A Stability Indicating High Performance Liquid Chromatographic Assay (HPLC) for the Determination of Terbinafine Hydrochloride in Bulk Drug Substance

Hamsa Kassem , Mohamed Amer Almardini, Heba Ghazal

Faculty of Pharmacy, Damascus University, Damascus, Syria.

*Corresponding Author E-mail: . Kassemhamsa@yahoo.com

ABSTRACT:

A simple, accurate, and stability- indicating reversed phase high-performance liquid chromatography (HPLC) method was developed and validated for the quantitative determination of terbinafine hydrochloride in bulk drug substance.

Chromatographic separation was performed using C18 column and an isocratic mobile phase consisting of methanol and acetonitrile (60:40, v/v) with (0.15% triethylamine and 0.15% phosphoric acid) at a flow rate of 0.4 ml/ min. The eluents were monitored by UV photodioide array detector at wavelength of 224nm.

The method was statistically validated for linearity, specificity, accuracy, precision, limit of detection, limit of quantification and robustness. The stability – indicating nature of the method had been proved by subjecting terbinafine hydrochloride to acidic hydrolysis, basic hydrolysis, oxidation, thermal and photo degradation. Degradation products resulted from the stress studies did not interfere with the detection of the drug and thus the assay is stability- indicating.

KEYWORDS: HPLC, stability-indicating, Terbinafine Hydrochloride

1- INTRODUCTION:

Terbinafine hydrochloride is a synthetic potent antimycotic agent of the allylamine class, (fig 1). It selectively inhibits fungal squaline epoxidase causing a fungicidal action due to the intracellular accumulation of the toxic sterol squaline, it also exerts a fungistatic action by depletion of ergosterol^1,
2.

Terbinafine hydrochloride now is the drug of choice in dermatophyte nail and skin infections because of its fungicidal mode of action over a short treatment duration.³

Fig (1) chemical structure of terbinafine hydrochloride

Stability testing forms an important part of the process of drug product development. The purpose of stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of variety of environmental factors such as temperature, humidity and light and enables recommendation of storage conditions, rest periods and shelf lives to be established ⁴. The main aspects of drug products that play an important role in shelf life determination are assay of active drug and degradation products generated during the stability study and these assays need to be performed by using stability-indicating methods as recommended by ICH guide⁵.

Terbinafine hydrochloride is officially listed in the British pharmacopoea⁶ and the USP⁷ .

The literature shows several methods to determine terbinafine hydrochloride in dosage forms and biological fluids, these methods include microbiology^{8, 9},electrochemistry¹⁰, spectrophotometry¹¹-¹⁵, titrimetry¹², high performance thin layer chromatography (HPTLC)^{16, 13}, high performance liquid chromatography (HPLC)^17-25and gas chromatography (GC)^26,27. However, only few methods described the determination of terbinafine hydrochloride in bulk drug substance^{12, 6, 7} and most of these methods used acid-base titration so they cannot be stability-indicating. Therefore, the aim of the present study was to develop a simple and sensitive stability-indicating reversed-phase HPLC method for analyzing terbinafine hydrochloride in bulk substance.

2- MATERIALS AND METHODS:

2ـ1 Materials:

Reference standard (RS) of terbinafine hydrochloride (assigned purity 99, 9%) was kindly provided by Al-Fares pharmaceutical Company (Damascus ـ Syria) which offered also samples of terbinafine hydrochloride bulk drug and all solvents and reagents which were needed in the study.

Methanol and acetonitrile were HPLC grade (Merck, Germany), triethylamine and phosphoric acid 85% were of pure analytical grade.

HPLC apparatus VWR Hitachi La Chrom Elite model with auto sampler and diode array detector.

2ـ1ـ1 Standard solution preparation:

Approximately (10) mg of terbinafine hydrochloride (RS) accurately weighed was transferred to (100) ml volumetric flask and diluted with methanol to the volume. Then (10) ml of this solution was transferred to (100) ml volumetric flask and diluted to the volume with methanol to make the final concentration (0.01) mg/ml of terbinafine hydrochloride.

2-1-2 Sample solution preparation

The previous steps were repeated using terbinafine hydrochloride bulk drug sample instead of (RS) sample.

2-1-3 Stock solution preparation for stress stability study:

The previous steps were repeated using (100) mg of terbinafine hydrochloride instead of (10) mg to make the final concentration of this solution (0.1) mg/ml of terbinafine hydrochloride.

