METHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OF RELATED SUBSTANCES IN TERIFLUNOMIDE BY RP HPLC METHOD

METHOD DEVELOPMENT AND VALIDATION FOR THE

DETERMINATION OF RELATED SUBSTANCES IN

TERIFLUNOMIDE BY RP-HPLC METHOD

Sushma G., Sridevi Pingali*, Bhagavan Raju M. and Vinutha K. Shaik Ashfaque

Sri Venkateshwara College of Pharmacy, Madhapur, Osmania University,

Hyderabad-500081, Telangana, India.

ABSTRACT

A Reverse Phase High Performance Liquid Chromatographic Method

(RP-HPLC) was developed and validated for the determination of

related substances in Teriflunomide. The method was carried out using

Zorbax SB Phenyl, 150 x 4.6 mm, 3.5µmwith mobile phase-A and

mobile phase-B, where mobile phase-A consisted of potassium

dihydrogen phosphate buffer and mobile phase-B consisted of

Acetonitrile in the ratio 60:40 (v/v) under gradient mode with an

injection volume of 10µL at flow rate of 1mL/min and the detection

was carried out at 209 nm. The retention time of Teriflunomide and its

related substance (RS) method were found to be 11.797min and

23.588min. This method produced linear responses for Teriflunomide

and its related substances in the concentration range of 0.012-0.304% with a correlation

coefficient of 0.999 and 0.999. The proposed method was validated as per ICH guidelines.

The method was found to be linear, specific, precise and accurate. Therefore, the proposed

method can be used for the routine analysis of drug in the process control of bulk and

pharmaceutical formulation.

KEYWORDS: RP-HPLC, Teriflunomide, Validation, related substances, pharmaceutical formulation.

INTRODUCTION

Immuno modulatory drugs modify the response of the immune system by increasing

(immunostimulators) or decreasing (immunosuppressives) the production of serum

antibodies.

Volume 8, Issue 12, 882-891. Research Article ISSN 2277– 7105

Article Received on 05 Sept. 2019,

Revised on 26 Sept. 2019, Accepted on 16 Oct. 2019

DOI: 10.20959/wjpr201912-16009

*Corresponding Author

Dr. Sridevi Pingali

Sri Venkateshwara College

of Pharmacy, Madhapur,

Osmania University,

Hyderabad-500081,

Teriflunomide: Teriflunomide is the active metabolite of Leflunomide, and it acts as an immunomodulatory agent/ immunosuppressive agent by inhibiting pyrimidine synthesis.

Figure. 1: Chemical Structure of Teriflunomide.

Mechanism of action: The exact mechanism by which Teriflunomide acts in MS is not known. What is known is that Teriflunomide prevents pyrimidine synthesis by inhibiting the

itochondrial enzyme dihydroorotate dehydrogenase, and this may be involved in its

immunomodulatory effect in MS.

Adverse Effects: Most common adverse reactions (≥10% and ≥2% greater than placebo): ALT increased, alopecia, diarrhoea, influenza, nausea, paresthesia and warning for

Hepatotoxicity, risk of Teratogenicity and Hepatotoxicity severe liver injury including fatal

liver failure has been reported in patients treated with Leflunomide, which is indicated for

rheumatoid arthritis.

MATERIALS AND METHODS

Chemicals: Teriflunomide API, Potassium Dihydrogen Phosphate, Tri Ethyl Amine, Ortho

Phosphoric Acid, Acetonitrile, Water.

Instruments

UV-Vis Spectrophotometer, HPLC, Analytical balance, Ultrasonic bath, Vacuum Filter, pH

meter, Milli-Q Water System.

Preparation of reference stock solution: Weigh accurately 3.0 mg of H-TFMRC01 and 2.0 mg

of TFM reference standards into a 100 mL volumetric flask, dissolve and dilute to the volume

with diluent and mix.

EXPERIMENTAL

Determination of Wavelength: The sensitivity of the HPLC method which uses UV detection depends upon the proper selection of wavelength. An ideal wavelength is one that

gives good response for all the drugs to be detected.

Procedure: 10mg of Teriflunomide working standard was weighed accurately and transfered to a clean dry 100ml volumetric flask. Sufficient volume of diluent is added, sonicated to

dissolve and volume is made up with diluent to get concentration (100µg/ml). From this 1mL

is pipetted out and make up to 10mL to get concentration (10µg/ml).

UV spectrum of Teriflunomide (10µg/ml) in diluents was recorded by scanning in the range

of 200nm to 400nm. From the UV spectrum wavelength selected as 209nm. At this

wavelength the Teriflunomide and related impurities shows good absorbance.

Selection of Chromatographic Methods: The proper selection of method depends upon the nature of the sample (ionic or ionisable or neutral molecule), its molecular weight and

stability. The drugs which were selected are polar and ionic in nature. Hence HPLC was

selected for separation because of its simplicity and suitability.

Selection of Mobile Phase: Initially the mobile phase tried was potassium dihydrogen phosphate buffer: methanol and Sodium perchlorate monohydrate buffer: acetonitrile+buffer

with various proportions. Finally, the mobile phase was optimized to potassium dihydrogen

phosphate buffer: acetonitrile+buffer with various proportions in gradient mode.

Selection of Column: The method was performed with various columns like Zorbax SB Phenyl, 150 x 4.6 mm, 3.5µm was found to be ideal as it gave good peak shape and

resolution at 1mL/min flow.

