Hindawi Publishing Corporation

Chromatography Research International

Volume 2012, Article ID 610427, 8 pages
doi:10.1155/2012/610427

Research Article
Development and Validation of Dissolution Test for Fluconazole
Capsules by HPLC and Derivative UV Spectrophotometry

Josilene Chaves Ruela Corrêa,1 Cristina Duarte Vianna-Soares,2

and Hérida Regina Nunes Salgado1
1 Schoolof Pharmaceutical Sciences, Universidade Estadual Paulista, Rodovia Araraquara-Jaú km 1, 14801-902 Araraquara, SP, Brazil
2 Department of Pharmaceutical Products, Federal University of Minas Gerais, Avenida Antônio Carlos 6627,
31270-901 Belo Horizonte, MG, Brazil

Correspondence should be addressed to Josilene Chaves Ruela Corrêa, [email protected]

Received 7 December 2011; Revised 26 January 2012; Accepted 27 January 2012

Academic Editor: Bengi Uslu

Copyright © 2012 Josilene Chaves Ruela Corrêa et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

The purpose of this study is to develop and validate a dissolution test for fluconazole, an antifungal used for the treatment of
superficial, cutaneous, and cutaneomucous infections caused by Candida species, in capsules dosage form. Techniques by HPLC
and UV first derivative spectrophotometry (UV-FDS) were selected for quantitative evaluation. In the development of release
profile, several conditions were evaluated. Dissolution test parameters were considered appropriate when a most discriminative
release profile for fluconazole capsules was yielded. Dissolution test conditions for fluconazole capsules were 900 mL of HCl 0.1 M,
37 ± 0.5 ◦ C using baskets with 50 rpm for 30 min of test. The developed HPLC and UV-FDS methods for the antifungal evaluation
were selective and met requirements for an appropriate and validated method, according to ICH and USP requirements. Both
methods can be useful in the registration process of new drugs or their renewal. For routine analysis application cost, simplicity,
equipment, solvents, speed, and application to large or small workloads should be observed.

1. Introduction drug are not well defined, for instance, its classification
as high or low soluble in water or its classification in the
The dissolution can be defined in a narrow sense as the biopharmaceutical classification system [4–6]. Well-defined
process by which a solid substance is incorporated into drug features, together with the dissolution studies, can be an
the solvent to form a solution. However, in a broad sense, important tool to justify the use of in vitro methods instead
it is more than a simple measurement of solubility rate of in vivo methods when they are requested. The aim of
and can be better described as physical test to predict the this dissolution study is to contribute to define fluconazole
drug release from a dosage form, for a given area for some dissolution conditions, what can be the focus for further
precise time. Fundamentally, this process is controlled by studies.
the affinity between the solvent and the solid substance A dissolution method should be discriminatory, and it
and the way by which the pharmaceutical system releases should allow evaluating the performance of the product and
the drug [1, 2]. According to Mehta and coworkers [3] possible changes it may suffer from during the stability study.
dissolution test provides an indication of bioavailability of According to Marcolongo [7], many variables can influence
a drug and, thus, pharmaceutical equivalence from batch the results of a dissolution test. Among these variables we
to batch. The dissolution test is an important tool in can find solubility, chemical nature of the drug, the dosage
quality control of drugs and it becomes more important for form, excipients and manufacturing technology employed,
drugs with relatively low water solubility, including broad the apparatus used, the stirring speed, the use of devices
spectrum antifungal fluconazole. Some characteristics of this for floating dosage forms (sinkers), the volume of media
2 Chromatography Research International

