INTRODUCTION:

Leflunomide is a pyrimidine synthesis inhibitor that is a member of the highly wide-ranging and diverse DMARD (disease-modifying antirheumatic drug) class of drugs.^1-2. chemically, leflunomide is 5-Methyl-N-[4(trifluoromethyl)phenyl] -isoxazole 4-carboxamide, with the chemical formula C12H9F3N2O2, is shown in Figure 1. Leflunomide is a disease-modifying antirheumatic medication (DMARD) that inhibits pyrimidine production³.

Figure 1: Structure of Leflunomide

According to the literature review, leflunomide can be found in pharmaceutical dosage forms, metabolites, and plasma using UV, TLC, LC-MS, and HPLC methods. The most popular technique for analysing leflunomide is HPLC. Not all of these, though, indicate stability. Therefore, the goal of the current study is to create and evaluate a stability-indicating HPLC approach for the measurement of leflunomide from tablets^4-11. In accordance with ICH Guidelines^12–14, this work also addresses the forced degradation of leflunomide under stress conditions such as acid hydrolysis, base hydrolysis, and oxidation, heat stress, and photolytic stress. The method is thus beneficial for determining stability as well as for normal quality control examination.

MATERIAL AND METHODS:

Instrumentation:

Shimadzu, Kyoto, Japan's LC-20AD binary pump, SPD-M20A photodiode-array detector, and rheodyne injector with 20-loop made up the chromatographic system. These components were connected to a multi-instrument data collecting and data processing system (LC Solution, software by Shimadzu). A Shimadzu model 1800 double beam UV/Vis spectrophotometer with a pair of 10mm matched quartz cells, a Sartorious analytical balance, an ultrasonicator, and a Hypersil BDS C18 column with a dimension of 250mm x 4.6mm, 5m, were also employed.

Reagents and Materials:

From the market, leflunomide standard and tablets containing 10mg of leflunomide were purchased. Acetonitrile, water, and Analytical Grade (MERCK Chem Ltd., Mumbai) chemicals were all utilized. The experiment employed sodium hydroxide, hydrogen peroxide, and hydrochloric acid AR grade.

Chromatographic Conditions:

With the mobile phase consisting of acetonitrile and 0.02M ammonium acetate buffer (60:40v/v), the chromatographic estimation was performed on a Hypersil BDS C18 column at a flow rate of 1.0mL/min. Before use, the mobile phase was degassed and filtered through a nylon 0.45m membrane filter. The measurement was made using a Photo Diode Array (PDA) Detector at 260nm wavelength. The overall run duration was 10minutes, and the injection volume was 20 L. Temperatures of 25 2°C were used to conduct the analysis.

Preparation of standard solution:

Accurately dissolving 10.0mg of leflunomide (100 g/mL) in a 100mL volumetric flask using acetonitrile-buffer (50:50, v/v) as a diluent allowed for the creation of the standard solution. Leflunomide was made at a concentration of 20g/mL by further diluting 2mL of stock solution in a 10mL volumetric flask with standard solution.

Preparation of hydrochloric acid (0.1N):

Transferred 0.85mL of concentrated hydrochloric acid (36%) precisely into a 100mL volumetric flask, then diluted it with distilled water to the appropriate strength.

Preparation of sodium hydroxide (0.1N):

0.4 gram of sodium hydroxide were precisely weighed, transported, and dissolved in 60mL of distilled water before being diluted to the appropriate strength with the same liquid.

Analysis of Tablet Dosage Form:

Weight 25mg of Leflunomide precisely, then transfer it to a 25mL volumetric flask with 15mL of diluent and sonicate it for 15 minutes. Through a 0.45 membrane filter, the diluted solution was filtered. Leflunomide was diluted to 20g/mL using standard solution after being further diluted from 2mL of stock solution in a 10mL volumetric flask.

Forced Degradation Study:

Leflunomide was produced by putting the sample under different stressful conditions. The examination into force degradation provides information on the circumstances under which the medicine is unstable.

