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Journal of the Chilean Chemical Society

versão On-line ISSN 0717-9707

J. Chil. Chem. Soc. vol.61 no.2 Concepción jun. 2016

http://dx.doi.org/10.4067/S0717-97072016000200021 

 

DEVELOPMENT AND VALIDATION OF HPTLC METHOD FOR SIMULTANEOUS ESTIMATION OF TERBINAFINE HYDROCHLORIDE AND MOMETASONE FUROATE IN COMBINED DOSAGE FORM

 

MEHUL M. PATELa*, HETA D. PATELa

a Pharmaceutical Chemistry Department, Ramanbhai Patel College of Pharmacy, Charotar University of Science and Technology, Gujarat, India.
* e-mail: mehulpatel.ph@charusat.ac.in


ABSTRACT

A new simple, precise, accurate, specific and selective high performance thin layer chromatographic (HPTLC) method has been developed for the simultaneous estimation of Terbinafine hydrochloride (TH) and Mometasone furoate (MF) in cream dosage form. The chromatographic separation was achieved on Merck precoated silica gel aluminium plate 60 F254 using Toluene: Ethyl acetate: Glacial acetic acid (8: 4: 0.1 v/v) as mobile phase. The densitometric scanning was carried out at 248 nm. Response was found to be linear in the concentration range of 1000-3000 ng/band with correlation coefficient (r2 = 0.999) for Terbinafine hydrochloride and 100-300 ng/band with correlation coefficient (r2 = 0.998) for Mometasone furoate. All parameters of method, repeatability, precision, LOD, LOQ, % recovery were validated as per ICH guidelines. The proposed procedure was successfully applied for the simultaneous determination of both drugs in commercial cream preparation. The advantage of the method is simplicity, reasonable sensitivity, rapidity, excellent resolving power, low cost and is a more effective option than other chromatographic techniques in routine quality control.

Keywords: Terbinafine hydrochloride (TH), Mometasone furoate (MF), High performance thin layer chromatography (HPTLC).


 

INTRODUCTION

Terbinafine hydrochloride (TH) is an antifungal and enzyme inhibitor drug. Chemically, it is (2E)-N, 6, 6-Trimethyl-N-(naphthalen-1-ylmethyl) hept-2-en-4-yn-1-amine hydrochloride, clinically it is use in the treatment of dermatophyte infections of the toenail or fingernail caused by susceptible fungi. Also for the treatment of tinea capitis scalp ringworm) and tinea corporis (body ringworm) or tinea cruris jock itch). Mometasone furoate (MF) is a glucocorticoid having anti-inflammatory activity and chemically it is 9,21-Dichloro-11p-hydroxy-16a-methyl-3,20-dioxopregna-1,4-dien-17-yl furan-2-carboxylate and it is also useful treatment of dermatological diseases. This combination of drugs is useful for relief of corticosteroid responsive dermatomes where fungal infections are present, suspected, or likely to occur. Both the drugs are marketed as combined dose cream formulation in the ratio of 10:1 w/w TH: MF. Literature survey reveals that Terbinafine hydrochloride (TH) can be estimated by spectrophotometrically, HPTLC and HPLC individually or with other drugs in bulk drugs and in human plasma1-5 while Mometasone furoate (MF) can be estimated by spectrophotometrically, HPLC and HPTLC in combination with other drugs6'10. However, there is no analytical method has been reported for the estimation of TH and MF in a combined dosage formulation. Present work describes HPTLC method4 for simultaneous estimation of TH and MF in cream formulation. Method validation was done according to ICH Q2 (R1) Guidelines.

MATERIALS AND METHODS:

Instrument

A CAMAG Linomat V sample applicator equipped with an applicator microsyringe (Hamilton, Bonaduz, Switzerland) and CAMAG UV cabinet with dual wavelength UV lamp (254 and 366 nm); densitometric scanning was performed at 248 nm with CAMAG TLC scanner IV operated in absorbance mode and controlled by Wincats 1.4.7 software.

