The consumption of psychoactive substances is increasing worldwide and remains an important social concern, both for consumers and for non-users potentially in contact with users, for example while driving or in the workplaces. Urine is the matrix of choice for workplace testing and mass spectrometry coupled with separation techniques, especially liquid chromatography (LC-MS/MS) is commonly used for detection, however sample preparation is still the main bottleneck.
The major goal of our proposed research is to develop innovative miniaturized approaches for the detection and quantification of traditional illicit drugs and novel psychoactive substances (NPS) in biological samples to be used in LC-MS/MS drug testing methods. The project involves the use of gel materials, traditionally used in the cultural heritage field, to obtain an efficient extraction of the analytes of interest alongside with a desirable clean-up of the complex matrix under investigation. The novel approach developed will be based on liquid phase microextraction (LPME) with the solvent being immobilized within the pores of the studied gels. Different geometries and configurations will be explored, also involving the use of stirring magnetic bars and the incorporation of the extracting devices in 96 well plate systems for high-throughput applications. The possibility of applying the novel extraction technique on dried urine spot (DUS) will be also explored; several advantages would be obtained by this approach such as reduction of sample volume, ease of sample collection, ease of transportation and storage.
The developed method(s) will be validated and applied to the analysis of psychoactive substances in the urine samples of workers provided by the Centre for Occupational Medicine (CMO). The obtained results may help to have a closer overview of the real diffusion of traditional illicit drugs and NPS, which are normally not included in toxicological methods, among worker populations.
In this project a novel sample preparation approach for the analysis of biological matrices by LC-MS/MS will be developed and tested. High throughput configurations, which are high desirable in modern analytical chemistry, will also be investigated.
Miniaturization of sample preparation is a recent trend in analytical chemistry and presents substantial advantages such as reduced sample and solvent volumes, and high enrichment factor. Among the new approaches for sample preparation LPME represent an interesting class of techniques, as they are cheap, quick and effective. The technique that will be explored in this project is a variant of HF-LPME: a significant novelty is related to unusual materials that will be tested and applied to support the extraction solvent. The gels that will be tested are used in the field of cultural heritage because of their characteristics of retentiveness that can be modulated for maximizing their selectivity.
Furthermore, their mechanical features make them practical to use and to apply on different typologies of samples (solids and liquid surfaces). To the best of our knowledge the application of these materials for biological sample extraction has never been reported.
These gels present a number of interesting features, when compared to the polymeric membranes used in HF-LPME applications. They can be prepared directly in the laboratory in high amount through simple and inexpensive procedures, so they result perfectly fitting for analyses on big sets of samples. In the case of agar hydrogel, in particular, the preparation requires only agarose, a natural polysaccharide, and water, resulting safe and eco-friendly. In addition, the gels result extremely versatile: they can be soaked in different classes of solvents, so they can be used for different extraction procedures. Finally, because of their mechanical properties, it is possible to model them in different configurations and geometries, and they can be used both for solid and liquid matrices.
The configurations that will be explored in this project are very promising to obtain a rapid and effective extraction. Similar approaches have recently been described in literature, but substantial improvements will be proposed in this project, involving the application of unusual materials, high-throughput set-ups and microvolume samples.
The principal extraction set-up that will be developed for single sample analysis involves the immobilization of the gel supported extraction solvent on a magnetic stirring bar, using a configuration similar to SBSE technique, but in this case a solvent extraction will be performed.
A further set-up that will be taken into consideration for high-throughput applications is the 96 well plate format, which may represent a new interesting tool for bioanalytical applications involving the analysis of many samples.
The use of DUS samples instead of traditional urine is another state-of-the art feature of the present project. Dried matrix spot, especially DBS have received great attention during recent years, because of several advantages such as reduction of sample volume, ease of sample collection, ease of transportation and storage etc. From an analytical point of view the filter paper can retain several matrix components, contributing to the clean-up of the sample. In this project we will test the possibility to extract the analytes of interest from the urine spot by applying the gel supported extraction solvent directly on the spot to obtain an effective and practical extraction. On-spot enzymatic cleavage of phase II metabolites will be also tested.
The developed method(s) will be then applied for the analysis of psychoactive substances in the urine samples of workers according to the "Accordo Stato-Regioni" of 2008 and subsequent amendments. The obtained results may help to have a closer overview of the real diffusion of traditional illicit drugs and also NPS, which are normally not included in toxicological methods, among worker populations.