Influence of Acid, Alkaline and Neutral Hydrolysis, Oxidation:

Accurately weighed 20mg Leflunomide was transferred to three different 100mL volumetric flasks and dissolved in diluent (20mL). Hydrochloric acid (0.1N, 10mL), sodium hydroxide (0.1N, 10mL) and water (10mL) were added to separate flasks containing drug samples and mixed properly for both degradation respectively and stored for 72 hr at room temperature.The samples were neutralized with base or acid as appropriate and diluted up to the marks with diluent to obtain stock solutions (200µg/mL). To obtain the deteriorated Leflunomide solutions (20g/mL), mobile phase dilutions were performed. Leflunomide 20mg powders was precisely weighed for oxidation, transported to a 100 mL volumetric flask, and dissolved using 20mL of diluent. 10mL of H2O2 (3%) was added, and it was left at room temperature for 72 hours. To create the stock solution (200g/mL), the sample was diluted with acetonitrile up to the desired level. To get the degraded Leflunomide solution (20g/mL), mobile phase was diluted.

Influence of Heat and Light:

The degraded Leflunomide solution (20g/mL) was obtained by dilutions with mobile phase using an accurately weighed powder equivalent to 20mg of Leflunomide placed onto a glass plate and stored in an oven at 60°C for 72 hours. Leflunomide solution (made by combining 20mg of Leflunomide-equivalent powder with 20mL of acetonitrile in a 100mL volumetric flask) was exposed to sunlight for 48 hours while Leflunomide-equivalent powder (20mg) was subjected to UV light for the same amount of time. Dilutions were created after exposure to produce the deteriorated Leflunomide solutions (20g/mL). As mentioned under chromatographic conditions, aliquots (20L) of the stressed samples were injected into the HPLC apparatus. Chromatograms were then recorded.

Method Validation:

The technique validation characteristics, such as specificity, linearity, accuracy, precision, limit of detection, limit of quantitation, and robustness, were examined in accordance with ICH guideline Q2 (R1).

Solution Stability:

The storage temperatures for the sample solutions were 25 2°C for 24 hours and 2 – 80°C for 3 days. These two time periods were compared to the assay percentage of the initial time period. It was computed how much the assay % had changed. For formulation, the test result discrepancy shouldn't exceed 2%, and for API, it shouldn't exceed 5%.

Specificity:

The capacity of an analytical method to measure an analyte precisely and specifically in the presence of components that may be anticipated to be present in the sample matrix is known as specificity. Leflunomide solution chromatograms and samples that had been degraded were examined to determine the method's specificity and stability indicating qualities. Acidic, alkaline, neutral, oxidative, thermal, and photolytic stress conditions were used, and the degraded samples were compared to freshly made sample solutions.

Linearity (Calibration Curve):

In a series of 10mL volumetric flasks, standard solutions (0.05, 0.1, 0.15, 0.2, 0.25, and 0.3mL, respectively, equal to 5.0, 10.0, 15.0, 20.0, 25.0, and 30.0g/mL of Leflunomide) were transferred and diluted to the target concentration with diluent. Under the previously mentioned operating chromatographic conditions, a 20-L aliquot of each solution was injected. Peak areas versus concentrations were plotted to create the calibration curve, and the regression equation was computed. The average of three determinations was used for each response.

Accuracy (% Recovery):

Accuracy of the method was determined by calculating percentage recovery of Leflunomide by the standard addition method. Known amount of standard solutions of Leflunomide (0, 5, 10 and 15µg/mL) were added to a pre-analyzed sample solution of Leflunomide (10 µg/mL). Each solution was injected in triplicate and the percentage recovery was calculated by measuring the peak areas and fitting these values into the regression equation of the calibration curve.

Precision:

Leflunomide solution (10g/mL) was repeatedly injected (n = 6) to test the repeatability, and the chromatogram was recorded each time. By measuring the equivalent responses three times on the same day and three other days over the course of a week for three different concentrations of Leflunomide (10.0, 20.0, and 30.0 g/mL), the intra-day and inter-day precisions of the devised approach were determined. Relative standard deviation was used to describe the outcomes.

