Int J Pharm Pharm Sci, Vol 14, Issue 9, 19-23Original Article



All India Shri Shivaji Memorial Society’s College of Pharmacy, Department of Quality Assurance, Kennedy Road, Near RTO, Pune 411001
Email: [email protected]

Received: 26 May 2022, Revised and Accepted: 06 Jul 2022


Objective: Teriflunomide is used for the treatment of multiple sclerosis and is available in 7 mg and 14 mg tablets. This study aimed to develop and validate a simple and economical HPTLC method for the estimation of Teriflunomide in human plasma.

Methods: HPTLC method was developed using toluene: ethyl acetate: acetic acid as the mobile phase and the stationary phase was a TLC plate precoated with silica gel 60 F254. The detection wavelength set was 294 nm. The sample preparation involved a simple protein precipitation technique with Acetonitrile as a precipitating protein agent; the internal standard selected was Rilpivirine. The validation was carried out as per bio-analytical method guidelines.

Results: The Rf value for Teriflunomide was found to be 0.46±0.04. The linearity range was observed from 10-60 µg/ml with a regression coefficient value of 0.9819. The developed method was validated for various parameters like specificity, linearity, accuracy, precision, recovery, and stability.

Conclusion: The developed method is simple, specific, accurate, and economical for the estimation of Teriflunomide in human plasma.

Keywords: Teriflunomide, HPTLC, Protein precipitation, Rilpivirine, Bioanalytical


Teriflunomideis the active metabolite of Leflunomide which is a pyrimidine synthesis inhibitor used for the treatment of relapsing forms of multiple sclerosis. Also, it has anti-inflammatory and immunomodulatory properties. Chemicallyit is (Z)-2-cyano-3-hydroxy-N-[4-(trifluoromethyl) phenyl] but-2-enamide [1].

Fig. 1: Chemical structure of Teriflunomide

Teriflunomide selectively and reversibly inhibits dihydro-orotate dehydrogenase, which is a key enzyme in the pyrimidine synthesis pathway. As Teriflunomide inhibits this enzyme leads to a reduction in the proliferation of activated T and B lymphocytes without causing cell death [2]. The oral bioavailability of Teriflunomide is 100%, with peak plasma levels achieved within 1-2 h of intake and Cmax ranging from 19-45µg/ml. Teriflunomide has a mean plasma half-life of 10-18 d [3].

The literature survey revealed that there are many papers [4-12] on the bioanalytical method of Teriflunomide using LC-MS/MS, LC-UV, and UPLC. But there is no paper reported on the bioanalytical method for Teriflunomide using HPTLC. Bioanalytical methods involve the determination of analytes of interest in various biological matrix-like plasma, serum, urine, etc [13]. Some of the advantages of HPTL Conclude, a short analysis time, cost-efficient analysis, prior treatments for solvents like filtration and degassing can be evaded, and a fresh stationary phase and mobile phase are used for analysis which helps to prevent carryover.


Chemicals and reagents

Teriflunomide was obtained as a gift sample from Natco Pharmaceuticals, Hyderabad, India. Rilpivirine was received as a gift sample from Mylan laboratories, Hyderabad, and was used as an internal standard. Methanol and ethyl acetate was of HPLC grade purchased from Merck Lifesciences, Pvt. Ltd., Mumbai, and toluene and acetic acid used were of AR grade purchased from Loba Chemie, Pvt. Ltd., Mumbai, India. Pooled plasma was obtained as a gift sample from Sassoon hospital, blood bank, Pune, India.


Camag HPTLC system with win CATS software version 1.4.2, Shimadzu UV 1780 UV-Visible spectrophotometer, Japan, Shimadzu AY 120 Analytical balance, Japan, Remi Cyclo-mixer and Remi Centrifuge R-302.


Chromatographic conditions

HPTLC method

The chromatographic resolution was performed on a Merck TLC plate precoated with silica gel 60 F254 using a Camag Linomat V sample applicator. Samples were applied on the plate in the form 6 mm band using a Camag 100 µl sample syringe. 20×10 twin trough chamber was used for ascending development with mobile phase toluene: ethyl acetate: acetic acid (6.5:3:0.5v/v/v). Saturation time was set at 20 min; the plate was allowed to develop up to70 mm of distance. Densitometric scanning was performed on a Camag TLC scanner III at 294 nm for all developments operated by Win cat’s software version 1.4.2. Deuterium and tungsten lamps were used as the radiation source.

