HPLC method for the simultaneous determination of Levocetirizine, Ambroxol and Montelukast in human Plasma employing response Surface Methodology

Multiple response simultaneous optimizations employing the Derringer’s desirability function for the development of reversed-phase HPLC methods for the simultaneous determination of Ambroxol(AMB) and Montelukast(MLS) with Levocetirizine (LCT) and Probenecid(PRO) as Internal standard in human plasma samples is described. The ranges of the independent variables used for the optimization were MeCN: 30-40%, buffer conc.: 10-20 mM and flow rate: 0.8-1.2 ml/min. The influence of these independent variables on the output responses: capacity factor of the first peak (k1), resolutions (Rs2,3), and Retention time (tR4) were evaluated. Using this strategy, mathematical model were defined and response surface were derived for the separation. The coefficient of determination R2 was more than 0.8972 for all the models. The three responses were simultaneously optimized by using Derringer’s desirability functions. Optimum conditions chosen for assay were MeCN, MeOH, 20.00 mM K2HPO4 (pH 7.0 ± 0.5) solution (32.7:30: 37.3 v/v/v) and flow rate 0.85 ml/min. The eluate was monitored using an UV detector set at 230 nm. Peak area ratio of the analyte and internal standard was used for the quantification of plasma samples. Total chromatographic analysis time per sample was approximately 8.665 min. The validation of the proposed analytical method was conducted in accordance to the recommendations of the guidelines ‘‘Bioanalytical method validation’’ [FDA-CDER, 2001]. The method was found to be simple, sensitive and hence it could be applied in bioavailability studies.

Author(s): Ramalingam Suresh, Rajappan Manavalan and Kannappan Valliappan

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