American Journal of Anal yt ical Chemistry, 2011, 2, 109-115
doi:10.4236/ajac.2011.22012 Published Online May 2011 (http://www.SciRP.org/journal/ajac)
Simple, Inexpensive and Ecologically Friendly Derivative
Spectrophotometric Fluconazole Assay from Nail Lacquer
Formulations
Alisa Elezović1, Amar Elezović2, Sabira Hadžović1
1Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Sarajevo, Sarajevo,
Bosnia and Herzegovina
2Agency for Drugs and Medical Devices, Control Laboratory Sarajevo, Sarajevo, Bosnia and Herzegovina
Received December 29, 2010; revised February 9, 2011; accepted February 14, 2011
Abstract
Nail lacquers represent new drug form specifically designed to treat infected nail plate. They are complex
organic solutions with specific assaying problems due to the high content of the polymer and plasticizer.
Furthermore, there is a lack of assaying methods of active substances from this type of formulations in scien-
tific literature. We developed derivative UV-spectrophotometric method for determination of fluconazole
content in antifungal nail lacquer formulations. The method was validated for specificity, linearity, precision
(repeatability), intermediate precision and accuracy (recovery). The method is specific, linear in the range of
99.53 - 497.65 μg/ml, precise and showed good recovery (98.79% - 101.77% from all six developed formu-
lations). Besides, it is inexpensive, simple and nontoxic, i.e. ecologically acceptable. This method can be
used for assaying fluconazole from this type of formulations.
Keywords: Nail Lacquer, Fluconazole Assay, Derivative Spectrophotometry
1. Introduction
Onychomycoses, fungal nail infections, are responsible
for 50% of all nail disorders [1].
Fluconazole is a potent triazole antifungal agent, very
effective in treatment of onychomycoses [2-4]. However,
it is underused in systemic treatment of the disease due
to its numerous drug interactions and side-effects [5] as
well as heightened susceptibility for development of
fungal resistance. These hindrances of fluconazole in
systemic therapy of onychomycoses can be solved by its
topical use [6].
Nail lacquers (named also transungual therapeutic
systems) represent new drug forms specifically designed
to enable longer release and sustained action of active
principle into the infected nail plate. These systems are
commonly solutions (or suspensions) containing anti-
fungal agent, film-forming polymer, plasticizer and suit-
able volatile solvent. These excipients give stable solu-
tion (or suspension), easily spread on nail plate, and after
evaporation leave homogenous film.
In our laboratory, we developed six formulations of
nail lacquer.
There are literature accounts of determination of few
substances from cosmetic nail lacquers, such as organic
paints [7,8], formaldehyde [9], phthalate esters [10,11].
However, methods of assaying of active substances from
this type of formulations are not available in scientific
literature. Thus, the derivative UV-spectrophotometric
method was developed for determination of fluconazole
content in nail lacquers.
2. Equipments
Shimadzu UV-1601 spectrophotometer was used for the
spectrophotometric measurements. Measurements were
performed in quartz cuvette (d = 1 cm) with fixed slit
width of 2 nm in the range of 225 - 400 nm. First deriva-
tive spectra were obtained in the same range, i.e. 225 -
400 nm (Δλ (N) = 4).
The inermediate precision study was performed under
the same conditions as described above on Shimadzu
UV-1700 spectrophotometer.

A. ELEZOVIĆ ET AL. 110
3. Reagents and Samples
Fluconazole, fluconazole working standard and Eudragit
RS100 were in accordance to the corresponding mono-
graphs’ specifications of Ph. Eur. 6.0. Fluconazole and
fluconazole working standard were gifts from Bosnalijek,
d.d., Sarajevo, Bosnia and Herzegovina; Eudragit RS100
was kindly provided by Degussa AG, Rhome-Pharma
GmbH, Germany. All other reagents were of analytical
grade or better.
