Técnicas miniaturizadas de tratamiento de muestra para el análisis dirigido y no dirigido de fluidos biológicos humanos

Abstract

The development of a new analytical method involves numerous stages, among which is sample treatment, probably the most crucial and complex one to ensure the success of the new method. Therefore, the research in the field of Analytical Chemistry is focused on the development of miniaturized techniques for sample treatment which has led to both the simplification of the sample and the preconcentration of the analytes contained in the samples. The development of the miniaturized techniques has brought about the beginning of what is known as microextraction techniques which allow the extraction and preconcentration of analytes contained in both liquid and solid phases in a reduced volume of extractant phase on the order of microliters. The Green Analytical Chemistry guidelines were followed in the development of the analytical methods proposed in the present Doctoral thesis. The sample treatment techniques applied are based on microextraction procedures, leading to an improvement in method sensitivity in comparison with the results obtained when traditional extraction techniques are used and the simultaneously cleaning matrix step achieved. Analytes detection was carried out by HRMS, more specifically by a hybrid combination of quadrupole with time-off flight mass spectrometry (Q-TOF-MS). Not only does this combination allow the identification and quantification of analytes under targeted approach, but also the possibility of carrying out a wider search for compounds under non-targeted approach is also allowed. Therefore, the presence of possible metabolites and other structurally related compounds with the target analytes has been studied by applying the same experimental conditions optimized for targeted analysis. The analytical methods developed in this Doctoral Thesis are focused on the analysis of different families of protoalkaloids in biological samples, namely, serum and urine human samples. These types of samples are widely used due to their possibility to detect a large number of organic compounds in them which provide information about the health state of individuals. The research carried out in this Doctoral Thesis has been organized in three chapters in which the three analytical methods developed has been described as well as presenting the successful results obtained when the microextraction techniques and LC-HRMS are combined. Therefore, in the first chapter, an analytical method for the determination of three capsaicinoids by dispersive liquid-liquid microextraction (DLLME) in human urine collected from patients who have been previously treated with capsaicin-based analgesic creams has been proposed. The results obtained were also studied under non-targeted approach with the purpose of expanding the search to the detection of possible metabolites derived from the capsaicinoids studied under the targeted approach and to a conjugated compounds resulting from the binding of the analytes to other biomolecules. In the second chapter, the knowledge of the three capsaicinoids studied in the previous chapter is expanded under another methodology. Thus, a new analytical method for its determination has been used, which consists in applying dispersive magnetic solid phase microextraction (DMSPE) and LC-HRMS. In this chapter, the non-targeted approach is also addressed. Additionally, thanks to the spectrum data provided by the mass spectrum, the fragmentation patterns of both the analytes and some metabolites derived from the capsaicinoids have been able to be elucidated. Finally, in the third chapter, a new method for the determination of four biogenic amines in human urine by DLLME and LC-HRMS. Prior to analysis, a derivatization step was applied in order to improve the sensitivity and selectivity of the method. In this chapter, a non-targeted analysis of the samples was also carried out to focus on the search for amine-related compounds as well as possible metabolites and bioconjugates whose most likely route of excretion is urine.