Materials

Standard gift sample of Terbinafine hydrochloride was provided by Aarti Drugs sales, Mumbai and Mometasone furoate was povided by Glenmark Generics pharmaceutical, Nasik. Combined dose Terbinafine hydrochloride and Mometasone furoate cream sebifin™ plus was purchased from local market. Methanol (AR Grade), Toluene (AR Grade), Ethyl acetate (AR Grade) and Glacial acetic acid (AR Grade) were used as solvents, procured from Loba Chemie pvt ltd., Mumbai.

Optimized Parameters for Chromatographic Condition

As stationary Phase, Silica gel 60F254 TLC Precoated Plates was selected and after trials of various mobile phase, final mobile phase selected was Toluene: Ethyl aceate: Glacial acetic acid ( 8: 4: 0.1 v/v/v ). Plate development technique was simple ascending at ambient room temperature and chamber saturation time was 20min. Run distance was 80mm and detection wavelength was 248 nm. Spotting Parameters were band width of 6 mm and syringe used was of 100 μl capacity. Scanning Parameters were set of slit dimension of 6 x 0.45 mm, detection was done at wavelength 248 nm and scanning speed was 100 mm/sec. In integration Parameters, baseline correction was done, peak threshold, height was 10AU, peak threshold, area was 50 and peak threshold, slope was kept 5.

Stock solutions

Stock solution: Standard stock solutions of TH (1000 μg/ml) and MF (1000 μg/ml) were prepared in methanol.

Working solutions

Working solution: Standard working solutions of TH (500 μg/ml) and MF (50 μg/ml) were prepared in methanol and used for the analysis.

Method validation

The developed method was validated for linearity, range, specificity, precision, accuracy and robustness as per ICH guidelines.

Linearity and range

Each concentration mixture in the range of 1000-3000 ng/spot for TH and 100-300 ng/spot for MF was spotted five times on individual plates and response was measured after scanning. For evaluation of linearity, peak area and concentrations were subjected to least square regression analysis to calculate calibration equation and correlation coefficient.

Specificity

The specificity of the method was ascertained by analyzing TH and MF in presence of excipients of TH and MF in cream formulations. The bands of TH and MF in the sample were confirmed by comparing Rf values and respective spectra of the sample with those of the standards. The peak purity of TH and MF was assured by comparing the spectra at three different levels, that is, peak-start (s), peak-apex (m) and peak-end (e) positions.

Precisión

Precisión was measured by using standard solutions containing TH and MF at concentrations covering the entire calibration range. The precision of the method was evaluated by calculating %RSD of mean peak areas obtained from each spot of TH and MF. Same procedure was performed at different time intervals on the same day and on different days.

Accuracy

The accuracy of the method was determined by recovery studies using standard additions at three different levels (approximately 80, 100 and 120% of label claim), i.e. multiple level recovery studies. This was done to check for the recovery of the drug at different levels in the formulations.

Robustness

Robustness was assessed by deliberately changing the chromatographic conditions and studying the effects on the results obtained.

Limit of detection and Limit of Quantification

Limits of detection and limit of quantification were determined on the basis of the mathematical terms mentioned in ICH guidelines for method validation from triplicate results of linearity.

LOD= 3.3 σ/s
LOQ= 10 σ/s

Limit of detection was determined using equation 3.3 σ/s and limit of quantification was determined using equation 10 σ/s, where o is the slope of calibration curve and s is standard deviation of responses.

Application of the proposed method for the determination of TH and MF in cream

1gm cream was weighed which is equivalent to 10 mg TH and transferred into 10 ml centrifugal tube, dissolved in methanol and centrifuged for 5 minute at 4000 rpm. The solution was filtered through whatman filter paper. Take 5 ml from this and adjust the volume up 1 ml with methanol. This solution contains 500 μg/ml of TH and 50 μg/ml of MF.

From this solution, 5 μl was spotted which gives 2500 ng/spot of TH and 250 ng/spot of MF concentration.