Method development

The RP-HPLC method for determination of related substances in Teriflunomide has been

developed and evaluated. The RP-HPLC method was tested for linearity, specificity,

accuracy, precision finally optimized RP-HPLC conditions used for the present studies are:

detection wavelength-209 nm, mobile phase Potassium dihydrogen phosphate:

acetonitrile+buffer, flow rate 1ml/min, run time 55 minutes, column temperature 45°C,

RESULTS AND DISCUSSION

Fig 1: Teriflunomide UV-vis spectrum.

Table 1: Chromatogram Data for Method Development.

Trail No. Chromatographic condition Retention Time

Run

time Results Column Mobile phase

1 Zorbax SB Phenyl,

150×4.6 mm, 3.5µm Buffer: Methanol

23.83 Minutes 55 minutes Broad Peak

2 Zorbax SB Phenyl,

150×4.6 mm, 3.5µm Buffer: Methanol

20.39 minutes 55 minutes Broad Peak 3

Zorbax SB Phenyl, 150 x 4.6 mm,

3.5µm Buffer:Acetonitrile + buffer 12.26 minutes 55 minutes Peak Fronting Optimized method

Zorbax SB Phenyl, 150 x 4.6 mm,

3.5µm Buffer:Acetonitrile + buffer 11.797 minutes 55 minutes Good Resolution

Fig. 2: Chromatogram of Optimized Method.

Method validation

Specificity: H-TFMRC01 solution and Teriflunomide standard solutions were prepared individually at target concentration of the test sample. A solution of all known impurities

solutions were analyzed using the PDA detector as per the HPLC method. No interference is

observed due to blank at the retention time of HTFMRC01 and Teriflunomide. So this

method was found to be specific.

Fig. 3: Chromatogram of Sample Teriflunomide.

PRECISION

System Precision: System precision was performed by injecting six replicates of 10µL reference solution of Teriflunomide, H-TFMRC01 and measured the area for all six injections

in HPLC. The %RSD for the peak area of each component from six replicate injections of

reference solution was within the limit.

Fig. 4: Chromatogram of system precision.

Method Precision: Teriflunomide test sample spiked with H-TFMRC01 at specification level with respect to the test sample concentration for six times and analyzed for the precision

study as per the procedure. The %RSD from the content of Teriflunomide of six replicate

obtained from method precision study were in the range of 1.40 to 3.57. So this method was

found to be precised.

Fig 5: Chromatogram of Method Precision.

Accuracy: Accuracy of the method was proved by checking the % recovery of H-TFMRC01 in test solution spiked with H-TFMRC01 at 50%, 100%, and 150% level. Each level solution was

prepared in triplicate and % recoveries of H-TFMRC01 were reported. The % recovery obtained

from 50% to 150% level was in the range of 106.7 to 113.3 for H-TFMRC01.

Fig 6: Chromatogram of accuracy.

Linearity: The linearity study was conducted for H-TFMRC01and Teriflunomide standard in the range of QL level to 200% level. Correlation coefficient values for H-TFMRC01 and

TFM standard were derived from respective linearity graph. Teriflunomide and H-TFMRC01

was found to be linear over the concentration range of 0.012-0.200µg/ml. Correlation

coefficient value for calibration plot of Teriflunomide and H-TFMRC01 was found to be

Fig 7: Linearity Graph of Teriflunomide.

Fig 8: Linearity Graph of H-TFMRC01.

Range: Established range for each component and proved that the method was precise and linear over the range. The % RSD obtained for all the components were in the range of 1.63

to 5.61 for lower and upper levels.

Limit of Detection: The detection limit (DL) is defined as the lowest concentration of an analyte in sample that can be detected, but not necessarily quantitated. The detection limit

was determined as the lowest concentration for which the response is approximately three

times greater than the baseline noise. The signal to noise ratio of each component was within

the limit. 0.004% for H-TFMRC01 and 0.004% for Teriflunomide.

Limit of Quantification: Quantification Limit of each component was established as per the procedure mentioned in the protocol. The QL solution was prepared based on the DL solution

and obtained the signal to noise ratio about 10:1 for each component. The signal to noise ratio

of each component was within the limit. 0.012% for H-TFMRC01 and 0.012% for

CONCLUSION

Reversed Phase High Performance Liquid Chromatographic method has been optimized and

developed for the determination of Teriflunomide and quantification of related substances in

API. The optimized chromatographic conditions for the determination of Teriflunomide using

waters HPLC 2695 system and schimadzu HPLC LC2010 CHT auto sampler, Zorbax SB

Phenyl, 150 x 4.6 mm, 3.5µm,in gradient mode using mobile phase containing Potassium

dihydrogen phosphate: acetonitrile and buffer. The flow rate maintained was 1ml/min at

column temperature of 45°C with PDA detection at 209nm and run time of 55min. The

retention time of Teriflunomide and its related substances was found to be 11.797 min &

23.588. The linearity for Teriflunomide and its related substances was obtained in the

concentration range of 0.012-0.304 with correlation coefficient 0.999 & 0.999. The methods

were validated for various parameters as per ICH guidelines and all the results obtained were

within the limits. Impurities (RS) present in the formulation were well separated, reported

and quantified. The developed method was found to be linear, specific and precise and

therefore, the proposed method can be used for routine analysis of drug in the process control

of bulk and pharmaceutical formulations.

ACKNOWLEDGEMENT

In the first place I would like to express my profound gratitude and deep regards to my

esteemed guide, Dr. P. SRIDEVI, Associate professor, SVCP, Madhapur, Hyderabad. We are very much thankful to the principal, management of Sri Venkateshwara College of Pharmacy,

Hyderabad, for giving permission to carry out my work.

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