used, pH and temperature of the media, the filtration, and 2.2. Preparation of Standard and Sample Solutions
analytical method employed.
To develop a dissolution method the characteristics of 2.2.1. Dissolution Performance. Fluconazole 150 mg capsules
the drug and its behavior in the chosen test media should be were dissolved in 900 mL of HCl 0.1 M at 37◦ C for 30 min,
taken in account. Moreover, the dissolution conditions must using baskets. The collected samples were filtered through
follow the sink conditions (final concentration equivalent to quantitative filter paper, when evaluated by FDS, or through
10% of saturation concentration) [7], and the quantitation 0.45 μm regenerated cellulose membranes, when evaluated
method should be sensitive, selective, accurate, and precise. by HPLC.
Although there are many published analytical meth-
ods for fluconazole, there are few dissolution studies for 2.2.2. LC and UV Determination Method. A stock solution
fluconazole, as it has been well compiled in a review by was prepared by dissolving 50.0 mg of fluconazole in a
Corrêa and Salgado [8]. Fluconazole dissolution has not been 100 mL volumetric flask using HCl 0.1 M with 30 min
recommended in any pharmacopeia until December 2010 sonication. Five standard solutions were prepared from stock
when it has been incorporated in the capsule monograph in solution in different concentrations (135, 150, 165, 180, and
the Brazilian Pharmacopeia, 5th edition [9]. However, FDA 195 μg/mL) by dilution with the same solvent. The range of
[10] has recommended dissolution methods since 2004 for concentrations chosen has the used concentration (150 mg
fluconazole suspension and since 2006 for tablets; the FDA- of fluconazole in 900 mL of dissolution media) as the central
recommended method for tablets was tested in this study concentration. The samples were filtered using quantitative
without good results. In the Brazilian Pharmacopeia [9], the filter paper, when they were evaluated by FDS, and using
dissolution method recommended for fluconazole capsule membranes of regenerated cellulose, 0.45 μm, when they
uses 900 mL of 0.1 M HCl at 37◦ C and baskets with 100 rpm were evaluated by HPLC. Sample, standard, and placebo-
for 30 min with quantitation by spectrophotometry at λ = enriched solutions were prepared in the selected central
261 nm. concentration in the same way.
The aim of this work is to develop and validate a
discriminative dissolution method for fluconazole capsules 3. Results and Discussion
employing two analytical methods to determine fluconazole
by high-performance liquid chromatography (HPLC) and by 3.1. Analytical Development
first-order derivative UV spectrophotometry (FDS). Prob-
3.1.1. UV Determination Method. The selectivity of the
lems encountered by the UV spectrophotometric method,
Brazilian-Pharmacopoeia-recommended method was evalu-
recommended by the Brazilian Pharmacopeia 5th edition [9]
ated using the compounding fluconazole and its excipients
are discussed.
(capsules and placebo). The calculations of interference were
performed in accordance with the recommendations of USP
2. Experimental 32 [11], since the Brazilian Pharmacopoeia does not cite this
calculation, using the following equation. The interference
2.1. Material and Equipment. Fluconazole chemical refer- must not exceed 2%:
ence (assigned purity 100%) was purchased from Sigma    
Ap V
Aldrich. Bulk drug was kindly donated (EMS, Hortolândia, 100C × × , (1)
As L
SP, Brazil) and was standardized against fluconazole chemical
reference. Capsules were purchased from local market with where C is the concentration (mg/mL) of standard solution,
150 mg drug label claim. A Hanson SR 8 Plus dissolution Ap and As are the absorbances of placebo and standard
system containing six vessels was used for dissolution tests. solutions, respectively, V is the volume of medium (mL), and
LC grade methanol was purchased from Tedia (Fairfield, L is the dosage of the product (mg).
USA). Purified water was prepared in-house by using Direct- Spectrum results showed a strong interference, caused
Q water system (Millipore, Billerica, MA, USA). Prior to use, by both capsule shells and placebo in the analytical result.
mobile phase solvents were degassed in an ultrasonic bath for Using the analytical method recommended by the Brazilian
30 min. Purified water (>18 MOhm cm) was used to prepare Pharmacopoeia the mean percentage of dissolution for six
the mobile phase. Solvents were filtered through a 0.45 μm fluconazole capsules was 115.21% and the mean percentage
membrane filter. of response for placebo and capsule was 3.94% and 10.70%,
An HP 8453 UV-Visible spectrophotometer (Agilent respectively, a total of 14.64%. These results are in accordance
Technologies, Inc., Santa Clara, CA, USA) with photodiode with those obtained by Oliveira and coworkers [12], who
array (PDA) and HP ChemStation software with automatic reported fluconazole capsules dissolution. They have shown
differentiation was used. A liquid chromatograph (Waters a huge interference of the excipients in fluconazole determi-
Corporation, Milford, MA, USA) equipped with a Waters nation by UV.
1525 binary pump, a Rheodyne Breeze 7725i manual injec- In order to eliminate the interference of the excipients
tor, and a Waters 2487 UV-VIS wavelength detector was (placebo and capsule) UV-FDS was tested. In general,
used. HPLC analysis was conducted in an RP C18 column the spectral derivation provides simultaneous drugs deter-
(Symmetry, 5 μm, 4.6 mm × 250 mm, Waters, Milford, MA, minations in association, as well as, increased selectivity.
USA). In addition, there are often an increased sensitivity and
Chromatography Research International 3

