Simple, Inexpensive and Ecologically Friendly Derivative Spectrophotometric Fluconazole Assay from Nail Lacquer Formulations

doi:10.4236/ajac.2011.22012

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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

E-mail: [email protected]

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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