Enzyme inhibition coupled to molecularly imprinted polymers for acetazolamide determination in biological samples

Several methods involving molecularly imprinted polymers (MIPs) devoted to extracting and analyzing sulfonamides from different matrices are reported in literature; however, the unresolved analytical issue is obtaining intra-class selectivity between sulfonamides. Here is presented for the first time a method coupling MIPs and enzymatic inhibition assay for the sensitive and selective determination of acetazolamide (ACZ) in biological samples. The MIPs were synthesized by thermal initiated polymerization in acetone, using acrylamide as functional monomer, ethylene glycol dimethacrylate as cross-linker and ACZ as template molecule. The developed MIPs/enzymatic inhibition based rapid colorimetric method was applied for the determination of ACZ in biological samples. The MIPs were used as sorbent phase in dispersive solid-phase extraction (MIPs-dSPE), and the optimal working parameters were selected. Liquid chromatography-tandam mass spectrometry (LC-MS/MS) analysis confirmed the MIPs ability to extract ACZ. Finally, to obtain a selective and sensitive method, the MIPs-dSPE was combined with an enzymatic inhibition colorimetric assay based on the carbonic anhydrase, an enzyme inhibited by specific sulfonamides. The developed combined method allowed the determination of ACZ in serum, blood and Diamox (a drug containing ACZ), with good recovery (85-96%). Furthermore, a significant correlation with LC-MS/MS analysis was achieved, with relative error ≤15%. In the proposed strategy, the double selectivity giving by MIPs and enzymatic inhibition allowed to obtain a method able to determine selectively ACZ in biological and pharmaceutical samples quantitatively.