0.06
0.04

d1 (Absorbance)
0.02
Placebo and capsule solutions
0
−0.02

−0.04 Standard and sample solutions

−0.06

−0.08

220 230 240 250 260 270 280

Wavelength (nm)
(a)

2.5

2
Absorbance (a.u.)

1.5
Capsule solution
1
Placebo solution Sample solution
0.5 Standard solution

383
390
392
216

0
200 220 240 260 280 300 320 340 360
Wavelength (nm)
(b)

Figure 1: (a) First-order derivative spectra overlay of fluconazole standard, dissolution sample, and placebo and capsule shells solutions. (b)
Zero-order derivative spectra overlay of standard solution, dissolution of fluconazole sample, and capsule shells and placebo solutions.

improved detection limits. The increased sensitivity observed The analytical development must be developed to obtain
in the derivative spectroscopy is based on the observation a simple and optimal method. Good results were obtained
that the amplitude of the absorbance derivative relative to using reversed phase C18 (250 × 4.6 mm, 5 mm) Water Sym-
the wavelength is inversely proportional to the bandwidth of metry endcapped column, 1.0 mL/min, water, and methanol
the ordinary spectrum. The order of the derivative must be (60 : 40, v/v), injection of 20 μL monitored at λ = 261 nm.
carefully selected since there is usually an increase in noise The samples used to test the method recommended
level with increasing order of differentiation [13, 14]. by the Brazilian Pharmacopeia [9] were now employed to
Figure 1 shows the first-order derivative of absorbance evaluate the HPLC method. The results showed that the
of fluconazole that has an intense and well-defined valley at HPLC method is selective, and it was able to separate the
268 nm. It was the wavelength chosen. Fluconazole capsule drug from the placebo and capsule (Figure 2).
samples were tested according to the method recommended Figure 2 shows that capsule shells and placebo absorb
by the Brazilian Pharmacopeia [9]. The same samples were energy in UV region (peaks at 5.15 min); however, they
employed to evaluate the derivative spectrophotometric could be well separated from fluconazole (peak at 3.65 min).
method. The average interference from placebo and capsules The capsule peak was observed in capsule shells and sample
was again calculated in the same way but using the derivative solutions. The peak at 2.45 min, present in all samples, refers
spectrophotometric method. The interference was equal to to the dissolution media (HCl 0.1 M), including the blank
0.1% due to placebo and 1.98% due to capsules shells. These solvent.
values have low analytical significance and are satisfactorily
acceptable. Thus, the interference of excipients (placebo and 3.1.3. Dissolution Performance. Fluconazole three different
capsule) was considerably reduced by using the differentia- pKa [15] values, 11.01 ± 0.29, 2.94 ± 0.10, and 2.56 ± 0.12,
tion. correspond to the groups alcohol (proton donor) and two
nitrogens (proton acceptors), respectively. Thus, the aqueous
3.1.2. HPLC Determination. HPLC is a widely used method solubility of fluconazole is greater in solutions with extreme
of separation with high precision and accuracy. It allows pH values (above 11.01 and below 2.94), situations in which
the separation between the drug and excipients, as well the drug would be fully ionized.
as degradation products, which is useful as an indicative However, dissolution of fluconazole capsules was evalu-
stability method. ated in deionized water, as recommended by FDA [10], and
4 Chromatography Research International

0.02
0.015 Fluconazole standard and sample

(a.u.)
0.01 Blank

0.005 Blank Capsule

Placebo
0

1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6

963 minutes, 0.01969 a.u. (minutes)

