INTRODUCTION:

Terbinafine hydrochloride (TFH) isanallylamine derivative ^1,13 and synthetic antimycotic agent from a new class of compounds, the allylamines ². Malaria is the world’s most important parasitic infection, ranking among the major health and developmental challenges for the poor countries of the world ³. Terbinafine hydrochloride (TBH) is a new antifungal agent widely used for the treatment of many contagious diseases⁴. Terbinafine Hydrochloride is acts by inhibiting squalene epoxidase, thus blocking the biosynthesis of ergosterol, an essential component of fungal cell membranes ^5-7.

1- aphthalenemethanamine, n-(6, 6-dimethyl-2-hepten-4-ynyl)-n methyl-, (E)-, hydrochloride, having molecular formula C₂₁H₂₅N.HCl and molecular weight 293. THF is very slightly or slightly soluble in water, freely soluble in anhydrous ethanol, methanol and in methylene chloride, slightly soluble in acetone . Forced degradation studies are used to identify reactions which may occur to degrade a processed product. Usually conducted before final formulation, forced degradation uses external stresses to rapidly screen material stabilities ⁸.

UV-spectrophotometry is one of the simplest techniques routinely used in pharmaceutical quality control laboratories because of its sensitivity, speed, fair selectivity, low cost and ease of performance. However, the literature on TFH is poor with regard to UV-spectrophotometric methods.⁹. Regulatory agencies recommend the use of stability-indicating methods for the analysis of stability samples. Thus, stress studies are required in order to generate the stressed samples, method development, and its validation ¹⁰. It is suggested that stress testing should include the effect of temperature, humidity, light, oxidizing agents as well as susceptibility across a wide range of pH values. It is also recommended that the analyses of stability samples should be carried out by the use of validated stability-indicating testing methods ^{11, 12}.

1. Terbinafine HCl [(E)-N-(6,6-dimethyl-2-hepten-4-ynyl)-N-methyl-1-naphthalenemethanamine hydrochloride]

2. Impurity-A [(Z)-N-(6,6-dimethyl-2-hepten-4-ynyl)-N-methyl-1-naphthalenemethanamine hydrochloride]

3. Impurity-B [(E)-N-(3-Chloro-3-propyn)-N-methyl-1-naphthalenemethanamine hydrochloride]

4. Impurity-C

[N-methyl-1-naphthalenemethanamine hydrochloride]

5. Impurity-D [(2E)-N,6,6-trimethyl-N-[(4-methylnaphthalen-1-yl)methyl]hept-2-en-4-yn-1-amine (4-methylterbinafine)]

MATERIALS AND METHODS:

Materials

Standard gift samples of Terbinafine and impurities were provided by Dr Benarji Patrudu, Associate Professor, Gitam University, Hyderabad. All the chemicals and reagents used were of analytical grade.

HPLC Chromatographic Parameters

Chromatographic separation was performed on The HPLC-PDA system used, consisted shimadzu high performance liquid chromatography with LC- 20AT pump and SPD-20A interfaced with LC solution software, equipped with a reversed phase C8 analytical column of 150 mm x 4.6 mm and particle size 5 µm (YMC-Pack ODS-AM, 120A) with flow rate of 1.0 mL/min.

An HPLC method was developed for Terbinafine and related impurities by using photo diode array detector. Terbinafine and all related impurities were injected into HPLC system by changing the different composition of Acetonitrile:Methanol: Water, pH 7.5 (adjusted with NaOH) in water. The Terbinafine and related impurities are separated in Acetonitrile: Methanol: Buffer pH 7.5::: 20:10:10 v/v/v. The absorption maxima for Terbinafine, Impurity –A, Impurity-B, Impurity –C and Impurity-D were found at 230 nm. Injection volume was set up to 20µL. By follow this analytical method conditions, Terbinafine and related impurities were separated. The representative blank and standard chromatograms were showed in Figure.1 and Figure 2.

Figure. 1. Representative chromatogram of diluent

Figure. 2. Representative chromatogram of standard

METHOD OF ANALYSIS

Assay and Related Substances –As such

Blank solution

As a blank solution, mobile phase was used.

Test solution

90.0mg of Terbinafine HCl sample were weighed in a 50ml volumetric flask, dissolved in mobile phase. 1.0 mL of this solution was transferred to a 50ml volumetric flask brought to volume with mobile phase and injected six consecutive times.

