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SPECTROPHOTOMETRIC DETERMINATION
OF TERBINAFINE THROUGH ION-PAIR
COMPLEX FORMATION WITH METHYL
ORANGE
MARINELA FLOREA*, CRINA-MARIA MONCIU
Department of Analytical Chemistry, Faculty of Pharmacy,
6th Traian Vuia str, Bucharest 2, 020956
*corresponding author: florea.marinela@gmail.com
Abstract
Considering basic drugs capacity to generate ion-pairs with azoic dyes there have
been investigated the experimental conditions in order to develop new spectrophotometric
method for the terbinafine assay in bulk and in dosage forms. The method is based on ionpair formation of terbinafine with methyl orange at pH 2.6. The resulted yellow ion-pair
complex was extracted with chloroform and the absorbance was measured at 422 nm. The
combination ratio of the ion pair was found to be 1:1 by Job’s method.The conditional
stability constant of the complex has been calculated. The proposed method was
successfully extended to dosage forms (tablets).
Rezumat
Având în vedere capacitatea substanţelor medicamentoase cu funcţie bazică de a
forma asocieri ionice cu indicatori azoici, au fost studiate condiţiile experimentale în
vederea instituirii unei noi metode de dozare a terbinafinei ca atare şi din forme
farmaceutice. Metoda are la bază proprietatea acesteia de a forma perechi de ioni cu
indicatorul metiloranj, la pH 2,6. Asocierea ionică este extractibilă în cloroform şi prezintă
un maxim de absorbţie la lungimea de undă de 422 nm. Raportul de combinare, precum şi
constanta condiţională de formare au fost determinate utilizând metoda Job. Metoda
propusă a fost aplicată cu succes la determinarea terbinafinei din comprimate.



ion-pairs
terbinafine
methyl orange
INTRODUCTION
Terbinafine hydrochloride, (E)-N-(6,6-dimethyl-2-hepten-4-ynyl)N-methyl-1-naphtalene methanamine hydrochloride (Fig.1), is an allylamine
derivative with antifungal activity. The drug has been found to be a potent
inhibitor of squalene epoxidasis which is an enzyme present in fungal and
mammalian cell systems, important in ergosterol biosyntesis [1].
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CH3
C(CH3)3
N
Figure 1
Chemical structure of terbinafine
Previously, the drug has been determined in biological fluids
(plasma, urine), tissues, nails and hair by HPLC [2-8], in tablets and creams
by HPLC [9-10] and in various dosage forms using UV-spectrophotometry,
spectrodensitometry [11-13]. The only colorimetric method available for the
determination of terbinafine is based on a charge transfer complex formation
with iodine [14].
Ion pair formation, initially investigated by the physical chemistry
has been found extremely interesting for the chemical analysis, including
pharmaceutical analysis [15]. The property of pharmaceutical substances to
form ionic association complexes is suitable for their assay using extractive,
titrimetric, spectrometric and chromatographic methods [16].
The present paper describes an extraction spectrophotometric
method for the determination of terbinafine hydrochloride (TBF) through
ion-pair complex formation with methyl orange (MO). The proposed
method was applied for the determination of terbinafine in bulk and in
pharmaceutical formulation. The proposed method is simple and suitable for
routine determination of terbinafine, provides economic procedures and it is
less time consuming.
MATERIALS AND METHODS
Instrument
All absorption spectra were registered using a UV-Vis Kary 100
Bio spectrophotometer (Varian Inc.) with a scanning speed of 600 nm/min,
equipped with 10 mm matched quartz cells. A Metrohm 716 DMS Titrino
was used for checking the pH of buffer solutions.
Material and reagents
All chemicals and reagents were of analytical grade and all
determinations were performed using bidistilled water.