Limit of Detection and Limit of Quantification:

The calibration curve's slope (S) and standard deviation of response () were used to calculate the calibration curve's limit of detection (LOD) and limit of quantitation (LOQ).

Robustness:

By deliberately varying the technique parameters, samples of Leflunomide were analysed and robustness was investigated. It was observed that Leflunomide reaction had changed. By adjusting the extraction duration of Leflunomide from the tablet dosage form by 2 min, the mobile phase's composition by 2% of organic solvent, the wavelength by 2 nm, the flow rate by 0.2 mL/min, and the column oven temperature by 2^oC, the robustness of the method was examined. Leflunomide response variations were noticed and contrasted with the initial reaction.

System-Suitability Test:

System suitability tests were done to ensure that the system's resolution and repeatability were sufficient for the required analysis. Retention duration, tailing factor, and theoretical chromatographic peak plates as RSD of peak area for replicate injections were the parameters employed in this assay.

RESULTS AND DISCUSSION:

Selection of Column and Mobile Phase:

Leflunomide can be analysed using reverse phase liquid chromatography (RP-HPLC), in accordance with the published literature and current knowledge of the molecule.

Different columns are available for RP-HPLC, however as the method's primary goal was to separate the compound from degradation products, Hypersil BDS C18 column (250mm x 4.6mm i.d. 5m particle size) was chosen in preference to the others. The most crucial criterion for the approach is resolution; it is essential to acquire good resolution between the chemical and degradation products. Different mobile phase compositions were tested in accordance with the compound's solubility and pKa value.

The chromatographic conditions were optimized with a view to develop a stability indicating assay method, which can separate the drug from its degradation products with good resolution. Mobile phase consisting of acetonitrile: ammonium acetate buffer (60:40% v/v) at a flow rate of 1.0mL/min, was found to be satisfactory to obtain well-resolved peaks with better reproducibility and repeatability for Leflunomide.

Method Validation:

Solution Stability:

It was assessed how the assay results changed after storage at 25°C for 24 hours and 2-8°C for 3 days. It was discovered that the variation in assay results for formulation and API was less than 2% and 0.5%, respectively, indicating stability of the leflunomide solution.

Specificity:

The developed analytical method was found to be specific as there was no inference of any related impurities after the stress degradation study.

Figure 3: Calibration curve of Leflunomide

Linearity:

The linearity of the calibration curve was validated by the value of the correlation coefficient of the regression (r), which is y = 38217x + 72833. The linear correlation was found between the peak area and the concentration of leflunomide in the range of 5-30 g/mL.

Accuracy (% Recovery)

Utilizing the traditional addition approach, the accuracy investigation was conducted. The accuracy of the approach was demonstrated by the percent recoveries, which were determined to be within the range of 98.86-101.12%.

Table 1: Results of recovery study (n=3)

Amount Taken (µg/mL)	Amount added (µg/mL)	Amount found (µg/mL)	Recovery ± S.D, %	% RSD
10	0	10.05	100.50 ± 0.92	0.92
10	5	14.83	98.86 ± 1.65	1.65
10	10	19.90	99.50 ± 1.37	1.37
10	15	25.28	101.12 ± 0.77	0.77

Precision:

Leflunomide repeatability was determined to have a % RSD of 1.25 (Table 2). The range of 0.93 to 1.15 percent RSD for intraday precision and 1.07 to 1.2 percent RSD for interday precision, respectively, showed that the approach was accurate. (Table 3)

Table 2 Results of repeatability (n=6)

Drug	Leflunomide
Drug	Peak area
1	443614.0
2	454953.1
3	443275.4
4	445228.7
5	448743.8
6	438564.2
Mean	445729.9
SD	5590.19
% RSD	1.25

Limit of detection and limit of quantification:

The Limit of detection (LOD) for Leflunomide was found to be 0.058µg/mLwhile the Limit of quantification (LOQ) was 0.165µg/mL

Robustness:

The approach was determined to be reliable because only minor variations in extraction time, mobile phase composition, and mobile phase flow rate had a noticeable impact on the outcomes.

