Extractive spectrophotometric determination of ketoconazole, clotrimazole and fluconazole by ion-pair complex formation with bromothymol blue and picric acid

Article abstract of DOI:10.4067/S0717-97072012000200010

The reactions of picric acid and bromothymol blue with three important antifungal drugs containing an imidazole ring ketoconazole (KC) and clotrimazole (CT) and fluconazole (FC) have been studied for the development of simple, rapid, sensitive an extracti

Full text of DOI:10.4067/S0717-97072012000200010

J. Chil. Chem. Soc., 57, Nº 2 (2012)
EXTRACTIVE SPECTROPHOTOMETRIC DETERMINATION OF KETOCONAZOLE,
CLOTRIMAZOLE AND FLUCONAZOLE BY ION-PAIR COMPLEX FORMATION WITH BROMOTHYMOL
BLUE AND PICRIC ACID
*NINA ALIZADEH, ZAHRA REZAKHANI
*Department of Chemistry, University of Guilan, Namjoo Street, Rasht, P.B. 41335-1914, Iran
(Received: July 15, 2011 - Accepted: March 15, 2012)
ABSTRACT
The reactions of picric acid and bromothymol blue with three important antifungal drugs containing an imidazole ring ketoconazole (KC) and clotrimazole
(CT) and fluconazole (FC) have been studied for the development of simple, rapid, sensitive an extractive-spectrophotometric method for determining the
concentration of these drugs. This method is based on the formation of yellow ion-pair complexes between the basic nitrogen of the drug and sulphonphthalein
acid dyes, namely; bromothymol blue (BTB) in phosphate buffer of pH =3 and picric acid (PA) in citrate buffer of pH=2.5. The formed complexes were extracted
with chloroform and measured at 410 and 415nm for KC, at 410 and 413nm for CT and at 373 and 415nm for FC using PA and BTB, respectively. The analytical
parameters and their effects on the reported systems are investigated. Beer’s law was obeyed in the range 1-60 , 1-58 and 3-60 µg/ mL with PA and 3-55, 2-50
and 5-55 µg/ mL with BTB for CT, KC and FC, respectively. The composition of the ion pairs was found 1:1 by mole ratio and job¢ s method in all cases. The
proposed methods have been applied successfully for the analysis of the studied drugs in pure forms and pharmaceutical formulations.The results are in good
agreement with those obtained by official methods.
Keywords: Ketoconazole; Clotrimazole; Fluconazole ; Bromothymol blue; Picric acid; Spectrophotometry
INTRODUCTION
Ketoconazole (KC), cis-1-acetyl-4-[4-2-(2,4-dichlorophenyl)-2-(1H-
imidazole-1-yl methyl)-1,3-dioxolan-4-yl] methoxy piperazine(I), is widely
used as a typical antifungal drug in the treatment of tinea infections [1]. KC is
a potent inhibitor of cytochrome P-450-dependent steroid hydroxylation in the
adrenals [2]. In addition, KC enhances microsomal epoxid hydrolase activity
with some epoxide substrates [3].
Clotrimazole (CT), 1-[(2-chlorophenyl) diphenyl methyl]-1H-
imidazole(II), is an imidazole antifungal agent with similar action and activity
to KC [4]. Most clinical studies indicate that systemic CT has little efficacy and
EXPERIMENTAL
considerable toxicity compared to other systemic imidazoles (miconazole and
KC) [4]. Fluconazole(FC), [2-(2,4-difluorophenyl)-1,3-bis(1H-1,2, 4-triazol-1-
Materials and Reagents
All chemicals used in this study were of highest purity available (Merck)
yl) propan-2-ol](ΙΙΙ), is an orally active antifungal agent, which is used in the
and used without further purification. Doubly distilled deionized water was
treatment of superficial and systemic candidiasis and cryptococcal infections
used throughout. The picric acid and bromothymol blue solution were freshly
in patients with the acquired immunodeficiency syndromes (AIDS). It acts by
prepared and standardized Reagent grade KC and its 200 mg tablet as well as
blocking the synthesis of ergosterol, an essential component of the fungal cell
2% cream samples and reagent grade CT, 100 mg vaginal tablet, 1% cream.
membrane. Thus, due to the vital importance of determination of these drugs in
were obtained from Behvazan Pharmaceutical Company (Rasht Iran) and
pharmaceutical preparations and biological fluids, several spectroscopic[5-12].
standardized. capsules containing fluconazole containing100 mg and 200 mg
high per- formance liquid chromatographic[13-/17] and electrochemical [18-
of the drug were obtained from Loghman Darou (Tehran Iran).