Terbinafine HCl – Effect of temperature at 80°C for 24 hours and 96 hours

Terbinafine HCl sample was stressed by exposing it to a temperature of 800C under vacuum for 24 hours and continued up to 96 hours.

Test solution- Assay

90.0mg of stressed Terbinafine HCl sample was weighed in a 50ml volumetric flask, dissolved and topped up with mobile phase. 1ml of this solution was transferred to a 50ml volumetric flask and also topped up with mobile phase and injected.

Test solution- Related Substances

10ml of Sol used for the assay were transferred in to a 20ml volumetric flask topped up with mobile phase and injected.

Terbinafine HCl – Effect of Hydrochloric Acid (1M)

The Terbinafine HCl sample was exposed to acidic conditions namely 1.0 M HCl.

Blank solution

5ml of 1M HCl left to sonicate for 3 hrs at 60oC.

Test solution-Assay

90.0mg of Terbinafine HCl sample was weighed in a 50ml volumetric flask together with 5ml of 1M HCl. This solution was left to sonicate together with the blank solution for 3 hrs at 60oC. Both the test solution and the blank were neutralized using 1ml of 5M NaOH and topped up to the mark with mobile phase and labelled as test solution A.

Then 1ml of the test solution A was transferred to a 50ml volumetric flask and also topped up with mobile phase.

Test solution- Related Substances

10ml of test solution A used for the assay was transferred to a 20ml volumetric flask and topped up with mobile phase and injected. The same procedure was performed for the blank solution.

Terbinafine HCl – Effect of Sodium Hydroxide (1M)

The Terbinafine HCl sample was exposed to basic conditions namely to 1M NaOH.

Blank solution

5ml of 1M NaOH in a 50ml volumetric flask left to sonicate for 3 hrs at 60oC.

Test solution-Assay

90.0mg of Terbinafine HCl sample was weighed in a 50ml volumetric flask together with 5ml of 1M HCl.

This solution was then left to sonicate together with the blank solution for 3 hrs at 60oC. Both the test solution and the blank were then neutralized by adding 1ml of 5M NaOH and topped up with mobile phase (Sol A)

Then, 1ml of the test solution A was transferred to a 50ml volumetric flask, topped up with mobile phase and injected.

Test solution- Related Substances

10ml of test solution A was transferred to a 20ml volumetric flask and also topped up with mobile phase and injected. The blank was performed using the same procedure.

Product – Effect of Hydrogen Peroxide (12%)

The product was exposed to 12% H202

Blank solution

5ml of mobile phase in a 50ml volumetric flask and topped up to the mark with 12% H₂O₂and left to sonicate for 3 hrs at 60^oC.

Test solution -Assay

90.0mg of Terbinafine HCl sample was weighed in a 50ml volumetric flask dissolved using 5ml of mobile phase and brought to volume with 12% H2O2 (Sol A). This solution was then left to sonicate together with the blank solution for 3 hrs at 60oC. Then, 1ml of this solution was transferred to a 50ml volumetric flask, topped up with mobile phase and injected.

Test solution- Related Substances

10ml of solution A was transferred to a 20ml volumetric flask and also topped up with mobile phase and injected. A blank solution was prepared similarly.

Product – Effect of UV light (powder)

The product was exposed to a UV light for 24 hours

Test solution-Assay

90.0mg of stressed Terbinafine HCl sample was weighed in a 50ml volumetric flask, dissolved using mobile phase. Then, 1ml of this solution was transferred to a 50ml volumetric flask and also topped up with mobile phase and injected.

Test solution- Related Substances

10ml of Sol A used for the assay were transferred in to a 20ml volumetric flask topped up with dissolution phase B and injected.

Results

Assay results show that there was no degradation of the sample since the assay value was found to have a value of (98.8%). It can be noted that compared to the untreated sample impurity A has increased from a percentage area of < 0.01% to a percentage area of 0.14% in the sample exposed to UV light.

Reviewing the PDA plot (230nm) namely the purity plot, it can be said that there are no impurities that fall under the Terbinafine HCl peak, this because the purity threshold is greater than the purity angle.

