Analytical Method Development and Validation of Teriflunomide Active Pharmaceutical Ingredient by RP-UHPLC

 

Arun Maruti Kashid1*, Pranali Prakash Polshettiwar2, Kshitija Maruti Bhosale2

1Department of Pharmaceutical Chemistry, Sinhgad Institute of Pharmacy, Narhe, Pune, Maharashtra, India – 41.

2Department of Pharmaceutical Quality Assurance, Sinhgad Institute of Pharmacy,

Narhe, Pune, Maharashtra, India – 41.

*Corresponding Author E-mail: arunkashid2006@gmail.com

 

ABSTRACT:

Drug analysis is crucial in the discovery, production, and therapeutic use of pharmaceuticals. Standard analytical procedures for newer medications or formulations may not be available in Pharmacopoeias; thus, newer analytical methods that are accurate, precise, specific, linear, simple, and rapid must be developed. A rapid, simple, sensitive, precise, and cost-effective RP-UHPLC method was developed and validated for the determination of Teriflunomide Active Pharmaceutical Ingredient (API) in this study. The method involved determination of Teriflunomide by resolving on RP-UHPLC using Sunniest C18 (250mm × 4.6mm, 5μm) column, utilizing a mobile phase of ACN: Water in the ratio of 60:40 v/v. The mobile phase was delivered with an isocratic flow rate of 1.0ml/minute. Ultra violet detection was carried out at 210nm. The retention time was optimized to 4. 4 minutes. The linearity range of Teriflunomide 35 to 247µg/ml was found to obey linearity with a correlation coefficient of 0.999. The LOD and LOQ were found to be 1.61µg/ml and 4.90µg/ml respectively and precision data was found to be <2 %RSD. The percentage recovery range was found to be satisfied which is represented in the results. The robustness studies were performed by changing the flow rate and mobile phase compositions. The method was validated for system suitability, specificity, linearity, precision, accuracy, Limit of Detection, Limit of Quantification and robustness. The developed method can be applied for the quality control of commercial teriflunomide API.

 

KEYWORDS: Teriflunomide, Method development, UPLC, Validation, ICH guidelines.

 

 


INTRODUCTION:

Teriflunomide was recently approved for the treatment of relapsing types of multiple sclerosis (MS) in the USA. It's second on the list, a drug that has been approved for the treatment of MS.

 

Teriflunomide has the advantage of previous expertise with its parent medication, leflunomide. It is an antiproliferative and anti-inflammatory, active metabolite of leflunomide that is orally bioavailable1. Teriflunomide is an immunosuppressive agent and it works as a dihydroorotate-dehydrogenase (DHODH) inhibitor, which is a crucial mitochondrial enzyme involved in the de novo synthesis of pyrimidines in rapidly growing cells. Teriflunomide inhibits the proliferation of proliferative T- and B-lymphocytes in MS patients. As a result, the overall inflammatory response is reduced. Pyrimidines play a role in a variety of biological processes. Protein and lipid glycosylation, as well as phospholipid synthesis, are all functions in addition to DNA/RNA synthesis and DNA repair, all of which result in a range of immunomodulatory effects downstream. It is a class II compound of biopharmaceutical classification system (BCS) due to its low solubility and high permeability. Teriflunomide is weakly acidic with pKa 3.1 at room temperature and has a pH dependent solubility2,3.

 

Ultra- High Performance Liquid Chromatography is a newer version of analytical separation technology that keeps the practicality and principles of HPLC while improving the overall interwoven features. To run separations using chromatographic principles, smaller particles in the columns and/or a higher flow rate enhanced speed, sensitivity, and resolution. In comparison to HPLC, UHPLC can decrease analysis times without affecting the quantity or quality of analytical data obtained4,5.

 

According to a thorough literature review, there are only a few analytical methods for determining Teriflunomide in bulk, pharmaceutical formulations, and biological fluids, which include, RP-HPLC, HPTLC High Performance Liquid Chromatography with tandem mass spectrometry, Visible spectrophotometric method, spectrophotometric methods (simultaneous determination), LCMS8,9.

 

The objective of this study to describe a simple, rapid, specific accurate, economical and RP-UHPLC method for the determination of Teriflunomide API. The parameters used to validate the method were linearity, specificity, precision, accuracy, limit of detection, limit of quantification and robustness10,11.

 

Drug Profile:

Below table 1 gives detailed information about Teriflunomide6,7.

