 1.41) and intermediary precision (1.38) showed the good precision of the method. Figure 4
shows the UV spectra of entacapone commercial sample solution of coated
tablets and entacapone standard solution.
The accuracy of the method ranged from 97.87 to 98.50%. These values
demonstrated the good accuracy of the purposed method.
The results of the experiments concerning robustness are presented in
Table 4. They are expressed in percentage of the drug in relation to the
nominal dose, calculated using standard solution in the nominal condition of
the method. The effects of the factors in analysis, the error estimated starting
from the factors dummy, and the value of t-calculated are shown in Table 5.
The analysis of the results of the robustness study demonstrated that the
factors in analysis did not have significant effects on the quantitation of the entacapone, indicating the robustness of the UV spectrophotometric method.
The comparison between the UV (101.33 + 1.75) and the HPLC
(100.48 + 1.10; mean + RSD) methods was performed through the statistical
t-student test at 0.05 significance level of the mean experimental values
obtained in the quantitation of entacapone in tablets by each method. The
test did not show statistical difference between the two techniques
(t-calculated 1.86 , t-theoretical 2.02). The developed and validated
methods provided similar results for entacapone quantitation.

Table 3. Repeatability and intermediate precision values obtained for entacaponecoated tablets by UV spectrophotometry at 305 nm
Repeatability
Sample (n)

Day 1

Day 2

Day 3a

1
2
3
4
5
6
7

103,30
103,62
103,94
101,35
100,10
100,84
100,97

97,59
100,52
99,55
99,05
99,87
101,46
100,00

103,00
101,24
100,44
100,59
103,12
103,66
103,68

Mean (%)
RSD

102,02
1,52

99,72
1,21

102,25
1,41

Analyst B.

Intermediate precision

102,02
99,72
102,25
101,33
1,38

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Validation of UV Spectrophotometric Method

579

Figure 4. UV spectra obtained through the analysis of entacapone commercial

sample solution of coated tablets (a) and standard solution (b).

Table 4. Results of the percentage of entacapone obtained in each experiment of the

robustness test by UV
Experiment
1
2
3
4
5
6
7
8

Percentage of entacapone
100.1
103.0
100.9
101.5
104.1
102.4
101.0
104.5

Table 5. Experimental values of the effects and t-calculated of

the factors analyzed
Factor

Effect

t-calculated

Mechanically shaken
Shaken in an ultrasonic bath
Wavelength of the detector

0.56
20.94
20.12

1.14a
21.94a
20.25a

No statistical difference to t(0,05;4); experimental error

(Ee) 0.487.

CONCLUSION
The results indicated that the UV spectrophotometric assay holds linearity,
precision, accuracy, specificity, and robustness. There is no significant difference between the previously validated HPLC method and the UV method,

580

C. S. Paim et al.

which confirms that the UV method is adequate and useful to the routine
quality control of entacapone in pharmaceutical dosage forms.

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