RESULTS AND DISCUSSIONS:

Assay and Related Substances – As such

The six injections of the assay solution were used as system suitability runs and to quantify the assay of the other stress stability tests. For the related substances method the product was injected as is. From the 2D plot at 230nm it can be noted that the unstressed sample contains three known impurities Impurity A, Impurity B and Impurity C present at very low percentage areas (<0.01%).

Impurities A and D are impurities which may result as by-products of the reaction synthesis while impurity B (TER E) and impurity C result from starting material used in the reaction synthesis.

Terbinafine HCl – Effect of temperature at 800C for 24 hours

From the results obtained, it was noted that there was hardly any degradation of the sample since the assay value obtained was 98.6%. The related substances plot at 230nm confirms the above statement in that it shows that only a few impurities were formed, the highest impurity being that eluted at 22.6min having a percentage area of 0.04%.

Reviewing the PDA plot at 230nm namely the purity plot, it can be said that no impurities fall under the Terbinafine HCl peak, this is because the purity threshold is higher than the purity angle.

From the results obtained, one can note that on exposing the sample for 96hrs under vacuum at 800C showed no degradation of the sample and the result assay value obtained was 101.6%.

On viewing the extracted chromatogram at 230nm, one can note that the known impurity C has increased slightly than that found in the untreated sample having a percentage area of 0.01%. The highest impurity is that found at retention time 37.077min having a percentage area of 0.04%.

On viewing the PDA plot at 230nm, one notes that for the Terbinafine HCl peak the purity threshold is higher than the purity angle and this implies that no peaks fall under the Terbinafine HCl main peak.

Terbinafine HCl – Effect of Hydrochloric Acid (1M)

The acid treatment of sample resulted in a very slight degradation of the product (4.7%) since the assay value decreased to 94.9%. However, any degradation less than 15%-20% is not considered as appreciable.

Infact on comparing the related substances chromatogram of the sample exposed to 1M HCl compared to that of the untreated sample one notes that the known impurities are present in approximately similar amounts showing that no degradation has occurred. Only a slight increase in the unknown impurity at retention time 25.7min can be noted.

On viewing the PDA plot at 230nm, one can note that the purity threshold is higher than the purity angle for the Terbinafine HCl main peak and thus it may be said that no peaks are coeluted with the Terbinafine HCl main peak.

Terbinafine HCl – Effect of Sodium Hydroxide (1M)

The basic treatment of Terbinafine HCl sample resulted in a very slight degradation of the product (~3.6%) since the assay value decreased to 96%. Since only a degradation of 15%-20% is considered as being appreciable, it can be said that hardly no degradation has occurred.

This is infact confirmed that by the small amount of impurities present. From the PDA plot (230nm), for the Terbinafine HCl peak it can be noted that the purity threshold is higher than the purity angle and thus it may be said that no peaks are coeluted with the Terbinafine HCl main peak.

Product – Effect of Hydrogen Peroxide (12%)

The treatment of the Terbinafine HCl sample with hydrogen peroxide resulted in no degradation of the sample. This was confirmed since the assay value found was 100.8% showing that there was no degradation whatsoever.

These results were further confirmed in that on viewing the extracted chromatogram at 230nm, impurities present have very low percentage areas, the highest being that eluted at 21.879min having a percentage area of 0.07%.

On viewing the PDA plot at 230nm one can note that the purity threshold is higher than the purity angle for the Terbinafine HCl main peak and thus it may be said that no peaks are coeluted with the Terbinafine HCl main peak.

Product – Effect of UV light (powder)

CONCLUSION:

From the results obtained in this study, one can conclude that the Terbinafine HCl is stable to all the conditions tested in this study, only slightly degrading in alkaline (1M NaOH) and acidic (1M HCl) conditions. However, since only a degradation of 15-20% is considered as appreciable, one can conclude that the product shows no appreciable degradation in the conditions tested in this study.

Results also show that the HPLC method used in this study is able to detect any degradation of the product and separate any degradation impurities formed, thus making it a suitable stability indicating method.

ACKNOWLEDGEMENT:

The authors are thankful to the Dr. Benerjee Patrudu, GITAM University , Hyderabad for providing the gift sample of Terbinafine HCl and providing necessary facilities to carry out the research work with keen interest and help.

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Received on 20.08.2016 Modified on 28.08.2016

Asian J. Research Chem. 2016; 9(11): 561-565.

DOI: 10.5958/0974-4150.2016.00076.6