The nail lacquer formulations were prepared as clear
solutions containing 0.9% (w/v) fluconazole as antifun-
gal agent, Eudragit RS 100 as film-forming polymer, and
acetone as solvent. The formulations varied with respect
to nature and concentration of plasticizer, and they con-
tain: di-butyl phthalate in concentration of 15% (formu-
lation 1 L) or 25% (2 L) compared to the polymer, poly-
ethylene glycol 400 in concentration of 15% (3 L) or
25% (4 L) compared to the polymer, and propylene gly-
col in concentration of 15% (5 L) or 25% (6 L) compared
to the polymer. The blank sample of each formulation
contained all components as the corresponding formula-
tion except for fluconazole.
4. Procedure
Nail lacquer sample (0.5 ml) was spread on filter paper
(blue ribbon) and left to dry in tilted position for 10 - 15
minutes. After acetone evaporated (it could not be
smelled), the filter paper was placed in Erlenmeyer flask
of 100 ml. Distilled water (25 ml) was added and the
paper was completely submerged in water. Erlenmeyer
flask was closed and sonicated for 60 minutes. The
content was filtered and filtrate was used for the measure-
ment of first order derivative of absorbance at 268.4 nm.
Fluconazole concentration was calculated using the
calibration curve.
The stock solution of fluconazole (c = 500 μg/ml) for
the construction of calibration curve was prepared as
aqueous solution of fluconazole standard by gentle heating
at 40˚C and was diluted afterwards to target concentrations.
For the recovery studies, the fluconazole standard was
added to each nail lacquer formulation in concentrations
of 10, 20 and 30 μg/ml.
5. Results and Discusion
Considering the nature of sample (nail lacquer), i.e. the
fact that it is organic solution with high content of film
forming polymer, there were very unique challenges to
solve. Namely, nail lacquers are very specific formula-
tions, since they are non-aqueous, they have high poly-
mer content (usually 12% - 36%) and plasticizer content
(10% - 40% compared to the polymer). These excipients
may interfere with the determination of active substance
present in comparatively low concentration (in this case
0.9%). When dry, the films are relatively thick and plas-
tic and the extraction of active substance from such ma-
trices is very difficult. We developed derivative UV-
spectrophotometric method, enabling relatively easy
fluconazole content determination from these six formu-
lations. This method was validated according to ICH gui-
deline Q2 (R1) [12]. Validation parameters were speci-
ficity, linearity, repeatability, intermediate precision and
accuracy (recovery).
6. Sample Treatment
First, the extraction of fluconazole was attempted by
pouring lacquer sample into water or different concentra-
tions of methanol (5% - 15%), leading to precipitation of
water insoluble acrylate polymer. However, spectropho-
tometry requires large sample volume for measurement,
and relatively large volume of extraction medium would
have to be used. Very large amount of nail lacquer sam-
ple would be needed for adequate precipitation to occur,
but this might not ensure complete dissolution of flu-
conazole and could pose another problem if trying to
extract it quantitatively from such suspension. Thus, by
using smaller amount of nail lacquer, instead of precipi-
tation, dilution resulted in strong opalescence. This pre-
vented obtaining UV spectra, and the opalescence could
not be removed by filtration through the 0.22 μm pore
filter.
Also, the presence of acetone, even in small concen-
trations (less than 5%), strongly destabilizes fluconazole
spectrum. This problem was solved by evaporating ace-
tone, i.e. by drying the lacquer. Firstly, we tried spread-
ing of lacquer on the bottom of Erlenmeyer flask and
evaporating the acetone at 40˚C. Acetone evaporation in
such way was disputable, depending on flask’s neck
width, and it might not be completed after 90 minutes of
drying. Recovery in this instance was around 70%, and
for 2 L being as low as 57%.
The solution was found in spreading the nail lacquer
on filter paper. In this manner, the acetone evaporates
fast and easily, the film is well bound to the filter paper
and there is double area from which fluconazole can be
extracted.
Water was chosen since Eudragit RS 100 is not solu-
ble in it (unlike most organic solvents including alcohols)
and it is also non-toxic and inexpensive. The volume of
extractant was set to give concentration of fluconazole in
the extract at the optimal position of calibration curve.
The sonication time was chosen to be 60 minutes,
since after 45 minutes or less the recovery was not suffi-

A. ELEZOVIĆ ET AL.111
cient for all samples.
