Development and validation of HPLC method for simultaneous determination of Leflunomide and folic acid in the nanoparticulate system by reversed-phase HPLC

Abstract

Objective

The main objective of this study was to develop a highly sensitive, accurate, and reproducible analytical method for the simultaneous detection of LEF and FA in polymeric nanocarriers.

Significance

Leflunomide (LEF), is widely employed in the treatment of rheumatoid arthritis (RA). However, long-term delivery of the drug is associated with systemic side effects. Therefore, folate (FA) conjugated LEF nanocarriers were fabricated for targeting the nanocarriers toward activated macrophages. HPLC is considered one of the most sensitive and precise analytical techniques for the simultaneous detection and estimation of different components in a particular sample.

Methods

Analysis was performed on HPLC (Shimadzu 10 A), having a reversed-phase C-18 column (Beckmen, 250 X 4.6 mm, 5 µm) equipped with a photodiode detector set at a wavelength of 260 nm (LEF) and 285 nm (Folic acid). The isocratic mobile phase was composed of acetonitrile, water, and trimethylamine in a ratio of 65:35:0.5 at pH 4. Rapid analysis of both agents was performed, with a total run time of 10 min (FA = 2.1 ± 0.1 min, LEF = 5.9 ± 1 min) at a 1 mL/min flow rate.

Results

The assay demonstrated good linearity of 0.9989 of 0.9997 for LEF and FA respectively with a recovery in the range of 95–100%. The method also depicted good specificity, and intra and inter-day precision based on relative standard deviation (RSD) values.

Conclusions

The study concludes, that the developed method was helpful in the detection and quantitation of lower values of both agents from polymeric nanocarriers.

Highlights

1. Optimization and validation of the RP-HPLC method were performed for the simultaneous detection of LEF and FA.

2. Validation was performed on the basis of linearity, accuracy, precision, LOD, LOQ, and robustness in accordance with ICH criteria.

3. Validated analytical procedure was employed for the simultaneous detection of LEF and FA from polymeric nanocarriers.

4. The proposed analytical method is reliable, fast, robust, and can be successfully applied for quantification of % EE, and % DL in polymeric nanocarriers.

Keywords:

• HPLC
• folic acid
• leflunomide
• nanocarriers
• validation

Disclosure statement

No potential conflict of interest was reported by the author(s).The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Additional information

Funding

Bazla Siddiqui was financially supported by Ph.D. Indigenous Scholarship (PIN no. 520-147090-2MD6-7 (50093071)) from Higher Education Commission (HEC) of Pakistan.