 

Table 1. Drug profile of Teriflunomide

Drug name

Teriflunomide

Structure

 

IUPAC Name

(Z)-2-Cyano-3-hydroxy-but-2-enoic acid-     (4-trifluromethyl phenyl)- amide (or)

2-Cyano-3-hydroxy-N-[4(trifluromethyl) phenyl]-2(Z)- butenamide

Mol. formula

C12H9F3N2O2

Mol. weight

270.21g/mol

Melting Point

229-232 ℃

Description

White Powder

Solubility

Sparingly soluble in acetone; slightly soluble in methylene chloride; very slightly soluble in acetonitrile; insoluble in water, ethanol and isopropyl alcohol

Category

Teriflunomide is an immunomodulatory medication that is the active metabolite of Leflunomide.

 

MATERIALS AND METHODS: 7

1.     Chemicals and Reagents:

Acetonitrile was used in mobile phase composition and it is also used as a diluent, which was purchased from Merck India Pvt. Ltd. Other chemicals used were Triethylamine and O- phosphoric acid of Analytical Grade purchased from local suppliers.

 

2.     Instruments:

Ultra- High Performance Liquid Chromatography (UHPLC) manufactured by Dionex with a gradient pump model UltiMate 3000 equipped with autosampler was used in the current study. The detector used was a PDA detector. There was a separate oven to adjust the temperature of the column. Column used was Sunniest C18 (250 x 4.6mm) containing particle size 5µm. Digital balance and pH meter manufactured by Mettler Toledo and ultrasonic bath manufactured by PCI- Analytics were used. Millipore filtration system with a vacuum pump and water purification system manufactured by Young Lin Instrument Co., Ltd. were used to make HPLC grade water and to remove particles from solvents, respectively.

 

3.     Selection of the optimum condition for UHPLC process:

In order to view the appropriate peak for the teriflunomide API the mobile phase used was ACN: water in the ratio of 60: 40 v/v. Other conditions were set as follows: C18 column with a length of 250mm, a diameter of 4.6mm and particle size of stationary phase 5µm, UV detection wavelength of 210nm, flow rate of 1 ml/min for mobile phase, column temperature 30°C and injection volume of 10ml.

 

4.     Preparation of solutions:

a.     Mobile Phase:

5ml Triethylamine (TEA) was added to 650ml water and pH was maintained to 3.4 with O- phosphoric acid for preparing water. This solution was sonicated for 5 min for degassing and filtered through 0.45μ Millipore filter. Final mobile phase was taken ACN: water (60: 40v/v).

 

b.    Diluent:

Acetonitrile was used as diluent.

 

c.     Teriflunomide Stock Solution (705µg/ml)

70.5mg Teriflunomide API was weighed and transferred into 100ml volumetric flask. After addition of 25ml of ACN in volumetric flask, solution was sonicated for 10 minutes by using ultrasonicator. Further volume was made uo to 100ml with same solvent (705µg/ml). Further working dilutions were prepared from prepared Standard Stock Solution.

 

 

Method Validation:

According to ICH Guidelines Method Validation is defined as, “establish the documented evidence which provides a high degree of assurance that a specific process will consistently produce a product of predetermined specifications and quantity attributes”8.

 

The HPLC method was validated in terms of System suitability parameters, Linearity, Specificity, Precision, Accuracy, LOD, LOQ and Robustness according to ICH guidelines.

 

1.     System suitability parameters:

System suitability parametersare commonly used by laboratories to ensure that the complete analytical systemto verify the chromatographic system is suitable for the intended analysis. Retention time, resolution, tailing factor and no. of theoretical plates should be within acceptable limit.

 

2.     Linearity:

Under developed experimental conditions the relationship between the peak area and concentration of drug is studied. The calibration curve is plotted against concentration vs. peak area by the prepared different aliquots of stock solution and the r2 value is determined. The test results show peak area directly proportional to the concentration of the analyte in the sample. The correlation coefficient value should be close to 1 which indicates good linearity.

 

3.     Specificity:

The specificity of an analytical method is to determine the effect of excipients and other additives thatare generally present in the formulation. It is evaluated by comparison between chromatogram of standard and test solutions. There should not be of any interfering peak with peak of analyte.

 

4.     Precision:

Method Precision: Method Precision is determined by injecting a series of standard or examining multiple samples from a homogeneous lot.

 

Intermediate precision:

It is also known as within-laboratory or within-device precision. It is a measure of precision under a set of parameters that include the same measurement process, same measuring apparatus, same location, and replicate measurements on the same or comparable objects across time.

 

5.     Accuracy:

Accuracy is referred as the closeness of test result to true value. The results were expressed as % recoveries of the particular components in the samples. The accuracy of the method is evaluated by the standard addition method. Recovery tests are carried out by analyzing drug with different compositions. Known amounts of standard drugs are added to pre-analyzed sample at three different levels, and the mixed standard solutions areanalyzed in triplicate at every level as per the suggested method.

 

6.     Limit of detection (LOD) and Limit of quantification (LOQ):

LOD is the lowest concentration in a sample that can be detected but not necessarily quantified under the stated experimental conditions and LOQ is the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy.

