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DETERMINATION OF TERBINAFINE
HYDROCHLORIDE BY ION-PAIR REVERSED
PHASE LIQUID CHROMATOGRAPHY
MARINELA FLOREA, CORINA-CRISTINA ARAMĂ, CRINAMARIA MONCIU
Department of Analytical Chemistry, Faculty of Pharmacy,
6th Traian Vuia str, Bucharest 2, 020956
*corresponding author: florea.marinela@gmail.com
Abstract
The present paper presents an ion-pair reversed phase liquid chromatographic
method (IP-RP-HPLC) for the assay of terbinafine. A C18 column was used as stationary
phase, and the mobile phase consisted of an aqueous acid solution (pH 2.0, adjusted with
orto-phosphoric acid) of sodium 1-heptanesulfonate (0.2%), as ion-pairing reagent, and
acetonitrile in a 60/40 (v/v) ratio. The detection wavelength was 220 nm. The method is
simple, accurate and reproducible. It was successfully applied for the assay of terbinafine in
pharmaceutical dosage forms (tablets).
Rezumat
Lucrarea de faţă prezintă o metodă de dozare a terbinafinei prin cromatografie cu
faze inversate, prin perechi de ioni. Ca fază staţionară s-a folosit o coloană C18, iar ca fază
mobilă, un amestec de soluţie apoasă, acidă (pH 2,0 creat de acid ortofosforic), de 1heptansulfonat de sodiu (0,2%), ca formator de perechi de ioni, şi acetonitril, în raport de
combinare 60/40 (v/v). Detecţia s-a realizat la lungimea de undă 220 nm. Metoda este
simplă, exactă şi reproductibilă, şi a fost aplicată cu bune rezultate la dozarea terbinafinei
din forme farmaceutice dozate (comprimate).
Keywords: terbinafine; ion pairs; IP-RP-HPLC
Introduction
Terbinafine hydrochloride, (E)-N-(6,6-dimethyl-2-hepten-4-ynyl)-Nmethyl-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 epoxidase (EC: 1.14.99.7) which is an enzyme present in
fungal and mammalian cell systems important in ergosterol biosynthesis [1].
There are only a limited number of techniques described for the
determination of terbinafine in its pharmaceutical formulations including
HPLC [2-4], UV-spectrometric methods [5-7], colorimetric methods [8,9],
agar diffusion method [10], capillary zone electrophoresis [11], and
electrometric method [12].
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83
We developed an ion-pair reversed
phase chromatographic method, with UV
C(CH3)3
CH3
detection for the assay of terbinafine. As
N
ion-pair forming reagent, the mobile phase
contains sodium 1- heptanesulfonate. The
major factors affecting the chromatographic
separation were studied, including the
mobile phase composition and pH, the ionFigure 1
pair forming agent concentration and
Chemical structure of terbinafine
column temperature.
The proposed method allows the quantitative determination of
terbinafine hydrochloride in pharmaceutical dosage forms.
Materials and methods
Reagents and equipment
All the reagents used were HPLC grade (acetonitrile, sodium 1heptanesulfonate, orto-phosphoric acid – Scharlau).
All the experiments were performed using a VARIAN – Prostar
equipment, with PDA Prostar 330 detector (detection wavelength was 220 nm
and the spectra recorded in the 200 – 400 nm range). An Inertsil 5 ODS 2, 250 x
4.6 mm i.d. (Chrompack Varian, USA), with 5 µm particles was used, at 25°C.
Sample injection was performed manually, using a Rheodine 7125
injector, with a 20 µL sample loop.
Terbinafine hydrochloride was a reference standard (SigmaAldrich, Germany).
For the assay of terbinafine in dosage forms, Terbinafine 250 mg®
(Actavis Group HF., Iceland) tablets were used.
The standard stock solution of terbinafine hydrochloride was prepared
by dissolving 10.68 mg of reference standard substance in 10 mL mobile phase
and diluted to 50.0 mL with the same solvent. Dilutions with concentrations
between 8.54 and 59.80 µg/mL were prepared using mobile phase as diluent.
The sample solutions were prepared from approx. 0.4 g powdered
tablets (equivalent to 250 mg terbinafine hydrochloride). The active
substance was extracted with 100 mL solvent A; 1 mL solution obtained
after filtration was diluted to 100 mL solvent A (solution 1) and 0.5 mL
solution 1 was further diluted to 50 mL with the same solvent.
Method
The mobile phase was a mixture of an aqueous solution containing
0,2% sodium 1-heptanesulfonate, as ion-pair forming reagent, adjusted to pH
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2,0 with orto-phosphoric acid (solvent A), and acetonitrile (solvent B), as
organic modifier, in a ratio 60/40 (v/v). The flow rate used was 1 mL·min-1.
The detection wavelength was 220 nm (with full spectra recorded
between 200 and 400 nm).
The column conditioning was performed using mobile phase, with
a 1 mL·min-1 flow rate, for 30 minutes. Stationary phase regeneration was
made using a mixture of acetonitrile/water 60/40 v/v, passed through the
column with a 1 mL·min-1 flow rate, for 60 minutes.