Method development

Selection of mobile phase

The mobile phase first tried was toluene: ethyl acetate: glacial acetic acid in the ratio of 7.5:2:0.5 v/v/v [14]. To obtain well-resolved peaks of internal standard and Teriflunomide, the mobile phase was optimized to toluene: ethyl acetate: glacial acetic acid in the ratio of 6.5:3:0.5 v/v/v.

Selection of internal standard

Based on the λmax of the drug, various internal standards like Ivabradine hydrochloride, Mifepristone, Nebivolol, Rilpivirine, and Paliperidone palmitate were tried. Rilpivirine showed better resolution, so it was selected as the internal standard.

Preparation of stock solution of teriflunomide

The stock solution was prepared by dissolving 10 mg Teriflunomide in methanol in a volumetric flask of 10 ml to obtain a concentration of 1000 µg/ml. Further dilution was made to obtain the concentration of 10 μg/ml.

Preparation of stock solution of internal standard

10 mg of Rilpivirine was dissolved in methanol in a volumetric flask of 10 ml to get a concentration of 1000 µg/ml.

Preparation of spiked plasma sample

In a test tube, 0.1 ml stock solution of Teriflunomide (10-60 µg/ml) and 0.1 ml of internal standard (1000 µg/ml) were added to 0.8 ml plasma to obtain the required QC (Quality Control) samples. The QC samples were vortexed and plasma proteins were precipitated using 1 ml of acetonitrile and centrifuged to obtain supernatant. This supernatant was spotted.

Selection of detection wavelength

The wavelength selected was 294 nm based on the UV spectrum taken at a concentration of 20 µg/ml. The UV spectrum is shown in fig. 2.

Fig. 2: UV spectrum of teriflunomide

Method validation

The developed method was validated as per the USFDA guidelines for bioanalytical methods [15-17].

QC samples

Different QC samples were prepared based on the Cmax of the drug and linearity range, namely, Low-Quality Control (LQC), Middle-Quality Control (MQC), and High-Quality Control (HQC) samples. These samples were used for the determination of method accuracy, precision, and stability of the sample.

Calibration curve

The calibration curve or linearity of the method exhibits direct proportionality between detector response and concentration of the analyte of interest. Three replicates of each QC sample were analyzed and peak areas were recorded. The response factor was calculated by taking the ratio of the area of Teriflunomide and the area of internal standard.

Specificity and selectivity

Selectivity of an analytical method is the ability of the method to differentiate and quantify the drug sample in the presence of other interfering substances. Specificity is the ability to discriminate and quantify the analyte of interest in presence of various other matrix components in the sample. The specificity of the method is demonstrated by analyzing blank plasma, zero plasma, and spiked plasma. No interfering peaks should be observed at the same Rf value of Teriflunomide.


The accuracy was estimated by analyzing three different runs of freshly prepared Quality Control samples. The accuracy was determined by analyzing peak areas and the % mean accuracy was calculated.


Precision includes the determination of Intermediate precision as well as repeatability. Intermediate precision represents within laboratory variation [18]. Precision should include three independent runs and the % CV was determined.


Recovery of method expresses the measures of the efficiency of extraction procedure within variation limit. It is performed by comparing the results obtained from the analyte or internal standard from the extracted samples at different concentrations with standard solutions [19].


The purpose of determining stability is to detect any degradation of the analyte that occurred during the entire process of sample collection, storage, extraction, and analysis. It is recommended to determine stability during short-term storage, long-term storage as well as freeze-thaw cycles. Stability samples should be compared with freshly prepared QC samples.


Method development

In the current work, the HPTLC method was optimized for the estimation of Teriflunomide from human plasma. This method involves use of less solvent as a mobile phase when compared to HPLC method. Also, the optimized method does not involve any preconditioning like maintenance of column temperature as reported in HPLC methods [4-12]. HPTLC has several advantages over the HPLC technique, which involves the use of a renewed stationary phase (a new TLC plate for every run), and time-cost saving because many samples can be analyzed in one run on a single plate using a few milliliters of the mobile phase.