 

LOD = 3.3*(SD) / S

 

LOQ = 10*(SD) / S

 

Where,

SD = Standard deviation of response

S= Slope ofthe calibration curve.

 

7.     Robustness:

Robustness refers to how sensitive the method is to uncontrolled small changes in parameters such as temperature, pH of solution, mobile phase composition, flow rate. There should not be interference from blank with the main peaks. Active ingredients peak in test preparation should be spectrally pure.

 

RESULTS AND DISCUSSION:

1.     Drug Identification

Melting point measurement, solubility studies, and IR spectra were used as preliminary work for drug identification, which yielded the following results.

 

a.     Melting point determination

Teriflunomide's melting point was confirmed to be within the acceptable range, as stated in the table 2.

 

Table 2. Drug Melting Determination

Drug name

Reported melting point

Observed melting point

Teriflunomide

227- 231 °C

228 °C

 

b.    IR spectra and interpretation:

It may be inferred from the IR interpretation data that prominent functional group peaks can be found in the IR spectra of the drug sample. The IR spectra of Teriflunomide and its interpretation is given below in fig. 1 and table 3.

 


Figure 1. IR spectra of Teriflunomide

 


Table3. IR interpretation of Teriflunomide

Sr. No.

Observed Peak (cm-1)

Functional Group

1.

1159.97

C–F Stretching

2.

2219. 67

C≡N Stretching

3.

1269.9

C–O Stretching

4.

3065.3

C=C Bending

5.

1323.89

C–N Bending

 

2.     Method Development:

The UPLC separation was achieved on Dionex UltiMate 3000 UHPLC instrument with Sunniest C18 column (250 × 4.6mm, 5μm) using a mobile phase of Acetonitrile: Water (60:40; v/v) at a flow rate of 1.0ml/min.

 

Typical chromatograph of optimized trial of teriflunomide API is shown in below fig. 2 and table 4.

 

Figure 2: Typical Chromatograph of Teriflunomide (700 µg/ml)

 

 

Table 4. Parameters of final chromatograph

Ret. Time (min)

Area (µAU*sec)

Area (%)

Asymmetry

Plates

4.437

28531247

100.00

1.37

7055

 

3.     Method Validation:

a.     System suitability:

All system suitability test parameters, such as RSD of Area, Resolution (Rs), Tailing Factor (T), and no. of Theoretical Plates (N), were examined and found to be within acceptable limits, indicating that the system and chromatographic conditions are adequate for this approach. The table 5 shows results of system suitability parameters.

 

Table5. System suitability data for Teriflunomide API

Injection No.

Retention time(min)

Peak Area

Tailing Factor

Theoretical Plates

1

4.433

5648599

1.33

11265

2

4.437

5661005

1.34

11095

3

4.463

5636706

1.35

11215

4

4.453

5666772

1.33

11265

5

4.447

5690711

1.33

11300

Mean

4.447

5660759

1.34

11228

SD

0.012

20370. 023

% RSD

0.27 %

0.36 %

 

b.    Linearity:

It was found that Beer-Lambert’s law was followed in the concentration ranges of 35-247μg/ml (35.5, 70.50, 105.75, 141.0, 176.25, 246.75μg/ml) for Teriflunomide API. Correlation co-efficient for calibration curve of Teriflunomide was found to be 0.999. The equation of line for Teriflunomide is obtained as y = 41558x+ 55602. Linearity data is given below in table 6.

 

Table 6. Linearity data for Teriflunomide

Sr. No.

Concentration (µg/ml)

Peak Area

1

35.5

1512257

2

70.50

3104587

3

105.75

4344983

4

141.0

5859649

5

176.25

7401661

6

246.75

10338995

 

Figure 3. Calibration curve of Teriflunomide

 

c.     Specificity:

A blank, standard and sample chromatograms are given in figure 4, 5 and 6. All the chromatographs are resolved properly. The sample chromatogram of Teriflunomide shows no interference at RT 4.4, so it is concluded that the method is specific.

 

 

Figure 4. Chromatogram of Blank

 

 

Figure 5. Chromatogram of Standard

 

Figure 6. Chromatogram of Sample

 

d.     Precision

Method precision and Intermediate precision was carried out in this study. The results are shown in table 7 and 8.

 

·       Method Precision

Table 7. Repeatability data for Teriflunomide

Sr. No.

Peak Area

1

5707410

2

5695211

3

5719412

4

5693618

5

5707921

Mean

5704714

SD

10571.337

%RSD

0.19 %

%RSD for 5 sets of preparation for Teriflunomide (705μg/ml) was 0.19 %. The results indicate a good degree of repeatability.

 

·       Intermediate Precision

Table 8. Intermediate precision data for Teriflunomide

Sr. No.