Figure 2: Chromatograms overlay of fluconazole dissolution samples, placebo and capsule shells, at λ = 261 nm.

in HCl 0.1 M at 37◦ C. Both baskets and paddles (with and

without sinkers) apparatuses were used in a total medium
90
volume of 900 mL in all tests to evaluate dissolution profile.
The dissolution media were degassed by sonication for

Dissolution (%)
30 min at 37◦ C before initiating the dissolution test. The 60
final concentration of fluconazole in 900 mL was nearly
165 mg/mL as capsule products contained 150 mg of the
drug. This is in agreement with required sink conditions, 30
desirable to prevent saturation. Thus, the concentration
165 mg/mL was included to be the central point of the range
used to validate the method. 0
0 30 60
In the dissolution profile, samples were collected after Time (min)
5, 10, 15, 20, 30, 45, 60, and 65 min, filtered through
quantitative filter paper, and fluconazole was quantified by Test 1 Test 3
UV-FDS. The dissolution medium was used as blank. The Test 5 Test 7
Test 2 Test 4
replacement of media was performed after each collection
Test 6 Test 8
using the dissolution media at 37◦ C. Both mass withdrawn
and the dilution made for each replacement of media were Figure 3: Dissolution profile obtained for testing fluconazole
taken into account in the calculations for the construction of 150 mg capsules by UV-FDS. Conditions: as in tests 1–8 specified
the dissolution profiles. in Table 1.
Rotation speeds 75 and 100 rpm were used in each test
with baskets and 50 and 75 rpm with paddles. During the last
5 min, the speed of rotation was changed to 150 rpm in all
tests. The speed can be increased at this point to verify if the 90
drug contained in the capsule was entirely released during
Dissolution (%)

the test. The results of dissolution profiles using the described 60

conditions are shown in Table 1 and Figure 3.
Water was used as dissolution medium in Tests 1 and 2, as 30
recommended by FDA [10]. It shows not to be an appropriate
medium for fluconazole capsules even after 65 min test at the 0
highest speed because the drug percentage release was below 0 30 60
57%. The HCl 0.1 M dissolution media tested showed better Time (min)
results.
Figure 4: Dissolution profile for fluconazole capsules. Samples
The paddle apparatus was tested at 50 and 75 rpm, with analyzed using 0.1 M HCl at 37◦ C as dissolution media, basket, and
and without sinker. The use of sinker made the dissolution 75 rpm. UV method was employed.
slower, and it is shown comparing Tests 4 and 6 to Tests 3
and 5. Tests 3 and 4, when 75 rpm was employed, showed fast
dissolution in the beginning and low discriminatory power.
Tests 5 and 6 showed slow dissolution in the beginning; 7 has shown fast initial dissolution with more than 90% of
however, at the end the drug was not released from the drug release after 5 min and therefore a low discriminatory
dosage form to the dissolution media. It could be realized power. In Test 8, there were small release increases in
after employing 150 rpm for 5 minutes in the end of test that fluconazole amount along the test, which has shown a
the concentration of drug increased rapidly. discriminative profile (Figure 4).
Baskets were employed in Tests 7 and 8 using HCl 0.1 M Therefore, basket apparatus, 75 rpm, 900 mL of 0.1 M
as dissolution media and 100 and 75 rpm, respectively. Test HCl at 37◦ C as dissolution media, and 30 min of testing
Chromatography Research International 5

Table 1: Dissolution profiles for fluconazole determination: tested parameters and results.

