INTRODUCTION:

Leflunomide [N-[4',5'-trifluoromethylphenyl]-5-methylisoxazole-4-carboxamide] is an isoxazole derivative used as an anti-rheumatic medication, with a molecular weight of 270.2 (fig. 1)¹.

Its mechanism of action involves the selective inhibition of dihydro-orotate dehydrogenase^2,3, a crucial enzyme in the de novo synthesis of pyrimidines, which subsequently suppresses the production of ribonucleic acid and deoxyribonucleic acid⁴. Leflunomide is particularly effective against activated T cells, which primarily produce pyrimidines through the de novo pathway⁵. Recent studies have explored leflunomide's immunomodulatory and anti-inflammatory effects, including the blockade of tumor necrosis factor⁶. The drug inhibits the activation of the transcription factor NF-κB, which in turn reduces reactive oxygen radicals⁷. Additionally, leflunomide increases the ratios of tissue inhibitors of metalloproteinases (matrix metalloproteinases) by preventing the migration of polymorphonuclear leukocytes into the rheumatoid synovial cavity and suppressing matrix metalloproteinases, thus affecting patients with its metabolism⁸. Plasma concentrations of leflunomide have been measured using methods such as LC-MS and HPLC. A recent study detailed the pharmaceutical determination of leflunomide using FIA-UV. The approach proposed in this study, which involves UV detection, is rapid and sensitive, making it suitable for frequent quality testing of leflunomide in pharmaceutical formulations⁹. The method was validated based on criteria such as linearity, accuracy, precision, and robustness, and the experimental design was confirmed for its robustness and intermediate accuracy.¹⁰

Figure 1. Structure of Leflunomide

HPLC methods for analyzing leflunomide have been well-documented¹¹. Literature reviews indicate that HPLC is the most widely used technique for this purpose, with previous studies reporting leflunomide's retention time ranging from 3 to 11 minutes. This study aims to reduce the retention time for leflunomide analysis, both in its pure form as an active pharmaceutical ingredient and in pharmaceutical dosage forms. We present a rapid and sensitive HPLC method with PDA detection for the systematic analysis of leflunomide in tablet formulations. This method achieves a shortened retention time of 2.65 minutes using a mobile phase of methanol and water (95:5).

MATERIALS AND METHODS:

HPLC System:

Chromatographic separation was performed using the RP-HPLC Waters system, specifically the Waters Model No. 2690/5 with a PDA detector, and data processing was handled by Waters Empower-2 software from Waters Corporation. The analysis utilized an Inertsil-ODS C18 column (250 x 4.6mm, 5μ).

Sonicator:

Sonication of solvents and various preparations was done by using the sonicator of the IKON Industries Ultrasonic Bath.

UV System:

The wavelength of leflunomide was determined using a Systronics-UV Model No. 119 equipped with a pair of 10mm matched quartz cells. A standard solution was scanned in a UV spectrophotometer in spectrum mode over the range of 200nm to 400nm to calculate the wavelength.

Reagents:

MERCK Limited, Mumbai, contributed HPLC-grade methanol and water, whereas Bharath Life Science Pvt. Ltd. provided a pure sample of working-standard leflunomide as a gift.

Experimental Work:

1. Preparation of standard solution:

Take 100mg of leflunomide working standard in 100ml of V.F., add methanol, and sonicate it for 30 minutes. (That is a 1000ppm solution.).

2. Preparation of stock solution preparation:

Take 100mg of leflunomide working standard in 100ml of V.F., add methanol, and sonicate it for 30 minutes. (That is a 1000ppm solution.).

Further Dilution (or) Optimized Method Solutions Preparation: Take 4ml of the above solution in 100 ml of V.F. and add methanol up to mark sonicate it for 10 minutes (that 40ppm solution).

Validation Parameters:

Validation Stock Solution Preparation:

Take 100mg of leflunomide working standard in 100ml V.F., add methanol, and sonicate it for 30minutes. That is 1000ppm. solution).

Validation Parameters, Solutions, and Preparation:

Prepare the following solutions by taking specific volumes from the above stock solution, diluting with methanol in a 100ml volumetric flask (V.F.), and sonicating for 10minutes:

· Take 2ml of the stock solution, add methanol to the 100ml mark, and sonicate (20ppm solution).

· Take 3ml of the stock solution, add methanol to the 100ml mark, and sonicate (30ppm solution).

· Take 4ml of the stock solution, add methanol to the 100ml mark, and sonicate (40ppm solution).

· Take 5ml of the stock solution, add methanol to the 100ml mark, and sonicate (50ppm solution).

