Simultaneous Separation, Quantitation, and Determination of the Dissociation Constant of Five Components of Ixeris sonchifolia by Microemulsion Electrokinetic Chromatography

This study established a highly effective and reliable method for the separation and quantitative determination of five pharmacodynamic active components of I. sonchifolia using microemulsion electrokinetic chromatography (MEEKC). The MEEKC conditions were investigated. The optimal running buffer (pH 9.25) consisted of 80 mmol/L Brij-35, 0.8% (v/v) n-heptane, 8% (v/v) 1-butanol, 25% (v/v) acetonitrile, 10 mmol/L sodium dihydrogen phosphate-10 mmol/L sodium borate at 25 kV voltage. The regression equations showed good linear relationships, with 0.61-7.76 mu g/mL limit of detection and 2.04-25.85 mu g/mL limit of quantitation. The established method was also accurate, with intra-day and inter-day relative standard deviations less than 4.78% and less than 6.27%, respectively. Recovery values ranged from 98.61% to 104.76%. The proposed method was successfully applied to determine the five compounds in Kudiezi injection samples. A MEEKC method without adding neutral markers was further established for dissociation constant (pK(a)*) measurement, and reliable pK(a)* values were obtained.