Investigation of the retention behavior of structurally diverse drugs on alpha1 acid glycoprotein column: Insight on the molecular factors involved and correlation with protein binding data

Abstract

Retention of 49 structurally diverse drugs on alpha1 acid glycoprotein column was investigated under different chromatographic conditions. Acetonitrile and 2-propanol were used as organic modifiers at different percentages and the pH was adjusted at 7.0 using PBS. Analysis of extrapolated and isocratic retention in terms of lipophilicity and electrostatic interactions revealed significant effect of the nature and percentage of organic modifier, which was attributed to the different shielding degree of the charged sites on the stationary phase by the buffer constituents. AGP retention factors were compared to HSA retention factors analyzed previously. Application of LSER analysis, extended to incorporate fractions ionized, demonstrated hydrogen bond acidity, dipolarity/polarizability and excess molar refraction as the most significant parameters for all AGP chromatographic indices, elucidating the differentiation of AGP retention from octanol–water partitioning and HSA retention. An attempt to correlate AGP chromatographic indices to AGP association constants, available in literature, supported the importance of stationary shielding in retention mechanism. Thus, isocratic retention factors logk10(ACN)AGP show a moderate but still better performance than lipophilicity in the case of A variant and may be a useful tool for the estimation of relevant association constants. For F1/S binding simulation lower stationary phase shielding is needed to obtain a significant two term regression equation, where logk20(ACN)AGP exerts a secondary contribution next to the most important bulk effect expressed by molecular weight.