/23] methods for assay of KC and also, various methods such as derivative
A working standard solution of 100 µg/ mL ketoconazole or clotrimazole
spectrophotometry
[24-25],colorimetry
[5-6-26-27],
HPLC[28-/30],
or fluconazole was prepared by dissolving 50 mg of pure drug in 50 ml of water
containing few drops of hydrocholoric acid (about 0.04 M for KC and 2M for
CT and FC) followed by further diluting of 5 ml of this solution to 50 ml.
electrochemistry[18] and differential scanning calorimetry (DSC) [31] have
been reported for the determination of CT in the literature. The official method
normally involves titration in non-aqueous solvent[32-33]. To the best of
our knowledge, despite the important ionassociation complex formation and
extractive spectrophotometric methods, there are no previous reports based on
the reaction of KC and CT and fluconazole with picric acid (PA) and reaction
this drugs with bromothymol blue (BTB) as ion pairing reagents.
In the present investigation, we report that the development of accurate,
reproducible, less time consuming and adequately sensitive three extractive-
spectrophotometric methods based on the formation of ion-pair complexes
between KC,CT and FC with anionic dye namely bromothymol blue (BTB)
and picric acid (PA) in acidic buffer compared with other reported methods.
The compositions of the complexes formed in proposed method have been
established and after optimizing various analytical parameters, the obtained
results were compared with those of official methods.
Apparatus
A Shimadzu 2100 UV-Vis spectrophotometer with matched quartz cells
of 1 cm path length, was used for all spectral measurements. The pH values
were determined with a model 825 metrohm using a combined electrod.
General procedure
Aliquots (1mL, 100 µg/ mL) of standard KC, CT and FC solution were
transferred into a series of 25 mL separating funnels containing 3 mL of 0.1%
w/v picric acid (or BTB) and 1 mL of citrate buffer of pH 2.5 (or phosphate
buffer of pH 3) were added. Then, 5 ml chloroform were added and the solution
was shaken for 10 min.The solution was allowed to stand for clear separation
of the two phases. The formed complexes were extracted with chloroform and
measured at 410 and 415 nm for KC, at 410 and 413 nm for CT and at 373 and
415nm for FC using PA and BTB, respectively.
Blood serum sample
0.25 mL of a 0.5 mol/L FC solution was added into 0.5 mL of a blood
serum samole. The volume of the mixture was made to 5 mL in a calibrating
volumetric flask and the general procedure of the proposed method was
followed.
e-mail address: nializadeh@yahoo.com
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J. Chil. Chem. Soc., 57, Nº 2 (2012)
Tablet sample solution
Five capsules of fluconazole were weighed accurately and their contents
were mixed and powdered thoroughly and Ten tablets of each drug (CT and
KC) after determining the average weight were powdered. Powder of each drug
equivalent to 25 mg was accurately weighed and shaken with water containing
a few drops of HCl (2M for CT, FC and 0.04 M for KC) and the volume was
filled up to 50 ml in a volumetric flask, then, the recommended procedures
for the determination of KC and CT were followed.The method of standard
addition was used for the accurate determination of the ketoconazole and
clotrimazole and fluconazole contents.
Cream sample solution
An accurately weighed portion of the cream or typical solution samples
equivalent to 25 mg of the drug was shaken and gently heated in ethanol
until they were completely dissolved and then filtered. The resulting clear
solution was diluted to 50 ml with ethanol and recommended procedure was
followed. The standard addition method was also employed for the accurate
determination of the drug contents.
RESULTS AND DISCUSSION
Fig. 1. electronic absorption spectra of drug (A) in chloroform and the
extracted chloroform solution of drug in the presence of excess amount of
picric acid solution (B)
Ion-pair complex extractive-spectrophotometry has been frequently used
for the quantitative analysis of many pharmaceutical compounds [34]. In this
study, we used PA and BTB as a suitable anionic dye, to form an intense yellow
color ion-pair complex with KC and CT and FC, in acidic pH, which is soluble
in chloroform and can be measured at at 410 and 415nm for KC, at 410 and
413 nm for CT and at 373 and 415 nm for FC using PA and BTB, respectively.
