NOVEL HPTLC-DENSITOMETRIC METHOD FOR THE ESTIMATION OF TERIFLUNOMIDE IN TABLET DOSAGE FORM

Objective: The current work is intended towards the development of a novel, simple and precise high-performance thin layer chromatographic (HPTLC) method coupled with a densitometer for the estimation of teriflunomide (TEF) present in the marketed formulation. Methods: The chromatographic development was performed on aluminum plates coated with silica gel 60 F 254 Results: Well separated band was observed with Rf value 0.46. The calibration curve plotted in the concentration range 100-700ng/band exhibited an excellent linear relationship with the r using toluene: ethyl acetate: glacial acetic acid (7.5:2: 0.5 v/v/v) as the mobile phase. Densitometric scanning was achieved at the absorbance maxima, UV 284 nm. 2 Conclusion: The method ensures minimal use of mobile phase with minimal run time compared to other reported analytical methods. This validated method can be used by quality control laboratories for the routine quantitative analysis of tablets consisting of Teriflunomide. value of 0.9928. The method was found to comply with all the validation parameters as per the ICH guidelines.


INTRODUCTION
Teriflunomide ( fig. 1) (TEF) is an orally administered, secondgeneration immunosuppressive/immunomodulatory agent which is an active metabolite of Leflunomide. It acts by inhibiting dihydroorotate dehydrogenase, an enzyme which is responsible for pyrimidine de novo synthesis [1][2][3][4]. It is permitted by both the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) as a disease-modifying therapy (DMT's) for adults with relapsing-remitting multiple sclerosis (RRMS). This drug, not official any pharmacopeias like IP, USP and BP.

Fig. 1: Teriflunomide
The literature survey disclosed various methods for the estimation of TEF in API, marketed formulations and biological fluids. The detailed information on the various methods available are as follows; chromatographic methods such as HPLC [5,6], UPLC [7], RP-HPLC [8,9], LC-MS [10][11][12][13]. In all the reported techniques, the overall solvent consumption, cost per analysis and overall time required for analysis were much more. Furthermore, No HPTLC method has been reported so far, for the estimation of TEF in the marketed formulation. Therefore, the current work is directed towards the development of a novel HPTLC method for the determination of TEF in the marketed formulation having sensitivity in terms of nanogram with minimal development time, minimal use of solvents and its validation according to ICH guidelines.

Materials and reagents
Teriflunomide pure drug (API) was procured as a gift sample from Glenmark Ltd., Noida. All the other reagents used were of analytical grade and were procured from Merck specialties, private limited, Mumbai, India. Marketed Formulation (Denopsy tablets, Natco Pharma, Hyderabad) was purchased from a local pharmacy.

HPTLC instrumentation and chromatographic conditions
CAMAG HPTLC system equipped with Linomat 5 sample applicator operated under a gentle stream of nitrogen, coupled with a 100 µl HAMILTON syringe and a CAMAG TLC scanner 3controlled by winCATS software was used for the application and detection of spots respectively. Aluminum sheets having a dimension of 20×10 cm, coated with silica gel 60 F254

Preparation of standard solutions
procured from Merck, Germany and a CAMAG twin trough TLC chamber having a dimension 27.0 cm width × 26.5 cm height × 7.0 cm was used for the chromatographic development.
For chamber saturation, 20 ml of the mobile phase was transferred into the development tank with the lid closed. The assembly was kept aside for 20 min under room temperature. The plates were air dried using an air dryer after the application of spots and after the development, prior to densitometric scanning. The chromatographic conditions were as described in table 1.
A stock solution containing 1000 ng/µl of TEF was prepared by using acetonitrile as a diluent. 0.5 ml of this solution was pipetted out into a 10 ml volumetric flask and the volume was made up, to get a working solution consisting of 50 ng/µl of TEF.

Preparation of sample solution (Formulation)
As specified in the label, each tablet contains 14 mg of TEF. The weight of 10 tablets was noted and crushed using a mortar and pestle, the quantity of powder equivalent to 10 mg of teriflunomide was transferred to a 10 ml volumetric flask and the volume was made up with acetonitrile. The required dilutions were made to get a final concentration of 50 ng/µl.

Method validation
The method was validated according to ICH guidelines for the following parameters: Linearity, accuracy, precision, the limit of detection (LOD) and limit of quantification (LOQ), specificity and robustness [14][15][16].