7. Spectrophotometric Measurements and
Method Validation
7.1. Specificity
For the specificity measurements, the aqueous solutions
of fluconazole, polyethylene glycol 400 and propylene
glycol were prepared in the same concentrations as in the
extract of nail lacquer formulations. Since Eudragit
RS100 is practically insoluble in water, the acetone solu-
tion was prepared with the same concentration of the
polymer as in the nail lacquer formulations. This solution
underwent the same extraction procedure as the nail lac-
quer formulations.
Fluconazole spectrum in range of 225 - 400 nm show-
ed two peaks, the first at 261.0 nm and the second 266.4 nm
(Figure 1(a)). Acrylic polymer contains components
which are also extracted, often variably, from the lacquer,
and show absorbance in the same range as fluconazole
(Figure 1(b)). Clear aqueous solution of dibutyl phtha-
late could not be obtained, since it is not miscible with
water. It was not possible to record its spectrum, because
of high light scattering from the emulsion. Hydrophilic
plasticizers in aqueous solution do not absorb in this re-
gion (225 - 400 nm), as can be seen in (Figures 1(c) and
(d)). The presence of all three plasticizers affects the
extraction of the acrylate polymer, thus affecting its
spectrum, and this can be seen in the spectra of blank
samples (Figures 1(e)-(g), formulations with higher
concentration of plasticizers).
These spectra differ, and since the aim was to obtain
method applicable to all six formulations, derivative
spectrophotometry method was chosen. Derivative spec-
trophotometry was already used in assays of fluconazole
in other pharmaceutical preparations, from which the
active principle was much easier to extract, such as cap-
sules, injection solutions [13] and syrups [14]. First de-
rivative of fluconazole spectrum showed maximum at
252.2 nm and minimum at 268.4 nm (Figure 2(a)).
Eudragit RS 100 as well as plasticizers, and thus blank
samples, show the first derivative at 268.4 nm (Figure 2)
to be dA/dλ = 0.000, recorded in Photometric mode of
the spectrophotometer. As can be seen on Figure 2,
Eudragit RS 100, unlike blank samples, does not have
the zero-crossing point at this wavelength and its first
derivative is less than dA/dλ = 0.0005, so the spectro-
photometer rounds its value to dA/dλ = 0.000. That small
offset from zero-crossing is not present in blank samples
implying that the first derivative value of absorbance at
268.4 nm does not originate from the excipients, but
solely from fluconazole.
(a)
(b)
(c)
(d)

A. ELEZOVIĆ ET AL. 112
(e)
(f)
(g)
Figure 1. UV spectra of fluconazole (a), Eudragit RS100 (b),
polyethylene glycol 400 (c), propylene glycol (d), blank
samples 2 (e), 4 (f) and 6 (g) in the region 225 - 400 nm.
The minimum of the first derivative of absorbance at
268.4 nm was chosen for quantification. In this manner,
the specificity of the method was proven.
7.2. Linearity
Linearity was shown by obtaining the first derivative of
absorbance of fluconazole at 268.4 nm in concentration
range 99.53 - 497.65 μg/ml, and the correlation coefficient
was R2 = 0.99985.(Figure 3)
(a)
(b)
(c)
(d)
(e)
Figure 2. The first derivatives of UV spectra of fluconazole
(a), Eudragit RS100 (b), blank samples 2 (c), 4 (d) and 6 (e)
in the region 225 - 400 nm.

A. ELEZOVIĆ ET AL. 113
7.3. Precision (Repeatability) standard in concentrations of 10, 20 and 30 μg/ml to all
six formulations and applying the method each time in
triplicate. Results are shown in Table 2.
Precision (repeatability) of the system was measured
using Dilutions 2 and 3 used for the construction of cali-
bration curve. The measurement was repeated six times
with the following results:
Dilution 2:
Average (n = 6) = 0.09219
SD = 0.00042
RSD = 0.45367%
Dilution 3:
Average (n = 6) = 0.15029
SD = 0.00039
RSD = 0.25977%
These dilutions were chosen because it was expected
that the nail lacquer extracts would have concentrations
around Dilution 2. Since these formulations contain the
lowest strength of fluconazole and they might be devel-
oped eventually in higher strength, Dilution 3 was also
tested.