Results and discussion
In order to design a reliable IP-RP-HPLC method for terbinafine
assay, we studied the effect of varying certain chromatographic parameters,
such as: mobile phase content in organic modifier, pH, concentration of ionpair forming reagent, column temperature.
Organic modifier percentage
The effect of the organic modifier was studied using various mobile
phases consisting of an aqueous acid solution (pH 2.0) of sodium 1heptansulfonate (0.2%), and acetonitrile. The content in acetonitrile was
varied over the range 50 to 35%. The results obtained (figure 2) show a
decrease of retention with the increasing of acetonitrile percentage, from
47.213 minutes, for 35% acetonitrile to 7.08 minutes, for 50% acetonitrile.
A mobile phase with 40% resulted in a convenient retention, k’= 20.89.
Mobile phase pH
The mobile phase pH value is one of the parameters with major
impact on the separation of ionizable molecules. Considering the pKa value
7.10 for terbinafine [13], to maintain it in protonated form, it is necessary to
work at lower pH levels.
Figure 2
The influence of organic modifier percentage on the retention time of terbinafine
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85
The effect of pH on retention was studied by changing the pH of
solvent A over the range 2.0 – 3.5 (in a 60/40 v/v mixture). The results
(figure 3) obtained show that the retention time increased with increasing
eluent pH. A convenient retention time, maximum peak area and good
symmetry peak was obtained at pH 2.0.
Figure 3
The influence of eluent pH on the retention time of terbinafine
Influence of the concentration of the ion-pair forming reagent
The effect of sodium 1-heptanesulfonate concentration was studied by
measuring the retention time of terbinafine, using a mobile phase consisting of
an aqueous solution (pH 2.0) containing variable amounts of the ion-pair
forming reagent (between 0.1 – 0.4%), and acetonitrile in 60/40 v/v ratio. The
retention increased with increasing of the ion-pair forming agent concentration,
from 16.667 minutes, for 0.1% ion-pair forming reagent, to 35.584 minutes, for
0.4% ion-pair forming reagent. A convenient retention time, 20.98 minutes,
was obtained at 0.2 % ion-pair forming agent concentration.
Influence of temperature
The effect of temperature was studied over the range 25 – 45°C.
The best results were obtained at 25°C. Higher temperatures resulted in a
decrease of the retention time, but with poor peak symmetry (figure 4).
Considering the results obtained, the optimum experimental
conditions for the IP-RP-HPLC assay of terbinafine are: the mobile phase
consisting of an aqueous solution of sodium 1-heptanesulfonate 0.2%,
adjusted to pH 2.0 with orto-phosphoric acid, and acetonitrile in a 60/40
(v/v) ratio, and a column temperature of 25°C.
The validation of the newly established method was performed
[14], and the results obtained are summarized up in table I.
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The selectivity of the method was proved checking the influence of
excipients used in tablets, such as sodium amidonglicolate, microcrystalline
cellulose, magnesium stearate, hydroxypropylmethyl cellulose and Aerosil.
The chromatograms showed no interference, proving the selectivity of the
method against excipents studied.
Analysis of pharmaceutical preparations
The proposed method was successfully applied for the
determination of terbinafine in commercial tablets. The results obtained are
shown in table II.
Figure 4
The influence of temperature on the retention time of terbinafine
Validation criterion
Precision
Linearity (figure 5)
Range
Detection limit***
Quantification limit***
Accuracy****
Table I
Summary of validation data obtained for the
new IP-RP-HPLC method for the assay of terbinafine
Parameter
Results
Repeatability*
RSD = 0.90%
Reproducibility**
RSD = 0.92%
Regression parameters
Y = 11536X - 13775
Correlation coefficient
0.9994
8.54 – 59.80 µg/mL
1.21 µg/mL
3.67 µg/mL
100.05 ± 0.63
* from 9 consecutive injections of standard solution
** in two different days
*** based on the standard deviation of the response and slope
**** in the range 80 – 120% from the declared amount
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800000
700000
600000
Peak area
500000
400000
300000
200000
y = 11536x - 13775
100000
0
0
10
20
30
40
50
60
70
Concentration (g/mL)
Figure 5
Linear correlation between chromatographic peak area
and concentration of terbinafine
Dosage
form
Terbinafine
Label content
250 mg
terbinafine
hydrochloride
*average of 9 measurements
Table II
Results obtained on terbinafine assay from tablets
Results*
Confidence interval
mg/tb
%
254.5
99.83
99.83 ± 0.29
Conclusions
A simple IP-RP-HPLC method for terbinafine assay was
developed. An Inertsil 5 ODS 2 column was used with a mobile phase
consisting in a mixture of an aqueous solution with 0.2% sodium 1heptanesulfonate as ion-pairing reagent, adjusted to pH 2.0 with ortophosphoric acid, and acetonitrile, in a 60/40 (v/v) ratio. The detection
wavelength was 220 nm. The method is simple, accurate and reproducible.
It was successfully applied for the assay of terbinafine in pharmaceutical
dosage forms (tablets).
Acknowledgements
This research was supported by a CNCSIS, PNII- Human
Resources –TD project (No. 18/01.10.2007).
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1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
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Manuscript received: 30.08.2008