System-Suitability Test:

The % RSD of system-suitability test parameters was found satisfactory. The results are listed in Table 4.

Table 4: System suitability test parameters (n = 6)

No.	Retention time	Tailing factor	Theoreticalplates
1	4.41	1.49	9282.12
2	4.41	1.49	9254.23
3	4.35	1.48	9237.48
4	4.27	1.49	9187.75
5	4.38	1.47	9265.58
6	4.41	1.45	9176.38
Mean	4.37	1.48	9233.92
SD	0.055	0.016	42.88
% RSD	1.26	1.08	0.46

Analysis of Tablet Dosage Form:

Leflunomide from tablet dosage form was effectively determined using the suggested RP-HPLC method. Leflunomide content was determined to be good; this result was consistent with the corresponding label claim.

Table 5: Analysis results of tablet dosage form (n=3)

Drug

No.

of Injection

Amount Taken

Amount found

Label Claimed

Amount Found per Tablet

% Label Claim

Lefluno-mide

30 mg

29.72 mg

15 mg

14.86 mg

99.07

Degradation Study:

Forced degradation study of Leflunomide was carried out under various stress conditions as follows:

Effect of Acid, Alkaline and Neutral Hydrolysis:

Effect of Oxidation:

In oxidation stress condition, almost 18.49% of Leflunomide was degraded and degradation peak appeared in chromatogram.

Effect of Heat:

Under dry thermal stress condition, Leflunomide was degraded about 1.13% with degradation product. Leflunomide was found to decompose 32.65% under acidic stress with a major degradation product at retention time of about 3.88 min and minor degradation product at retention time of about 2.30 min. Under basic stress, leflunomide decomposed about 6.32% minutely with a degradation product at retention time of about 2.30 min. No degradation was noticed under the neutral degradation condition.

Effect of light:

No degradation was seen when leflunomide in its solution form was exposed to sunlight or when leflunomide in its powder form was exposed to UV radiation.

The samples that were subjected to heat, photolytic, oxidative, neutral, acidic, alkaline, and neutral conditions were colourless. Leflunomide was discovered to be stable in photolytic stability and showed no deterioration. The percentage of degradation for each condition showed that all degradates were separated from the peak of leflunomide, proving that there was no interference from them in determining the dosage of leflunomide in tablet form. Thus, the proposed, method was found to be "Stability Indicating".

Table.6: Results of forced degradation study

Stress conditions/duration	% Degradation
Acidic/0.1N HCl	32.65
Alkaline/ 0.1N NaOH	6.32
Oxidative/ 3% H202	18.49
Thermal 60°C	1.13

CONCLUSION:

For the estimation of Leflunomide in tablet dosage form, a stability-indicating reverse phase liquid chromatographic method has been created and validated. After conducting a stress degradation study, .the method was found to be specific because there was no interference from any co-eluting impurities. The proposed approach was discovered to be straightforward, precise, accurate, sensitive, and resilient. As a result, it can be used to analyse stability samples obtained during an expedited stability study as well as for the routine analysis of leflunomide in pharmaceutical dosage forms.

ACKNOWLEDGMENT:

We are thankful to Principal Dr. P. K. Deshmukh to provide necessary library facilities to carry out literature searches on Leflunomide.

CONFLICT OF INTEREST:

Authors do not have any conflict of interest.

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Received on 15.06.2022 Modified on 13.09.2022

Asian J. Pharm. Ana. 2023; 13(2):93-98.

DOI: 10.52711/2231-5675.2023.00016

Lefluno-mide (µg/mL)	Intra-day precision		Inter-day precision
Lefluno-mide (µg/mL)	Mean peak area ± SD	% RSD	Mean peak area ± SD	% RSD
10	443614.0 ± 4152.12	0.91	448745.3 ± 4836.18	1.04
20	875777.0 ± 9638.35	1.08	878418.6 ± 9858.21	1.07
30	1200410.0 ± 13864.16	1.13	1201059.6 ± 14728.26	1.12