Average %
Test Time (min) Medium Medium volume (mL) Apparatus Rotation (rpm) % R.S.D.
dissolution
5 39.05 10.58
10 52.79 3.26
15 55.73 3.14
1 20 water 900 Basket 100 56.73 1.16
30 56.65 0.38
45 56.82 0.31
60 56.74 0.60
65 150 56.69 1.11
5 32.29 0.07
10 48.09 4.78
15 50.31 3.21
2 20 water 900 Paddle 75 51.01 2.02
30 51.77 1.57
45 52.48 1.25
60 53.52 1.26
65 150 56.99 0.89
5 86.79 6.23
10 94.14 3.47
15 95.89 2.72
3 20 0.1 M HCl 900 Paddle 75 97.32 3.43
30 98.64 2.40
45 99.19 1.58
60 98.24 1.68
65 150 100.24 2.37
5 85.45 9.69
10 93.92 1.13
15 95.70 0.26
4 20 0.1 M HCl 900 Paddle + sinker 75 96.63 0.73
30 96.34 1.31
45 97.06 1.71
60 97.57 1.37
65 150 100.00 1.43
5 76.67 5.84
10 86.41 1.35
15 88.70 2.58
5 20 0.1 M HCl 900 Paddle 50 89.78 3.47
30 90.44 3.65
45 91.79 2.94
60 92.36 3.81
65 150 101.24 2.77
5 58.10 4.62
10 68.09 6.98
15 71.54 7.02
6 20 0.1 M HCl 900 Paddle + sinker 50 73.34 5.75
30 76.28 4.53
45 78.76 4.70
60 80.19 4.78
65 150 99.05 4.55
6 Chromatography Research International

Table 1: Continued.
Average %
Test Time (min) Medium Medium volume (mL) Apparatus Rotation (rpm) % R.S.D.
dissolution
5 92.96 6.34
10 98.96 0.32
15 100.07 0.20
7 20 0.1 M HCl 900 Basket 100 100.56 0.39
30 100.47 0.38
45 98.09 0.15
60 98.66 0.23
65 150 98.92 0.87
5 59.50 15.07
10 82.07 4.45
15 93.61 2.19
8 20 0.1 M HCl 900 Basket 75 97.00 1.93
30 97.64 1.36
45 96.43 1.35
60 96.38 1.23

were the conditions chosen for the dissolution of fluconazole intraday precision tests showed R.S.D. of 0.81%, HPLC and
capsules. This method was validated by FDS and HPLC. 0.75%, FDS and interday precision tests showed R.S.D. of
2.29%, HPLC and 2.55%, FDS. These results indicate good
3.2. Method Validation. The quantitative methods were vali- precision.
dated according to the ICH [16] and USP [11] guidelines for The accuracy was performed in triplicate using the stan-
development and validation of dissolution methods. Because dard addition method (enriched placebo). Known amounts
nonuniform drug distribution may affect the dissolution test of standard of fluconazole were added to placebo in order
in the single-dose units, fluconazole capsule samples were to reach five established concentration levels. The mean
evaluated regarding uniformity content (UC). All fluconazol percentage recovery of fluconazole standard found was
capsules UC results obtained by HPLC (between 91.42– 98.56 ± 0.82% for HPLC and 98.35 ± 0.88% for UV-FDS
100.2% of labeled value) were within accepted specifications. (Table 2). These results indicate an agreement between the
The calibration curves were obtained at five fluconazole true values and found values.
concentration levels from 135 to 195 μg/mL for HPLC (λ = This paper compares the methods to determine flu-
261 nm) and UV-FDS (λ = 268 nm). Lambert-Beer law was conazole after dissolution test regarding their precision
observed at this concentration range. Linearity was evaluated accuracy, and repeatability (Table 3). Both methods showed
by the least square method with determinations in triplicate to be specific, precise, accurate, and linear in the range of
at each concentration level. Both methods were linear with concentration tested.
this model. Regression equations were y = 2.5 × 106X − 1.1 ×
104 (r 2 = 0.996) for HPLC and y = 0.51439X − 2.9 × 10−3 , 3.2.1. Dissolution Performance Validation. After several con-
(r 2 = 0.998) for UV-FDS. The standard deviations of the ditions tested for the dissolution test development, appro-
regression were 0.83% for HPLC and 0.62% for UV-FDS. priate parameters were considered optimized whether pro-
The validity of the assay was verified by means of ANOVA. vided most discriminatory dissolution profile for fluconazole
According to ANOVA there is a statistically significant linear capsules. That means test conditions must be able to show
regression (Fcalculated > Fcritical ; P = 0.05) and there is no differences in drug release from batch to batch products, as
deviation from linearity (Fcalculated < Fcritical ; P = 0.05) for well as to distinguish possible changes that may occur during
both methods. stability studies or shelf-life of the product. The optimal
The precision of the methods was determined by repeata- parameters for fluconazole capsules dissolution are 900 mL
bility (intraday) and intermediate precision (interday). For of HCl 0.1 M, 37 ± 0.5◦ C using baskets with 50 rpm during
repeatability test three curves were constructed with the 30 min.
established five concentration levels using standard solutions Validation of dissolution performance was carried out
in the same day; for intermediate precision three curves by the two methods of quantification, HPLC and FDS.
were constructed with three concentration levels (high, The concentration of 150 mg of fluconazole in 900 mL of
intermediate, and low) using standard solutions in a different dissolution media is nearly equal to the central concentration
day. An interval of two days between repeatability and inter- (165 mg/mL) at the range established.
mediate precision was observed. The results were expressed The precision was determined by repeatability (intra-
as percentage of relative standard deviation (R.S.D.). The day) and intermediate precision (interday). The repeatability
Chromatography Research International 7

