Chemometric Assisted Ion-Pair Chromatography of Metaxolone and Diclofenac in Binary Mixture: A Mechanistic Study

  A reversed-phase high-performance liquid chromatographic (RP-HPLC) method based on ion pair formation is demonstrated for the simultaneous determination of Metaxalone (MTX) and Diclofenac potassium (DCP) in commercial formulations. MTX (pKa=12.24) and DCP (pKa=4.00) are hydrophilic ionic substances that make the separation critical due to distinct pKa values. Addition of ionic additives (ion-pairing reagents or chaotropic agents) to the mobile phase allowed a significant improvement in retention of the ionic analytes. However, finding the suitable condition of the Ion-Pair Chromatography (IPC) to achieve desirable separation was challenging and often rebellious to influential chromatographic parameters. The judicious selection of eluent pH, flow rate, acidic modifiers and organic modifier, detector wavelength was performed with the aid of Plackett–Burman design and Box–Behnken design. Desired separation of MTX and DCP was achieved using an Inertsil ODS2 (250x4.6mm; 5µm) column equilibrated with 10mM phosphate buffer (pH-5.0): acetonitrile (42: 58%v/v) containing 0.4% triethylamine and 0.5% tetrabutyl ammonium hydroxide as an eluent at a flow rate of 1.20 mL/min. Under optimal condition, the detection limits are 0.306µg/mL and 0.807µg/mL and limits of quantification 0.927µg/mL and 2.44µg/mL for MTX and DCP respectively. Subsequently, the plausible retention behavior of both analytes was predicted in the ion interaction condition.