Development and validation of LC method for the determination of leflunomide in pharmaceutical formulations using an experimental design

Abstract

A rapid and sensitive RP-HPLC method with UV detection (260 nm) for routine analysis of leflunomide in a
pharmaceutical formulation was developed. Chromatography was performed with mobile phase containing a
mixture of acetonitrile and phosphate buffer (60:40, v/v) with flow rate of 0.8 ml min−1
. Quantitation was
accomplished with internal standard method. The procedure was validated for linearity (correlation coefficient =
0.9999), accuracy, robustness and intermediate precision. Experimental design was used for validation of
robustness and intermediate precision. Plackett-Burman design was used to screen the essential factors for
optimization. To test robustness, three factors were considered; percentage v/v of acetonitrile in mobile phase,
flow rate and pH; an increase in the flow rate results in a decrease of the drug found concentration, while the
percentage of organic modifier and pH have no important effect on the response. For intermediate precision
measure the variables considered were: analyst, equipment and number of days. The RSD value (0.93%, n = 24)
indicated a good precision of the analytical method. The proposed method was simple; highly sensitive, precise,
accurate and retention time less than 6 min indicating that the method is useful for routine quality control.
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