- Terbinafine reference standard (Hetero Labs Limited, India)
- A stock solution was prepared by dissolving 0.1206 g terbinafine
reference standard in methanol and completed to 100 mL with the
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-
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395
same solvent. The stock solution was stored at 4oC. Diluted standard
solutions were prepared from stock solution with a methanol/water
(40/60 v/v) solution
Methyl orange (Fluka) solution was prepared by dissolving 0.2204 g
methyl orange in water and completed to 250 mL with the same solvent
HCl 10-2M, 10-1M, were prepared by diluting a 38% HCl (Merck) solution
Series of Britton-Robinson buffer solutions (pH 2.5 and 3) were
prepared by adding proper amounts of NaOH 10-1M to a CH3COOH
0.02M/H3PO4 0.02M/B(OH)3 0.02M solution
Series of potassium hydrogen phthalate-HCl buffer solutions (pH 3.5
and 4) were prepared by dissolving 1.020 g potassium hydrogen
phthalate in water and completed to 100 mL with the same solvent
and adjusting the pH by addition of 10-1M HCl
Chloroform (Merck)
Methods
Procedure for calibration curves
Into a series of 250 mL separating funnels there were measured
accurate aliquots of (2–5.5 mL) the standard drug solution - prepared by
diluting 7 mL stock solution in methanol/water (40/60 v/v), 9 mL HCl 10-2 M,
3 mL dye solution and water to 20 mL. The mixture was extracted three
times with 5 mL chloroform by shaking for 1.0 minute, then allowed to
stand for clear separation of the two phases. The chloroform layer was
passed through anhydrous sodium sulphate. The collected extract was
transferred and brought to 50 mL with chloroform in a volumetric flask. The
absorbance of the yellow colored complexes was measured at 422 nm,
against corresponding reagent blank prepared in the same manner except
addition of the examined drug (Fig. 2). The calibration graph is linear over
the concentration range 6 – 17 μg/mL (Fig. 3).
Procedure for the assay of dosage forms
The contents of 10 tablets (Terbisil® containing 250 mg terbinafine
corresponding to 281.25 mg terbinafine hydrochloride – Gedeon Richter)
were weighed, ground into a fine powder and mixed. An accurately weighed
portion of the powder equivalent to one tablet was transferred into a 50 mL
volumetric flask. The volume was made up to the mark with methanol.
After 20 min of mechanically shaking, the solution was filtrated in a 50 mL
calibrated flask through Whatman No. 42 filter paper. Necessary amounts of
filtrate were diluted to 100 mL with a methanol/water (40/60 v/v) solution
and the same procedure was applied as described for the calibration curve.
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1,2
Absorbance
1
0,8
0,6
0,4
0,2
y = 0,0617x - 0,0415
0
0
5
10
15
20
Concentration (m g/L)
Figure 2
Absorbance for TBF-MO ion-pair method
Figure 3
Calibration curve for TBF-MO
RESULTS AND DISCUSSION
The optimization of the method was carefully studied to achieve
complete reaction formation, highest sensitivity and maximum absorbance.
In the proposed method, some variables in the reaction conditions were
studied and the influence of these variables on the reaction was tested.
Selecting of the optimum wavelength
Absorption spectra of pure terbinafine in methanol/water (40/60
v/v) solution was obtained at 200 – 800 nm, against blank. Absorption
spectra of methyl orange in water was recorded in the same manner.
The absorption spectra of the ion-pair complexes, formed between
terbinafine and methyl orange was measured at 220-800 nm against the
blank solution (Fig.4). The yellow chloroformic extracts show maximum
absorbance at 422 nm. The measurements were made at this wavelength.
Figure 4
Absorption spectra of (1) methyl orange (12.1 mg/L) in aqueous solution, (2)
terbinafine (12 mg/L) in methanol / water (40/60 v/v) solution, (3) TBF-MO ionpair (12 mg/L) in chloroform
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Effect of pH on the ion-pair formation
The effect of pH was studied by extracting the ion-pair at various
pH values obtained using HCl 10-1M (pH 1.5), HCl 10-2M (pH 2.0 – 2.6),
Britton-Robinson buffer solutions (pH 2.5 and 3) and potassium hydrogen
phthalate-HCl buffer solutions (pH 3.5 and 4). The maximum color intensity
and highest absorbance value was observed at pH 2.6 created with HCl 10-2 M
(Fig. 5). Further, 9 mL HCl 10-2M were used to obtain maximum
absorbances and reproducible results.