Table 2: Recovery data for fluconazole standard solutions added to Table 4: Recovery data of dissolution performance obtained by
the placebo by using the proposed HPLC and UV-FDS. HPLC and UV-FDS methods.

Added amount Founda amount Bias Recoverya Added amount Founda amount Bias Recoverya
Method Method
(μg/mL) (μg/mL) (%) (%) ± R.S.D. (μg/mL) (μg/mL) (%) (%) ± R.S.D.
135 133.92 0.80 99.20 ± 0.77 135 133.55 1.07 98.93 ± 0.23
150 148.00 1.33 98.67 ± 1.36 HPLC 165 160.33 2.83 97.17 ± 0.55
HPLC 165 161.28 2.25 97.75 ± 0.30 195 191.14 1.98 98.02 ± 0.63
180 177.30 1.50 98.50 ± 0.20 135 135.11 0.08 100.08 ± 0.42
195 192.41 1.33 98.67 ± 0.65 FDS 165 163.40 0.97 99.03 ± 0.32
135 134.54 0.34 99.66 ± 1.02 195 190.05 2.54 97.46 ± 0.34
a
150 147.51 1.66 98.34 ± 0.17 Average of three replicates.
FDS 165 161.93 1.86 98.14 ± 0.08
180 175.68 2.40 97.60 ± 0.22
4. Conclusions
195 190.96 2.07 97.93 ± 0.15
a
Average of three replicates. A discriminative dissolution test for fluconazole capsules
determination was presented in this study. Selective, sen-
sitive, precise, and accurate analytical methods were used
Table 3: Validation parameters for different analytical methods, for quantitation. The results showed that the determination
UV-FDS and HPLC, to determine fluconazole in capsules. of fluconazole capsules using direct UV spectrophotometry,
Parameters FDS HPLC recommended in the Brazilian Pharmacopeia, is not enough
selective; however, the developed first-order UV derivative
Analytical curve 0.51439X −2.9 × 10−3 2.5 × 106 X − 1.1 × 104
spectrophotometry and the HPLC showed to be selective
Intercept values −2.9 × 10−3 −1.1 × 104 and meet requirements for an appropriate validated method.
Standard error of Both methods are useful for the registration of new drugs or
8.8346 × 10−7 19.63
slope their renewal. The application of each method, as a routine
Correlation analysis, should be observed considering cost, simplicity,
0.998 0.996
coefficient (r 2 ) equipment, solvents, speed, and application to large or small
R.S.D. of workloads.
0.75 0.81
repeatability (%)
R.S.D.
2.55 2.29 Acknowledgments
intermediate (%)
Accuracy (%) 98.35 98.56
The authors wish to thank the EMS Pharmaceutical Com-
R.S.D. of pany (Hortolândia, Brazil) for the kind supply of the raw
0.88 0.82
accuracy (%)
material and thank the Fapesp, CNPq, FUNDUNESP, and
LOQ 4.9 × 10−3 1.28 × 10−8 PADC-UNESP for financial support.
−3
LOD 1.4 × 10 3.84 × 10−9

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Q2 R1/Step4/Q2 R1 Guideline.pdf.
 Photoenergy
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