ABSTRACT
We report a novel, fast, and automatic SPME-based method capable of extracting a small molecule-drug conjugate (SMDC) from biological matrices. Our method relies on the extraction of the drug conjugate followed by direct elution into an electrospray mass spectrometer (ESI-MS) source for qualitative and quantitative analysis. We designed a tool for extracting the targeting head of a recently synthesized SMDC, which includes acetazolamide (AAZ) as high-affinity ligand specific to carbonic anhydrase IX. Specificity of the extraction was achieved through systematic optimization. The design of the extraction tool is based on noncovalent and reversible interaction between AAZ and CAII that is immobilized on the SPME extraction phase. Using this approach, we showed a 330% rise in extracted AAZ signal intensity compared to a control, which was performed in the absence of CAII. A linear dynamic range from 1.2 to 25 µg/ml was found. The limits of detection (LOD) of extracted AAZ from phosphate-buffered saline (PBS) and human plasma were 0.4 and 1.2 µg/ml, respectively. This with a relative standard deviation of less than 14% (n = 40) covers the therapeutic range. Graphical abstract.
Subject(s)
Acetazolamide/isolation & purification , Enzyme Inhibitors/chemistry , Small Molecule Libraries/isolation & purification , Solid Phase Microextraction/methods , Spectrometry, Mass, Electrospray Ionization/methods , Acetazolamide/chemistry , Automation , Limit of Detection , Reproducibility of Results , Small Molecule Libraries/chemistryABSTRACT
Molecularly imprinted polymers (MIPs) were computationally designed and synthesized for the selective extraction of a carbonic anhydrase inhibitor, i.e. acetazolamide (ACZ), from human plasma. Density functional theory (DFT) calculations were performed to study the intermolecular interactions in the pre-polymerization mixture and to find a suitable functional monomer in MIP preparation. The interaction energies were corrected for the basis set superposition error (BSSE) using the counterpoise (CP) correction. The polymerization solvent was simulated by means of polarizable continuum model (PCM). It was found that acrylamide (AAM) is the best candidate to prepare MIPs. To confirm the results of theoretical calculations, three MIPs were synthesized with different functional monomers and evaluated using Langmuir-Freundlich (LF) isotherm. The results indicated that the most homogeneous MIP with the highest number of binding sites is the MIP prepared by AAM. This polymer was then used as a selective adsorbent to develop a molecularly imprinted solid-phase extraction procedure followed by differential pulse voltammetry (MISPE-DPV) for clean-up and determination of ACZ in human plasma.
Subject(s)
Acetazolamide/blood , Acetazolamide/isolation & purification , Drug Design , Molecular Imprinting , Polymers/chemistry , Acetazolamide/analysis , Acrylamides/chemistry , Adsorption , Binding Sites , Computer-Aided Design , Computers , Electrochemistry/methods , Humans , Hydrogen-Ion Concentration , Kinetics , Solid Phase Extraction , Solvents/chemistryABSTRACT
In this paper we report a systematic recovery study based on reversed phase high performance liquid chromatography (RP-HPLC) separation and mass spectrometric (MS) based fractionation. Factors including a compound's physicochemical properties, column mass loading and presence of impurities were investigated through commercially available compounds. Results suggest that the delay time between MS peak detection and fraction collection, fraction detector's signal-to-noise ratio and compound's base peak width in the chromatogram have the biggest impacts on purification recovery. In an effort to assess sample recovery within our high throughput purification process, re-purification was performed on four compound libraries that were synthesized in-house. Reproducible recoveries (>80%) were achieved in all tests.