· Take 6ml of the stock solution, add methanol to the 100ml mark, and sonicate (60ppm solution).

· Take 7ml of the stock solution, add methanol to the 100ml mark, and sonicate (70ppm solution).

RESULTS AND DISCUSSION:

HPLC method optimization:

For method optimization, various mobile phases were tried in different ratios.

Optimized Method Stock Solution Preparation:

Weigh 100mg of leflunomide working standard and dissolve it in a 100ml volumetric flask (V.F.) with methanol, then sonicate for 30minutes to prepare a 1000 ppm solution.

For further dilution (or) optimized method solutions preparation, take 4ml of this 1000ppm solution, dilute it with methanol to the 100ml mark in a V.F., and sonicate for 10minutes to obtain a 40ppm solution.

Chromatographic Conditions:

Table 1. Chromatographic condition for optimized trial.

Parameter	Method
Stationary phase (column)	Inertsil-ODS C18(250 x 4.6mm, 5µ)
Mobile Phase	Methanol: Water (95:5)
Flow rate	1.0ml/min
Run Time	6min
Column of Temperature	Ambient
Volume of Injection loop	20
Detection Wavelength	274nm
Drug RT (min)	2.65min

Figure 2. Optimized Method Development Trial Chromatogram of Leflunomide

Validation Parameters:

When method development and optimization are complete, it is necessary to accomplish method validation¹⁴.

1. System suitability:

A standard solution was prepared using the leflunomide working standard according to the test method and injected into the HPLC system five times. System suitability parameters were assessed from the standard chromatograms by calculating the relative standard deviation (RSD) from the five replicate injections for leflunomide, including retention times and peak areas.

Table 2. System suitability data

Injection	RT	Peak Area	USP Plate Count	USP Tailing
1	2.649	674753	10953.609	1.15353
2	2.650	674261	10951.014	1.15527
3	2.652	675298	10003.278	1.15774
4	2.649	679221	10986.906	1.15949
5	2.652	688636	10946.878	1.15282
Mean	2.6504	678433.8	10768.34	1.15577
SD	0.001517	6031.135	-	-
% RSD	0.057221	0.888979	-	-

Specificity:

Solutions of the standard and sample were prepared as per the test method and injected into the chromatographic system

Figure 3. Blank Chromatogram

Figure 4. Standard Chromatogram

1. Precision:

Repeatability:

System precision: A standard solution was prepared according to the test method and injected five times.

Method precision: Six individual sample preparations were made according to the test method, and each solution was injected separately.

System Precision:

Table 3. System precision data

Concentration 40 ppm	Injection	Peak Areas of Leflunomide	% Assay
	1	671753	101.5
	2	671261	101.4
	3	671298	101.4
	4	670221	101.2
	5	670636	101.3
Statistical Analysis	Mean	671033.8	101.36
	SD	603.647	0.114018
	% RSD	0.089958	0.112488

Method Precision:

Table 4. Method precision data

Concentration 40 ppm	Injection	Peak Areas Leflunomide	% Assay
	1	663495	100.2
	2	665992	100.6
	3	669828	101.2
	4	661098	99.85
	5	663241	100.2
	6	661322	99.88
Statistical Analysis	Mean	637312	100.3217
	SD	5988.879	0.509133
	% RSD	0.0891	0.507501

Intermediate precision:

Table 5. Intermediate precision data:

Concentration 40 ppm	Injection	Peak Areas of Leflunomide	% Assay
	1	666792	100.71
	2	664360	100.34
	3	655696	99.03
	4	664147	100.31
	5	664127	100.30
	6	652525	98.56
Statistical Analysis	Mean	644607.8	99.875
	SD	6392.59	0.863313
	% RSD	1.183	0.864394

2. Accuracy:

Accuracy was assessed through recovery studies of leflunomide by adding a known amount of standard to a pre-analyzed sample and performing the proposed HPLC analysis13. An accuracy study was conducted by performing the drug assay in triplicate according to the test method, with an equivalent amount of leflunomide in each volumetric flask for each spike level to achieve concentrations corresponding to 50%, 100%, and 150% of the labeled amount. The average recovery of leflunomide was then calculated.

