Síntese, caracterização e emprego de Polímeros Molecularmente Impressos na extração de fármacos em amostras humanas de plasma e urina

Abstract

Analysis of organic compounds in complex matrices requires the application of methods with good selectivity and detectability, to lead reliable results. Based on this demand, analytical chemistry has proposed viable and innovative solutions to make these tests more dynamic. Among them, we can highlight the chromatographic techniques, mass spectrometry and multidimensional systems. Following this development, selective sorbents such as molecularly imprinting polymers (MIPs), restricted access materials (RAM), restricted access molecularly imprinting polymers obtained by covering the surface with hidrofilic comonomers (RAMIP) and restricted access molecularly imprinting polymers obtained by covering the surface with hidrofilic comonomers and bovine serum albumin (RAMIP-BSA), have been successfully used in sample preparation. In this context, this PhD thesis aimed to synthesize, characterize and evaluate the different types of molecularly imprinted polymers potentialities for drugs extraction from biological samples and associate these materials with online sample preparation techniques, combined to chromatography and/or mass spectrometry. In paper one we proposed a comparative study of different molecularly imprinted polymers selective to beta-blockers. The polymers were synthesized by precipitation method using methacrylic acid as functional monomer, hydroxy-ethyl methacrylate and glycidyl dimethacrylate, as hydrophilic co-monomer, ethylene glycol dimethacrylate as crosslinking agent, 2,2-azoisobutironitrile as radical initiator and acetonitrile as solvent. One part of the obtained materials was coated with BSA (bovine serum albumin). The polymers were characterized and it was possible to conclude that the synthesis procedure influences the size and shape of particles and that hydrophilic co-monomer addition as well as coating with BSA do not alter the chemical recognition ability of the material. The difference between imprinted and non-imprinted polymers’ adsorption was evident (suggesting that imprinted polymers have a better capacity to bind the template than the non-imprinted ones). The Langmuir model presents the best fit to describe the materials’ adsorption profile. The polymer covered with hydrophilic monomers presented the best adsorption for the template in an aqueous medium, probably due to a hydrophilic layer on its surface. We also concluded that an association of the hydrophilic monomers with the BSA coating is important to obtain materials with higher capacity of macromolecule exclusion. In paper two, the RAMIP selective to oxprenolol was chosen as sorbent in a online method for beta blockers analyses from human urine for doping monitoring. A column filled with RAMIP was coupled to an LC-MS/MS instrument under the multidimensional configuration, with 10.0 mmol L−¹ ammonium formate buffer (pH 5.0) as the loading and reconditioning mobile phase and a 0.01% formic acid aqueous solution – methanol (30:70 v/v) as the elution mobile phase. The system was used for on-line extraction and quantization of oxprenolol (from 1.0 to 75.0 μg L−¹), atenolol, propranolol, nadolol, pindolol, labetalol and metoprolol (all from 3.0 to 50 μg L−¹) simultaneously, from urine samples. The correlation coefficient was higher than 0.99 for all the analytes. Suitable precision and accuracy were obtained. In paper three a RAMIP, selective to tricyclic antidepressant, was synthesized by the in bulk method. Amitriptyline was used as template molecule, methacrylic acid as the functional monomer and glycidyl methacrylate as hydrophilic co-monomer. Afterwards the epoxide ring opening was made and the polymer was covered with BSA. A column filled with RAMIP-BSA was coupled to an MS/MS instrument under the multidimensional configuration, with water as the loading and reconditioning mobile phase and a 0.01% acetic acid aqueous solution - acetonitrile at 30:70 as the elution mobile phase. The system was used for online extraction and quantization of nortriptyline, desipramine, amitriptyline, imipramine and clomipramine, simultaneously, from plasma samples. The correlation coefficient was higher than 0.99 for all the analytes. The RSD (relative standard deviation) values ranged from 1.34% to 19.13% for intra assay precision and 1.32% to 19.77% for inter assay precision. The E% (relative error) values ranged from -19.15% to 19.51% for intra assay accuracy and from -9.04% to 16.22% for inter assay accuracy.