2ـ2 Methods:

2ـ2ـ1 chromatographic conditions:

The chromatographic conditions which were used in the method are displayed in table (1)

Table (1) Chromatographic conditions of HPLC method

Column	Intersil :L1ODS ( 150×4.6mm) particle size 5 µm
Mobile phase	Methanol - acetonitrile (60:40, v/v) with (0.15% triethylamine and 0.15% phosphoric acid ) pH=7.68
Detection and wavelength	Photodiode array detector monitored at 224 nm
Flow rate	0.4 ml/minute
Injection volume	10 µl
Oven temperature	25°C

2ـ2ـ2 Preparation of solutions for validation study:

2ـ2ـ2-1 Solutions for linearity:

Five sequential concentrations were prepared containing 50%, 75%, 100%, 125% and 150%, respectively of the standard solution.

2-2-2-2 Solution for specificity:

Terbinafine hydrochloride standard solution spiked with methanolic solutions of impurities A, B, C and D was prepared (impurities were defined by the BP).

2-2-2-3 Solutions for accuracy:

Nine samples of terbinafine hydrochloride bulk substance covering the working range 50% to 150% were prepared.

2ـ2ـ2ـ4 Solutions for precision:

Nine samples of terbinafine hydrochloride bulk substance covering the working range 50% to 150% were prepared.

2ـ2ـ2ـ5 Solutions for robustness:

Samples of terbinafine hydrochloride bulk solutions containing 100% of standard solution were prepared.

2-2-3 Preparation of solutions for stress stability studies:

2-2-3-1 Solution for acidic hydrolysis:

(10) ml of stock solution was treated with 0.5 N HCL and heated in a water bath for an hour. Then the solution was allowed to cool and neutralized to pH=7 by 0.5N NaOH and the concentration was modified to 0.01mg/ml.

2-2-3-2 Solution for basic hydrolysis :

(10) ml of stock solution was treated with 0.5N NaOH and heated in a water bath for an hour .Then the solution was allowed to cool and neutralized to pH=7 by 0.5N HCL and the concentration was modified to 0.01mg/ml.

2-2-3-3 Solution for oxidative degradation:

(10) ml of stock solution was treated with H ₂O ₂10 % and stirred well for half an hour then the concentration was modified to 0.01mg/ml.

2-2-3-4 Solution for thermal degradation:

(10) ml of stock solution was incubated at a temperature of 60°C for a week and then the concentration was modified to (0.01) mg/ml.

2-2-3-5 Solution for photodegradation:

(10) ml of stock solution was exposed to white light for a week at a temperature of 25°C and then the concentration was modified to (0.01) mg/ml.

3ـ RESULTS AND DISCUSSION:

3ـ1 Chromatographic conditions:

It was reported that lipophilic compounds with alkalyted nitrogen can easily be separated on reversed ـphase column using acetonitrile as a mobile phase and if triethylamine is added, peak tailing due to analyte interaction with free silanol groups could be avoided. In our study a mobile phase consisting of water – acetonitrile (60 :40, v/v ) with (0.15% triethylamine and 0.15% phosphoric acid ) was tested first and the chromatography was performed on C18 column and the eluents were monitored with photodiode array UV detector at 224 nm.

By using these chromatographic conditions, a peak of terbinafine hydrochloride appeared at a retention time of (4) minute but this peak was not suitable for analysis.

The composition of mobile phase was modified using methanol instead of water, then the peak of terbinafine hydrochloride appeared at a retention time of (8.5) minute and this peak was suitable for analysis, Fig (2).

Column efficiency was determined by calculating the number of theoretical plates, tailing factor and the relative standard deviation RSD for five sequential injections of the standard. The number of theoretical plates was 4729 (more than 2500) and the tailing factor was 1.65 (it did not exceed 3.0) and RSD for five sequential injections of the standard was 0.65 (it did not exceed 1.5%).

3-2 Forced degradation studies:

Forced degradation studies were performed on terbinafine hydrochloride to prove the stability indicating property of the method²⁸. The stress conditions employed for degradation study of terbinafine hydrochloride included acidic hydrolysis, basic hydrolysis, oxidation, thermal and photo-degradation. The results of these studies and the chromatograms of degraded samples are displayed in figures (3, 4, 5, 6) and table (2). The results indicate that the method can differentiate between terbinafine hydrochloride and its degradation products.