Peak Area

1

5942205

2

5937720

3

5936774

4

5954485

5

5943628

Mean

5942962

SD

7062. 984

%RSD

0.12 %

%RSD for 5 sets of preparation for Teriflunomide (705μg/ml) was 0.12 %. The results indicate a good degree of precision.

 

e.     Accuracy:

The results of the accuracy study are shown in table 9 for Teriflunomide. Individual recovery at each level meets the established acceptance criteria. Hence, the developed method is accurate.


 

Table 9. Accuracy data for Teriflunomide

Sr. No.

Conc. Level (%)

Sample amount (µg/ml)

Amount added (µg/ml)

Peak Area

% Recovery

Average

% RSD

1

50%

100

10

7635307

98.07

97.85

0.32

2

100

10

7637380

97.63

3

100%

100

20

10275191

97.25

96.96

0.43

4

100

20

10249220

96.66

5

150%

100

30

13927853

99.31

99.32

0.02

6

100

30

13918890

99.33


f.      Limit of Detection and Limit of Quantification:

LOD and LOQ was found to be 1.61 and 4.90µg/ml, respectively.

 

g.     Robustness:

5 determinations of standard preparation were injected and results are recorded with respect to Teriflunomide peak in the table 10.


 

Table 10. Robustness study of Teriflunomide

Sr. No.

Conditions

Peak Area of 5 determinations

Mean area

SD

%RSD

Theoretical Plate

Tailing Factor

RT

1

MP composition

Water: ACN

(35: 65 v/v)

5945422

5913469

5957126

5964450

5915198

5939133

23641.992

0.40

10486

1.34

3.8

2

MP composition

Water: ACN

(45: 55 v/v)

5809610

5851803

5825615

5813421

5846784

5829447

19140.179

0.33

12671

1.31

5.2

3

Flow Rate: 0.9ml

6319561

6349266

6354355

6345122

6371040

6347869

18640.037

0.29

11098

1.31

4.9

4

Flow Rate: 1.1ml

5112150

5104037

5085962

5100101

5102240

5100898

9512.900

0.19

11703

1.35

4.0

 


CONCLUSION:

In this study, a unique and reliable method for teriflunomide quantification was developed. The developed RP-UHPLC approach was proven to be simple, economical, precise, linear, sensitive, and accurate for estimating Teriflunomide API and its marketed preparation. The procedure was thoroughly tested and yielded positive results. The approach was devoid of interference from the formulation's other active components and additives12.

 

The mobile phase was ACN: water (60:40) and the peak was obtained at 4.4 retention time. The developed method was tested for system suitability, accuracy, precision, linearity, specificity, and robustness, all of which were verified according to ICH guidelines13. As a result, it can be stated that this method can be used for routine quality control analysis of Teriflunomide API and its formulations.

 

List of Symbols:

mm     :  milimeter

ml       :  mililitre

µm      :  micrometer

v/v      :  volume/volume

nm      :  nanometer

ml/min:  mililitre/minute

µg/ml  : microgram/mililitre

R2      : Correlation coefficient

%        : Percentage

°C       : Degree Celcius

Rs       : Resolution Factor

T         : Tailing Factor

N        :  Number of Theoretical Plates

g/mol  :  gram/mole

 

List of Abbrevations:

RP-UHPLC: Reverse Phase- Ultra High Performance Liquid Chromatography

ACN           : Acetonitrile

SD              : Standard deviation

RSD           : Relative Standard Deviation

MS             :  Multiple Sclerosis

USA           :  United States of America

DHODH    :  Dihydroorate- dehydrogenase

DNA          :  Deoxyribose Nucleic Acid

RNA          :  Ribose Nucleic Acid

BCS           :  Biopharmaceutical Classification System

pKa            :  Partition Coefficient

pH              :  Potential of Hydrogen

HPLC         :  High Performance Liquid Chromatography

HPTLC: High Performance Thin Layer Chromatography

LC-MS: Liquid Chromatography- Mass Spectroscopy

API            :  Active Pharmaceutical Ingredient

UV             :  Ultra Violet

PDA           :  Photo Diode Array

TEA           :  Triethanolamine

ICH            :  International Council of Harmonization

LOD           :  Limit of Detection

LOQ           :  Limit of Quantitation

 

ACKNOWLEDGEMENT:

The authors were thankful for STES, Sinhgad Institute of Pharmacy, Narhe, Pune for providing necessary infrastructural facilities to carry out research work.

 

DISCLOSURE STATEMENT:

No potential conflict of interest was reported by authors.

 

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Received on 06.05.2022       Modified on 18.05.2022

Accepted on 02.06.2022   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2022; 12(3):166-172.

DOI: 10.52711/2231-5675.2022.00028