RESULTS AND DISCUSSION

Selection of best solvent system is the critical step in HPTLC method development. From the different solvent systems tried, the mobile phase consisting of toluene, ethyl acetate and glacial acetic acid in ratio of 8:4:0.1 v/v/v gave good separation between TH and MF selected as optimized mobile phase. Rf value of symmetric peaks of TH and MF were obtained at 0.62 and 0.35 respectively (Fig.3). Well-defined bands were obtained when the chamber was saturated with mobile phase for 20 min at ambient temperature. Reproducible responses were obtained. For quantitative purpose, the densitometric scanning was carried out at wavelength 248 nmThe proposed method could be employed for routine quality control of TH and MF in combined cream dose formulation.

 

Figure 3: Typical HPTLC chromatogram of 2500 ng/spot of TH and 250 ng/spot MF

 

Linearity and Range

Linearity was observed over the concentration range 1000-3000 ng/spot for TH and 100-300 ng/spot for MF confirming adherence of the system to Beer’s law. Calibration curve was taken (Fig.4). The regression analysis equation was y=3.688x+1559 for TH and y=13.09x+88.35 for MF and, correlation coefficient (r2) was 0.999 for TH and 0.998 for MF respectively. Linearity data were taken (Table 1, 2 and 3).

 

Figure 4: 3-D view of calibration curve of TH and MF

 

Table 1: Linearity data of TH (n=6).

 

Table 2: Linearity data of MF (n=6).

 

Table 3: Linear regression data of TH and MF for HPTLC method.

 

Specificity

Specificity of the method for TH and MF were proved from the spectral scan (Fig 5 and 6) for TH and MF in bulk and in cream formulations indicate that there is no merging or co-elution of interfering peaks with TH and MF, so there is no interference from any excipients present in cream formulation.

Precision

For determination of precision of TH and MF by the proposed method, same homogeneous samples of TH and MF (real samples) were prepared repeatedly and analyzed. Intermediate precision was evaluated at different times on same day, on different days. Low values of RSD (less than 2%) obtained in the precision studies (Table 4, 5 and 6) indicate that the method is precise and reproducible.

 

Table 4: Intraday precisión data for TH and MF (n=6).

 

Table 5: Interday precisión data for TH and MF (n=6).

 

Table 6: Repeatability data of TH and MF(n=6).

 

Accuracy

Accuracy of the proposed method was studied by standard addition to sample solution at three levels 80, 100 and 120% of the label claim. % Mean recovery for TH (Table 7) and MF (Table 8) were between ±2% indicating that the developed method was accurate for the determination of TH and MF in pharmaceutical formulations.

 

Table 7: Accuracy data of TH (n=3).

 

Table 8: Accuracy data of MF (n=3)

 

Limit of detection and Limit of Quantification

LOD and LOQ were found by using equations (Table 9).

 

Table 9: LOD and LOQ

 

Robustness

Acceptable %RSD values obtained after making small deliberate changes in the developed HPTLC method indícate that the method is robust for the intended purpose (Table 10 and 11).

 

Table 10: Robustness study of TH

 

Table 11: Robustness study of MF

 

Method application

The proposed, developed and validated HPTLC method was successfully applied for determination of TH and MF in marketed formulation. The assay results obtained were satisfactory, accurate and precise as indicated by %RSD values (Table 12). The good performance of the method indicates that it can be used for the determination of TH and MF in drug substances and pharmaceutical preparations.

 

Table 12: Assay of marketed formulation by HPTLC method (n=5).

 

CONCLUSION

The developed methods were validated as per ICH guidelines and were found to be within the prescribed limit. It concludes that the developed method is simple, accurate, sensitive and precise and suitable for routine quality contol analysis for both authentic and cream dosage form.

ACKNOWLEDGEMENTS

The authors are very thankful to Dr. R.H PARIKH; Principal, Associate Prof. Dr. Manan Raval, Assistant Prof. Mr. Niraj Vyas, Ramanbhai Patel College of Pharmacy, Changa, for providing the research facilities. We are also thankful to Aarti Drugs sales, Mumbai and Glenmark Generics pharmaceutical, Nasik for providing the gift samples of Terbinafine hydrochloride and Mometasone furoate respectively.

 

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