Table (2) Results of degradation studies on terbinafine hydrochloride.

Degradation condition	Result of degradation
Acidic hydrolysis	27%
Basic hydrolysis	44%
Oxidation	50%
Thermal degradation	20%
Photo degradation	No degradation

Fig (7) calibration curve of terbinafine hydrochloride.3-3

Validation of analytical method:

Validation was based on the requirements of the USP⁴ by studying the following parameters: linearity, specificity, precision, accuracy and recovery, limit of detection, limit of quantification and robustness.

3-3-1Range and linearity:

A series of terbinafine hydrochloride concentrations containing 50% -150% of the standard concentration (0.01 mg/ml) was injected in HPLC apparatus and the analyses were performed triplicate. Calibration curve of peak areas versus concentrations was plotted (fig 7) and it showed good linearity. The equation of the regression line was:

y= 1367706200x -594568 with a correlation factor of 0.9993.

3-3-2 Specificity:

Specificity is the ability of the method to measure the analyte response in the presence of interference including impurities and degradation products. The specificity of the developed method was carried out in the presence of its impurities namely; A, B, C and D as defined by the BP. The results are displayed in fig (8) and they showed no interference and good resolution between the peaks of the impurities and the drug.

Also applying forced degradation studies on terbinafine hydrochloride demonstrated that the method could differentiate between terbinafine hydrochloride and its degradation products and this confirm the specificity of the method.

3-3-3 Accuracy:

Nine samples of terbinafine hydrochloride bulk substance covering the working range 50-150% were injected into HPLC apparatus. The practical concentration for each sample was calculated from the equation of the calibration curve then the percentage for each sample was calculated according to the formula:

The percentage = (practical concentration / theoretical concentration).100

then the relative error (RE) was calculated according to the formula :

RE % = (A - P) / P .100

Where A is the average of the percentages of the nine samples, P is the purity of reference standard and it did not exceed 2%. The results of accuracy are displayed in table (3).

Table (3) Accuracy of HPLC method.

The average of the percentages	101.90%
Purity of reference standard	99.96%
RE %	1.95

3-3-4 Precision:

The precision of the method was determined with respect to both repeatability (intraday) and intermediate precision (intraday) studies.

3-3-4-1 Repeatability:

Nine samples of terbinafine hydrochloride bulk substance covering the working range 50% to 150% of the standard solution were injected in HPLC apparatus in the same day. The RSD of the results was calculated and it did not exceed 2% indicating good repeatability.

3-3-4-2 Intermediate precision:

Nine samples of terbinafine hydrochloride bulk substance covering the working range 50% to 150% of the standard solution were injected in HPLC apparatus in the same day by different analysts. The RSD of the results was calculated and it did not exceed 3%.

The results of repeatability and intermediate precision are displayed in table (4).

Table (4 ) Repeatability and intermediate precision of HPLC method.

	Repeatability	Intermediate precision
Number of samples	9	9
Average of test results	98.61%	98.66%
Relative standard deviation RSD	1.98 %	1.76%

3-3-5 Limit of detection and limit of quantification:

The limit of detection (LOD) and the limit of quantification (LOQ) were determined based on signal to noise ratios and were based on analytical response of 3 and 10 times the background noises, respectively. The (LOD) was found to be (0.1) µg/ml and the (LOQ) was found to be 0.3µg /ml.

3-3-6 Robustness:

Robustness is the capacity of the method to remain unaffected by small and deliberate variations in method parameters .In our study flow rate was increased from 0.4 ml/min to 0.6 ml/min and this small change in flow rate did not cause important differences on assay value and relative retention time.

The results are displayed in table (5).

Table (5) robustness of HPLC method

	Flow rate 0.4 ml / minute	Flow rate 0.6 ml / minute
Assay value	99.66%	100.85%
Relative retention time	1	1.001

4 - CONCLUSION:

The HPLC method developed in this study for analyzing terbinafine hydrochloride in bulk substance is specific, sensitive, accurate, precise, robust and stability- indicating because it can differentiate between terbinafine hydrochloride and its degradation product. Moreover, it offers advantages in terms of simplicity in mobile phase, isocratic mode of elution, easy sample preparation and comparative short run time encouraging its application in stability studies.

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Received on 30.09.2013 Modified on 20.10.2013

Research J. Pharm. and Tech. 7(1): Jan. 2014; Page 23-28