Method validation

Calibration curve

The method was found to be linear over the range of 10–60 µg/ml when concentration was plotted against the response factor. The regression coefficient was found to be 0.9819.

Table 1: Linearity

Plasma concentration (µg/ml) Response factor* % CV
10 0.15±0.0075 5.05
20 0.24±0.0198 8.25
30 0.31±0.0008 0.26
40 0.45±0.0049 1.10
50 0.62±0.0091 1.48
60 0.72±0.0240 3.34

*Data given in mean±SD, n = 3.

Specificity and selectivity

The method was found to be specific as no interfering peaks were observed at the Rf of Teriflunomide.


The method was found to be accurate as the % mean accuracy at each concentration was found to be in the acceptable range, as shown in table 2.

Fig. 3: Calibration curve of teriflunomide spiked plasma

Table 2: Accuracy

Concentration(µg/ml) (n=5) LQC (4.5) MQC (27) HQC (50)
Spotted amount(ng/band) 90 540 1000
Amount found (mean±SD) 100.72±2.96 549.56±10.03 939.37±0.29
% CV 2.93 1.82 0.03
% Mean accuracy 111.91 101.77 93.94

Table 3: Precision

Intermediate precision (n=5) Concentration(µg/ml) LQC (4.5) MQC (27) HQC (50)
Spotted amount (ng/band) 90 540 1000
Amount found (mean±SD) 100.22±3.18 538.21±9.12 937.22±3.18
% CV 3.17 1.69 0.33



Concentration (µg/ml) LQC (4.5) MQC (27) HQC (50)
Spotted amount (ng/band) 90 540 1000
Amount found (mean±SD) 100.72±2.96 528.84±8.88 936.24±3.62
% CV 2.93 1.67 0.38

Table 4: Recovery

Concentration (ng/band)


Peak area (mean±SD) % CV % Mean recovery % Overall mean recovery Overall SD (Recovery)

Overall % CV


32 460.22±3.4539 0.75 76.85 75.5 3.42 4.53
598.8±4.5782 0.76
90 675.54±10.7123 1.58 74.19
910.54±10.9680 1.20
200 1000.26±7.9060 0.79 72.71
1375.64±17.0468 1.23
320 1101.8±5.0275 0.45 73.53
1498.26±6.5435 0.43
540 1714.38±54.1313 3.15 71.68
2391.6±6.0573 0.25
760 2308.62±9.4414 0.40 76.95
2999.98±5.1878 0.17
1000 2956.76±8.2432 0.27 82.59
3579.74±17.5449 0.49

Table 5: Stability

S. No. Stability (n=6) % Mean stability % CV
1 Freeze-thaw stability (Three cycles) LQC 96 2.79
HQC 96.33 1.77
2 Short-term stability (for 4h at RT) LQC 96.99 3.98
HQC 98.92 1.02
3 Long-term stability (for 14 d at 4 °C) LQC 95.67 1.88
HQC 98.20 2.14
4 Stock solution stability (for 5 d) MQC 96.60 2.01
IS 98.53 0.43
5 Post preparative stability (for 5hat RT) MQC 97.61 0.71
IS 99.09 0.26

n = 6.


The %CV for each concentration was calculated and was found to be within the limit based on the result given in table 3.


The recovery was determined by comparison of standard samples with that of spiked plasma samples and the overall % CV was calculated, which was found within the limit.


By comparing the peak areas of spiked stability QC samples with peak areas of freshly prepared QC samples, the % mean stability was calculated and the results are summarised in table 5.


The optimized method was found to be simple, economic, and linear over a wide concentration range. A simple, easy protein precipitation technique was used for sample pre-treatment. The high throughput advantage of HPTLC makes the method rapid. It may be used for therapeutic drug monitoring.


The authors are thankful to AISSMS College of Pharmacy, Pune, for providing the necessary analytical instruments to carry out this work.


No funds were received from any agency.


Pooja Gurav studied, carried out the research work, and drafted the manuscript under the guidance of Mrinalini Damle. The manuscript was checked and approved by Mrinalini Damle.


The authors declared that they have no conflict of interest.


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