Optimization of chromatographic conditions
The ideal mobile phase of the method was selected after several experimental trials. Solvents like acetonitrile, methanol, toluene, ethyl acetate used initially; did not provide a clear spot. Therefore, various permutations and combinations of these solvents were used for the trials. Out of all the combinations, toluene and ethyl acetate was found to give a spot. For better resolution, 0.5% glacial acetic acid was added. Thus, the final chromatographic condition, toluene: ethyl acetate: glacial acetic acid in the ratio 7.5: 2: 0.5 (v/v/v) was able to achieve a clear spot with good Rf value. Increasing the volume of glacial acetic acid leads to an increase in the Rf The wavelength for the densitometric scanning of the spots was selected after obtaining the UV spectra of TEF ( fig. 2), which showed the maximum absorbance at 284 nm.
value and vice versa. The chamber saturation time was found to show a similar effect.

Linearity
The calibration curve ( fig. 3) was plotted between peak areas versus concentration. The linearity of TEF was constructed by applying 7 bands in the concentration range of 100-700 ng/band. The regression equation (r2= 0.9928) demonstrated the good linearity of the method. The slope and intercept of the regression equation were 15.471 and 3505.7 respectively for TEF. The linearity was found to be satisfactory and reproducible. The calibration curve of linearity and overlay densitogram of TEF is depicted in fig. 3 and 4 respectively.

Limit of detection and limit of quantitation
LOD and LOQ were calculated with the aid of standard deviation (σ) and slope (s) from the calibration curve (n=3), by using the formula LOD = 3.3 σ/s and LOQ = 10 σ/s. LOD and LOQ of TEF were found to be 21 ng/band and 65 ng/band respectively, which indicates the good sensitivity of the method towards the analyte.

Specificity
The specificity of the method was assessed by comparing the UV absorption spectra ( fig. 2) and densitogram of standard ( fig. 4) TEF with the formulation (fig. 5). The densitogram of both standard and formulation showed the same Rf

Robustness
value indicating the good specificity of the method.
The robustness was assessed by making small but deliberate changes in the method parameters such as; mobile phase ratio, chamber saturation time, scanning wavelength and finding out its effect on the peak area by calculating %RSD. The % RSD was found to be within 2%, which indicates the reliability of the method. The robustness of the method are depicted in table 4.

Analysis of formulation
Mean for five independent analyses, SD=Standard deviation, RSD=Relative standard deviation.
The % of a drug found in the formulation of the currently developed method was found to be 98.9%. The densitogram obtained from the formulation ( fig. 5) exhibited a single spot at Rf 0.46 without any interference of the excipients. The close agreement of the percentage of the drug found with label claims depicted the application of this method for the routine analysis of TEF present in its formulation.

Fig. 5: Densitogram of the formulation DISCUSSION
A novel, simple and precise high-performance thin layer chromatographic (HPTLC) method coupled with densitometer was developed for the estimation of TEF present in the marketed formulation. There are some HPLC methods [7,8] reported in the literature, but they have certain restrictions like require a large quantity of sample and/or organic solvents or sensitive to microgram concentration. Few methods like Liquid chromatography-mass spectroscopy for determination TEF in human or animal plasma [9][10][11][12] etc. are found in the literature, but these methods are not preferred for routine analysis of TEF in bulk and formulation studies, because of the high cost of analytical technique and the skilled requirement for sample treatment. A detailed evaluation of the analytical method procedures discussed with the present method was given in table 1. Accuracy results displayed good reproducibility with % RSD values below 2. This was found to be accurate as percent recovery observed was high i.e. within the range of 98.6-98.94 %, suggesting that the proposed method showed good agreement between the standard and the observed values and demonstrate an adequate accuracy within the specified limits as shown in the table 2. Furthermore, detection limit depends upon the instrument sensitivity as low detection limits give high sensitivity. The LOD and LOQ of developed for HPTLC method was found to be 21ng/ml and 65ng/ml respectively confirms method is sensitive. Results of intra and inter-day precision studies were expressed as %RSD, the results were given in table 3. The low values of %RSD showed that the method is precise. The results of robustness are tabulated in table 4, the method is not altered by changing the method parameters like mobile phase composition, chamber saturation time and scanning wavelength which proves that the method is robust. In addition, the estimation of the marketed preparation of TEF with the validated methods showed that the drug contents separated with no interfering peaks generated by the excipients in the marketed formulation as shown in the fig. 5. The method is versatile and simple for the analysis TEF in pure and pharmaceutical formulations. The method was found to obey all the validation parameters as per the ICH guidelines. The method confirms minimal use of the mobile phase with a short run time compared to other reported analytical methods. This validated method can be used for quality control laboratories for the routine quantitative analysis of tablets consisting of TEF.

CONCLUSION
A novel, simple, rapid and precise HPTLC method coupled with densitometer scanning has been developed for the determination of TEF in the marketed formulation. This validated method can be used by quality control laboratories for the routine quantitative analysis of tablets consisting of TEF. The additives used in this formulation were also not interfering with the analysis. The method ensures minimal use of mobile phase with minimal run time compared to other reported analytical methods. Non-requirement of skilled personnel to operate the instruments involved is an added advantage of this method.