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Miniaturized tools development for trace analysis in biological matrices
Valérie Thibert, doctorante. Crédits : ESPCI ParisTechAn initial extraction procedure is usually needed for the trace analysis of biological fluids to reduce the amount of interfering compounds. Thus, solid phase extraction (SPE) is often applied before the separation, but its lack of selectivity can lead to the co-extraction of interfering compounds compromising the analysis. In order to increase the selectivity of the analysis, selective tools, based on molecular recognition, can be used such as immunosorbents, using antibodies, or molecularly imprinted polymers (MIPs). MIPs are synthetic polymers possessing cavities which are complementary in size, shape and functionality of the imprint molecule. Sensitivity, speed and efficiency of the analysis can be improved by downscaling the separation system, therefore, miniaturization of the extraction with a MIP can be interesting to couple it with the separation on the same miniaturized device. MIP miniaturisation was thus studied for the extraction from biological matrices of cocaine and its main metabolites, benzoylecgonine (BZE) and ecgonine methyl ester (EME) as model molecules.
The performances of a chip with an enrichment channel, a separation channel and a metalized nanospray tip were initially assessed. The optimized analysis method showed excellent results in terms of sensitivy (limits of quantification of a few pg mL-1). It was therefore successfully applied to cocaine and BZE trace analysis in urine and hair extracts. Nevertheless, the lack of selectivity of the SPE limited the potential of the chip analysis, stressing the need for the integration of a selective tool for the extraction.
The feasibility of a MIP selective for cocaine and its metabolites was evaluated on a conventional format. After the optimization of the synthesis and the extraction protocol, an excellent selectivity was obtained in organic, hydro-organic and aqueous media for cocaine and EME, but BZE was not retained in aqueous and hydro-organic media. Selective cocaine extraction from hair extracts, serum and urine was successfully obtained. The MIP could not retain cocaine metabolites in serum, while a preliminary liquid-liquid extraction allowed their selective extraction from urine samples prior to analysis.
The last part of this work was dedicated to the synthesis of a miniaturized cocaine-selective MIP. The first approach consisted in the synthesis of MIP microparticles by precipitation polymerization. Many synthesis key parameters were studied to obtain MIP and NIP microspheres of about 3 µm in diameter. Unfortunately, the obtained MIP microspheres were not selective for cocaine and its metabolites neither in organic nor in aqueous media. The second approach consisted of monolithic MIPs synthesized in situ on silica capillaries (100 µm i.d.). Various parameters were studied to obtain a MIP monolith with a morphology appropriate to its coupling with liquid nanochromatography. The selected MIP exhibited a higher affinity for cocaine in organic media than the non imprinted polymer, and the synthesis was repeatable in terms of morphology and selectivity. The coupling was thus performed and, even though a deeper study of the device is necessary,very interesting results were obtained for the on-line extraction of cocaine from spiked urine samples.