Table 6. Accuracy data

Concentration % of Spiked level	Amount Added (ppm)	Amount found (ppm)	% Recovery	Statistical Analysis of % Recovery
50 %	20	20.04	99.49	Mean- 99.44 % RSD- 0.0628
50%	20	19.97	99.46
50%	20	20.02	99.37
100 %	40	40.01	101.4	Mean- 100.57 %RSD-0.721534
100%	40	40.05	100.3
100%	40	40.05	100.03
150%	60	60.8	99.81	Mean-99.8366 %RSD-0.02520
150%	60	59.97	99.84
150%	60	59.98	99.86

3. Linearity:

A range of solutions is prepared using the leflunomide working standard across concentration levels spanning from 20ppm to 70ppm of the intended target concentration.

Table 7. Linearity data (concentration vs peak area)

Concentration (ppm)	Average Area
0	0
20	328546
30	488296
40	670413
50	830308
60	992582
70	1158499

Statistical Analysis:

Table 8. Statistical analysis of Linearity

Slope	16594
y-Intercept	1648
Correlation Coefficient	0.9998

Figure 5. Plot of Linearity (concentration vs peak area)

4.Ruggedness:

System to system variability:

A variability study between different HPLC systems was conducted under similar conditions at various times. Six samples were prepared and analyzed according to the test method. Comparing the results obtained from two different HPLC systems indicates that the assay test method demonstrated robustness against system-to-system variability.

For system 1, refer to system suitability data in Table No. 2.

Table 9. Ruggedness data for system 2

Sr No	Peak Area	Assay % of Leflunomide
1	664360	100.34
2	664098	100.30
3	665696	100.54
4	663289	100.18
5	664147	100.31
6	663495	100.21
Mean	664180.8	100.3133
% RSD	0.127783	0.126673

5. Robustness:

Effect of variation of flow rate:

The method's robustness was confirmed by intentionally making minor adjustments to the flow rate and mobile phase composition. A study was conducted to assess the impact of varying the flow rate. A standard solution, prepared according to the test method, was injected into the HPLC system using flow rates of 0.8ml/min, 1.0 ml/min, and 1.2ml/min. System suitability parameters were evaluated and found to be within acceptable limits for all three flow rates (0.8ml/min, 1.0ml/min, and 1.2 ml/min)¹².

Table 10. Data for robustness (Flow rate 0.8ml)

Flow 0.8ml	Std. Area	Tailing factor
	620286	1.322089
	619282	1.331920
	621337	1.296438

	620456	1.315454
	620765	1.326551
Avg	620425.2	1.31849
SD	754.0018	0.013728
% RSD	0.12153	1.0411

Table 11. Data for robustness (Flow rate 1.0ml)

Flow 1.0ml	Std. Area	Tailing factor
	664322	1.604878
	665792	1.0584354
	664360	1.543805
	665696	1.568590
	663147	1.559986
Mean	664663.4	1.572323
SD	1100.917	1.486118
% RSD	0.165635	1.486118

Table 12.Data for robustness (Flow rate 1.2ml)

Flow 1.2	Std. Area	Tailing factor
	602077	1.285372
	601854	1.319385
	602403	1.292055
	603421	1.304561
	602465	1.294621
Mean	602444	1.299199
SD	599.8833	0.013223
% RSD	0.099575	1.017792

LOD and LOQ (limit of detection and limit of quantitation):

LOD = 3.3 X Standard deviation of the response of the blank (σ) /Slope

= 3.3 X 6031.135/1659

= 1.2µg/ml

LOQ = 10 X Standard deviation of the response of the blank (σ) /Slope

= 10 X 6031.135/1659

= 3.6µg/ml

Table 13. Values of LOD AND LOQ

LOD	1.2µg/ml
LOQ	3.6µg/ml

Marketed Sample Analysis:

Table 14. formation of marketed sample of Leflunomide

Drug name	Brand name	Company
Leflunomide	Lefno-20	IPCA Laboratories Ltd.

Table 15. Marketed sample analysis data

Injection	Peak areas of Leflunomide	% Assay
1	658795	99.50
2	659678	99.63
3	655692	99.03
4	659382	99.59
5	660584	99.77
Mean	658826.2	99.504

CONCLUSION:

The proposed high-performance liquid chromatography method was evaluated for its linearity, precision, accuracy, and suitability, demonstrating its practicality and effectiveness for leflunomide quality control in pharmaceutical formulations. With a correlation coefficient of 0.9998, it was established that the measured signal was precise, accurate, and linear across the concentration range (20–70mcg). Additionally, the chromatographic technique is cost-effective and environmentally friendly due to its minimal solvent usage and short analytical runtime of 6.0 minutes. The data indicate that the proposed method can reliably detect leflunomide with high sensitivity and without interference. Therefore, the suggested approach is rapid, selective, and involves straightforward sample preparation, making it a highly efficient method for analyzing leflunomide tablets.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENTS:

I wish to express my gratitude to P. R. Pote Patil College of Pharmacy, Amravati, for their support. I am especially thankful to Prof. Prashant J. Burange, my mentor, for his steadfast encouragement throughout my endeavors. I am privileged to acknowledge the unwavering support of my friends and family on this occasion.

