Direct determination of free state low molecular weight compounds in serum by online TurboFlow SPE HPLC-MS/MS and its application

Abstract

The free concentration of low molecular weight compounds (LMWCs) in serum play important roles in both the environmental health and pharmacokinetic fields. A novel and broad spectrum method for direct determination of free LMWCs in serum was established by online TurboFlow SPE HPLC-MS/MS. Macromolecules, including proteins and protein-bound LMWCs, were removed by the TurboFlow SPE column. Meanwhile, free LMWCs were retained on the TurboFlow SPE column and eluted by the mobile phase for HPLC-MS/MS detection. 16 representative LMWCs with low, medium and high affinities to serum proteins were selected for method optimization and validation, and their free concentrations in serum detected by present method showed fairly consistent results with the equilibrium dialysis (r = 0.9714, p < 0.01) and ultrafiltration (r = 0.9054, p < 0.01) method. Then, present method was successfully applied to detect free LMWCs in human serum as well as to evaluate the dissociation constants (Kd) of LMWC–HSA complexes. Kd values of some emerging pollutants, such as 9Cl-PF3ONS, PFEtCHxS and 8:2 diPAP, were firstly evaluated by the present method. The rapid processing time (18 min), small sample amount requirement (25 μL) and convenient operation (direct injection) made present method be promising in studies on the bioavailability, bioaccumulation and elimination of LMWCs in serum, real-time detection of free LMWCs for therapeutic drug monitoring during clinical drug research and development, and the degradation kinetic processes of free LMWCs in serum.