Absorption spectra of 2.5× 10-5 mol.L-1 of drugs (CT, KC and FC ) and
PA ion-pair complexes in chloroform are shown in Fig. 1 with a maximum
absorbance at 410 nm for CT, KC and 373 nm for FC with PA.The colorless
blanks have practically negligible absorbance. Absorption spectra of 2.5× 10-5
mol.L-1 of drugs (CT, KC and FC) and BTB ion pair complexes in chloroform
are shown in Fig.2
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J. Chil. Chem. Soc., 57, Nº 2 (2012)
Fig.4: Absorbance-mole ratio plots for KC-BTB, CT-BTB, CT-BTB and
FC-BTB systems inc.
In preliminary experiments, a number of immiscible organic solvents were
examined in order to provide an applicable extraction procedure. Chloroform
was found to be the most suitable extractant as it was observed that a single
extraction was adequate to achieve a quantitative recovery of the complex.
Despite the higher molar absorptivity of the complexes in methylene chloride,
chloroform was preferred for its selective extraction of the drug-dye complex
from the aqueous phase, mainly due to the lower volatility in comparison with
methylene chloride. It is important to mentioned that the colored ion-pair
complex in the chloroform phase is quite stable for several days. There was no
appreciable change in the absorbance or colour of the product if the order of
addition of the reactants is varied.
Moreover, the optimum volume of citrate buffer at pH =2.5 and volume of
phosphate buffer at pH =3 was investigated and 1 mL of citrate and phosphate
buffer was used for further studies.
The influence of the volume of picric acid (0.1% w/v) solution and
bromothymol blue solution on the extraction of CT, KC and FC was studied,
as well. The absorbance of drug-picric acid complex and drug-bromothymol
blue complexes in the organic phase increases with increasing amount of picric
acid and bromothymol blue in the aqueous phase. Maximum extraction occurs
when the volume of reagent added is 3 ml. A further excess of the reagent has
no considerable effect on the fraction of the complex extracted.
Fig. 2. electronic absorption spectra of drug (A) in chloroform and the
extracted chloroform solution of drug in the presence of excess amount of
Bromothymol blue solution (B)
Such pronounced spectral changes are presumably due to the occurrence of
both charge-transfer and proton-transfer during the formation of the resulting
molecular complex, as pointed out previously[36,38].
The absorbance vs. bromothymol blue or picric acid /drug mole ratio plot
obtained at 410 nm for CT, KC and 373 nm for FC with PA (Fig.3) at 413 nm
for CT, 415 nm for KC and FC with bromothymol blue is shown in Fig. 4.
The resulting mole ratio plots show a distinct inflection point at a dye-to-drug
mole ratio of about 1, emphasizing the formation of a relatively strong 1:1
molecular complex in solution. The 1:1 stoichiometry was further supported
by the method of continuous variations.
The optimum volume of the organic phase and the number of extractions
required were also studied. Maximum absorbance values were obtained by
using 5 ml of chloroform during a single stage extraction. In addition, the
extraction of drug with dye under the conditions recommended was found to
be rapid. A shaking time of 8-10 min is sufficient for the complete extraction
of the resulting 1:1 ion pair complexes. Absorbances of the separated extracts
were stable for more than 1 h. The blanks did not absorb at λmax
.
Linearity of calibration graphs
After the optimization of all the variables, the calibration graphs were
constructed for the three drugs by plotting absorbance versus concentration
in µg/mL. Beer’s law was obeyed over the concentration range 1-58, 1-60 and
3-60 µg/mL for KC, CT and FC by using picric acid indicator and 3-55, 2-50
and 5-55 µg/ mL for KC, CT and FC by using bromothymol blue. Conformity
with Beer¢s law was evident in the concentration ranges shown in Table 1.
Regression analysis of the Beer¢s law plots at λmax reveal a good correlation,
(R² = 0.981-0.995). Regression equations, intercepts, slopes and correlation
coefficients for the calibration data are presented in Table 1. The following
equations correspond to the linear ranges for KC and CT and FC with picric
acid:
KC : A=0.008C (µg/mL)+0.002, R² = 0.991, n = 6 CT : A=0.001C (µg/
mL)-0.007, R² = 0.992, n = 6
FC : A=0.0144C (µg/mL)+0.245, R² = 0.981, n = 6
The following equations correspond to the linear ranges for KC and CT
and FC with bromothymol blue:
Fig. 3: Absorbance-mole ratio plots for KC-PA, CT-PAand FC-PAsystems
in chloroform at 25 °C.
Optimization of conditions
The optimum conditions for quantitative estimation of the associated
ion-pair formed were established via a number of preliminary experiments.