REFERENCES:

1. Srinivas RV, Narasimha RM, Allam AR, Maheswari I, Srinubabu G. Development and validation of LC method for the determination of leflunomide in pharmaceutical formulations using an experimental design. Afr J Pure Appl Chem. 2008; 2(2):10-7.

2. Adhao Vaibhav S., Ambhore Jaya P., Thenge Raju R. Development and Validation of Stability Indicating High Performance Liquid Chromatography Method for Determination of Leflunomide. Asian Journal of Pharmaceutical Analysis. 2023; 13(2): 93-8. doi: 10.52711/2231-5675.2023.00016

3. Rastogi RP, Mehrotra BN. Compendium of Indian medicinal plants. Vol. 2. Lucknow and New Delhi: Central Drug Research Institute and National Institute of Science Communication; 1980. p. 473-5.

4. Schiff MH, Whelton A. Renal toxicity associated with disease-modifying antirheumatic drugs used for the treatment of rheumatoid arthritis. Semin Arthritis Rheum. 2000; 30(3): 196-208. doi: 10.1053/sarh.2000.16641. PMID: 11124283.

5. Herrmann ML, Schleyerbach R, Kirschbaum BJ. Leflunomide: an immunomodulatory drug for the treatment of rheumatoid arthritis and other autoimmune diseases. Immunopharmacology. 2000; 47(2-3): 273-89. doi: 10.1016/s0162-3109(00)00191-0. PMID: 10878294.

6. Viviano KR. Pharmacotherapeutics of immune-mediated disease. Pharmacother Vet Dispensing. 2019: 3: 39-60.

7. Gupta S, George M, Singhal M, Sharma GN, Garg V. Leaves extract of Murrayakoenigii Linn for anti-inflammatory and analgesic activity in animal models. J Adv Pharm Technol Res. 2010; 1(1): 68-77. PMID: 22247833.

8. Stomrud E, Bjorkqvist M, Janciauskiene S, Minthon L, Hansson O. Alterations of matrix metalloproteinases in the healthy elderly with increased risk of prodromal Alzheimer’s disease. Alzheimers Res Ther. 2010; 2(3): 20. doi: 10.1186/alzrt44. PMID: 20576109.

9. Nussbaumer S, Bonnabry P, Veuthey JL, FleurySouverain S. Analysis of anticancer drugs: a review. Talanta. 2011;85(5):2265-89. doi: 10.1016/j.talanta.2011.08.034. PMID: 21962644.

10. Srinubabu G, Raju Ch A, Sarath N, Kumar PK, Rao JV. Development and validation of a HPLC method for the determination of voriconazole in pharmaceutical formulation using an experimental design. Talanta. 2007; 71(3): 1424-9. doi: 10.1016/j.talanta.2006.04.042. PMID: 19071468.

11. Md. Ahsanul Haque, Mohammad Shahriar, Most. Nazma Parvin, S. M. Ashraful Islam. Validated RP-HPLC Method for Estimation of Ranitidine Hydrochloride, Domperidone and Naproxen in Solid Dosage Form.Asian J. Pharm. Ana. 2011; 1(3): 59-63.

12. Akhilesh Gupta, Swati Rawat, Mayuri Gandhi, Jaydeep Singh Yadav. Method Development and Acid Degradation Study of Doxofylline by RP-HPLC and LC-MS/MS. Asian J. Pharm. Ana. 2011; 1(1): 10-13.

13. L. Satyanarayana, S.V. Naidu, M. Narasimha Rao, C. Ayyanna, Alok Kumar. The Estimation of Raltigravir in Tablet dosage form by RP-HPLC. Asian J. Pharm. Ana. 2011; 1(3): 56-58.

14. B. Thangabalan, M. Salomi, N. Sunitha, S. ManoharBabu. Development of validated RP-HPLC method for the estimation of Itraconazole in pure and pharmaceutical dosage form. Asian J. Pharm. Ana. 2013; 3(4): 119-123.

Received on 08.06.2024 Revised on 11.09.2024

Accepted on 19.10.2024 Published on 10.12.2024

Available online on December 30, 2024

Asian Journal of Pharmaceutical Analysis. 2024; 14(4):241-246.

DOI: 10.52711/2231-5675.2024.00043