0,8
0,7
Absorbance
0,6
0,5
0,4
0,3
0,2
0,1
0
1,5
2
2,6
3
3,5
4
pH
Figure 5
Effect of pH on the formation of ion-pair
Effect of reagent concentration
The effect of the reagent concentration was studied by measuring the
absorbances of solutions containing the same concentration of terbinafine (12
mg/mL) and varied amounts of the methyl orange 0.1% solution.
Maximum color intensity of the complex was achieved with 3.0
mL of dye solution. Larger volumes of reagent had no pronounced effect on
the ion-pair complex formation. The volume of 3.0 mL of methyl orange
solution was used for the further determinations.
Stoichiometric relationship
Job’s method of continuous variation [16] of equimolar solutions
was employed; 5x10-4M, 4.5x10-4M, 4x10-4M standard solution of drug and
5x10-4M, 4.5x10-4M, 4x10-4M solution of methyl orange were used. Series
of solutions were prepared, in which the total volume of drug and reagent
was 10 mL and the ion-pair complex was extracted using the described
experimental conditions. The absorbance was measured at 422 nm against
blank reagent. The results indicate that 1:1 (drug: dye) ion-pairs are formed
(Fig. 6).
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Conditional stability constant (Kf) of the ion-pair
The stability of the ion-pair complex was evaluated. The
conditional stability constant (Kf) of the ion-pair formed by terbinafine with
methyl orange, was calculated from the continuous variation data using the
following equation [17]:
A
Am
Kf 
A n 2
[1 
] C (n) n
M
Am
where A is the maximum observed absorbance and Am is the absorbance
value when all the amount of drug is associated. CM is the mole
concentration of drug at the maximum absorbance and n is the combination
ratio of the ion-pair considered. The log Kf value obtained for the TBF-MO
ion-pair, on five determinations (α = 0.05) is 5.28 ± 0.25.
Absorbance
0.8
0.6
0.4
0.2
0
0.3
0.4
0.5
0.6
0.7
[TBF]/[TBF]+[MO]
Figure 6
Continuous variation plots
(●)solutions of terbinafine and methyl orange 4x10 -4M
(■) solutions of terbinafine and methyl orange 4.5x10-4M
(▲)solutions of terbinafine and methyl orange 5x10 -4M
Analytical data
In described experimental conditions for terbinafine assay, standard
calibration curve was constructed by plotting absorbances versus
concentrations. The linear regression equation, molar absorbtivity, standard
deviation, correlation coefficient and linearity range for the proposed
method are listed in Table I.
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Table I
Regression characteristics of the proposed method
Parameters
Values
λmax
422 nm
Beer’s law limits
6 – 17 μg/mL
Molar absorptivity
18935,67
(L·mol-1·cm-1)
Linear regression equation (y)
0.0617x – 0.0415
Correlation coefficient
0.99977
Analysis of pharmaceutical preparations
The proposed method proved to be selective against excipients
used in the chosen dosage form and has been successfully applied for the
determination of terbinafine in commercial tablet. The obtained results are
shown in Table II.
Dosage
form
Terbisil®
Label content
281.25 mg
terbinafine
hydrochloride
Table II
Results obtained on terbinafine assay from tablets
Results*
RSD%*
Confidence
interval
mg/tb
%
280.63
99.78
1.22
99.78 ± 1.28
*average of 6 measurements
CONCLUSIONS
A significant advantage of the extractive spectrophotometric
methods is that they can be applied for the assay of individual substances in
a complex mixture.
The method proposed for the assay of terbinafine is simple and has
a low cost. The reagents used in the proposed method are cheaper, readily
available and the procedures do not involve any critical reaction conditions
or tedious sample preparation.
The developed method may be used for routine and quality control
analysis of the investigated drugs in dosage forms.
Acknowledgements
This research was supported by the Romanian Ministry of
Education, Research and Youth - CNCSIS, PNII- Human Resources –TD
project (No. 18/01.10.2007).
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