The effect of pH for the quantitative extraction of KC, CT and FC with PA
in chloroform was studied over the range 1-5, as it was established by using a
proper citrate buffer. The resulting data at higher pH values show that the extent
of ion-pair extraction decreases drastically, most probably due to the decreased
amount of protonated form of drugs. At pH below 1-5, ion-pair extraction
decrease . Thus, pH 2.5 was chosen for further studies. The optimum pH value
for the quantitative extraction of of KC, CT and FC with bromothymol blue
in chloroform was studied over the pH range of 2.5-7.0. According to the
results a pH below 4.5 is recommended. At higher pH values, the fluconazole
existed mainly in its neutral form (pKa=2.03) [37]. and the extent of ion pairing
between anionic bromothymol blue and cationic fluconazole decreased, thus
decreasing the color developed in chloroform phase. A pH value of 3.0 was
used for further studies. .
KC : A=0.007C (µg/mL)-0.004, R² = 0.984, n = 6 CT : A=0.002C (µg/
mL)-0.006, R² = 0.995, n = 6
FC : A=0.015C (µg/mL)+0.218, R² = 0.993, n = 6
A= absorbance; C= drug concentration. The detection limit, calculated
following the expression a+3Sxy [35], where a=/intercept and Sxy=/error
standard deviation, was 1.32 , 0.53 and 4.1 µg/mL for KC, CT and FC with
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J. Chil. Chem. Soc., 57, Nº 2 (2012)
picric acid and 1.02, 0.41 and 3.6 µg/mL for KC, CT and FC with bromothymol blue respectively.
Quantification, accuracy and precision
The validity of the proposed procedure for the determination of the studied drugs in their pure state and in their pharmaceutical formulations, was tested by
analyzing these products using the proposed procedure and the official method [33]. The results obtained for pure drugs (Table 1) were reproducible with low
relative standard deviations and the mean recoveries were comparable to these obtained using the official method (Table 2) for each of the studied drugs. The
results of analysis obtained for pharmaceutical forms obtained by the proposed and the official methods are in good agreement.
Table 1. Analytical data in the determination of the studied drugs using the proposed methods(n=6).
Parameters
Ketoconazole
Clotrimazole
Fluconazole
PA
BTB
415
PA
BTB
413
PA
BTB
415
Wavelengthsλmax(nm)
410
410
373
Linear range (µg/mL)
1-60
3-55
1-58
2-50
3-60
5-55
Molar absorptivity
(L (L.mol-1cm-1×10 3)
correlation coefficient(R²)
3.4
0.991
4.8
0.994
9.3
0992
9.9
0.995
3.2
0.981
4.8
0.993
Detection limit (µg/mL)
Quantitation
0.53
1.76
0.41
1.37
1.32
4.40
1.02
3.40
4.1
13.66
3.6
12.00
limit(µg/mL)
R.S.D%*(Relative
standard deviations)
1.72
1.85
1.87
1.5
1.8
1.71
1.77
1.91
2.05
RE%(Relative error)
1.7 6
1.56
2.02
* confidence limit (95%), 6 replicate measurements.
Effects of interference
determination of the fluconazole in biological fluids, it was applied to the
recovery of the fluconazole from a blood serum sample. The recovery of the
fluconazole using the proposed method and the standard addition technique[12]
was found to be 84 % ± 4.
The effects of common excipients that often accompany the studied drugs
in pharmaceutical dosage were tested for possible interference in the assay. An
attractive feature of the procedure is its relative freedom from interference by
the usual tablet diluents and excipients such as talc, sucrose, starch, gelatin,
and magnesium stearate. Amounts far in excess of their normal occurrences in
dosage forms were added, and no effects due to these excipients were noted in
the experimental procedure. Moreover, no interference due to yhe degradation
products of the studied drugs was observed.
ACKNOWLEDGMENTS
Financial support from the Research Affairs of Guilan University is
acknowledged. The authors are grateful to the Behvazan Pharmaceutical
Company, ( Rasht industrial city, Iran) for the donation of pure drugs and its
preparations.
Application to dosage forms
In order to establish the validity of the proposed analytical methods, the
assay of KC, CT and FC in their formulations were carried out on proprietary
drugs. The same samples were analyzed by the official non-aqueous
potentiometric method (Table 2) [32,33]. The recovery experiments shown in
Table 2 indicated a good agreement between the KC and CT and FC contents
determined by the proposed and official methods as well as the declared amount
of drug in the preparations used. This is indicative of non-interference of the
other ingredients and the excipients, which are present in the formulations.
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