Figure 3. Calibration curve of the first derivative of absor-
bance of fluconazole.
Table 1. Intermediate precision study.
7.4. Intermediate Precision
Formulation A*(% w/v) B*(% w/v) % I - II
1 L 0.90 0.91 100.77
2 L 0.94 0.92 98.27
3 L 0.89 0.90 100.92
4 L 0.88 0.89 101.71
5 L 0.93 0.94 100.68
6 L 0.90 0.91 101.08
For intermediate precision study, the method was per-
formed by Analyst I on UV-1601 spectrophotometer and
by Analyst II on UV-1700 spectrophotometer on all six
formulations in triplicate. The results are shown in Table 1.
The usual acceptable limit range for the intermediate
precision set by pharmaceutical industry and regulatory
bodies is (100 ± 2)%. The intermediate precision for all
six formulations was within this range (Table 1).
7.5. Accuracy (Recovery) A* Analyst I, Shimadzu UV-1601 spectrophotometer, average fluconazole
content (n = 3); B* Analyst II, Shimadzu UV-1700 spectrophotometer, ave-
rage fluconazole content (n = 3).
Accuracy (recovery) was tested by adding fluconazole
Table 2. Recovery for the nail lacquer formulations 1 L – 6 L.
sample standard
Average recovery per standard (%)
(n = 3)
Average recovery per sample
(%) SD per sample RSD per sample
10 g/ml 99.02
20 g/ml 99.00 1 L
30 g/ml 98.89
98.97 0.07 0.07
10 g/ml 99.36
20 g/ml 100.89 2 L
30 g/ml 98.79
99.68 1.09 1.09
10 g/ml 100.73
20 g/ml 100.79 3 L
30 g/ml 99.71
100.41 0.60 0.60
10 g/ml 101.33
20 g/ml 100.05 4 L
30 g/ml 101.18
100.85 0.70 0.70
10 g/ml 100.63
20 g/ml 98.99 5 L
30 g/ml 101.77
100.46 1.39 1.39
10 g/ml 101.42
20 g/ml 100.70 6 L
30 g/ml 100.34
100.82 0.55 0.55

A. ELEZOVIĆ ET AL. 114
Table 3. Content of fluconazole in formulations 1 L – 6 L,
and the percent compared to the declared content.
Formulation Fluconazole content
(% w/v)
Percent (%) of the
declared value
1 L 0.90 100.47
2 L 0.94 103.94
3 L 0.89 99.24
4 L 0.88 97.54
5 L 0.93 103.66
6 L 0.90 99.99
The usual acceptability limits set by pharmaceutical
industry and regulatory bodies for recovery are (100 ±
5)%, or if set more stringently (100 ± 2)% allowing for
analytical error, with RSD maximum of 2.0%. The re-
sults of this recovery study (Table 3) for each formula-
tion were inside these limits. Moreover, when consider-
ing all six formulations, the lowest recovery was 98.79%
(2 L), and the highest 101.77% (5 L), thus it was within
more stringent boundary range of (100 ± 2)%.
7.6. Content Determination
The concentration of fluconazole was determined in all
six formulations, and the results are shown in Table 3.
The content of the formulations assayed by previously
validated spectrophotometric method was in the accept-
able limit range (100 ± 5)% compared to the declared
value (0.9%).
8. Conclusions
The matrix of fluconazole formulation medium, being
organic solution with large amounts of acrylic polymer
and plasticizer, caused problems with the extraction of
the active substance. The only possible way to obtain
clear solutions was by drying the nail lacquer (i.e.
evaporation of the solvent), and then extracting the flu-
conazole from thus formed solid films over the double
area of filter-paper, serving here also as a supporting
medium for the film itself. Overlapping effect of the ma-
trix on UV-spectrum of fluconazole was removed by
derivative spectrophotometry, i.e. by recording first de-
rivative spectra of the nail lacquer extracts.
This method was validated for all six tested formula-
tions, and the validation parameters were specificity,
linearity, repeatability, intermediate precision, accuracy
(recovery). Method is inexpensive, simple, nontoxic, i.e.
ecologically acceptable and can be used for the assay of
fluconazole from this type of formulations.
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