The development and the characterization of new materials for sample preparation is a sector at the cutting edge of analytical chemistry. Over the last few years, attention has mainly been focused on carbon nanostructured materials because of their potential as superior sorbents in solid phase extraction (SPE) operations on conventional and miniaturized scale. Recently, attention of our research group has been attracted by buckypaper (BP), a nanoporous flexible felt of about 0.2 mm thickness, composed of entangled unoriented oxidized carbon nanotubes (CNTs), which is suitable to be used as a sorbent membrane.
The aim of this research is to develop an oxidized BP-based device for micro SPE of biomolecules of interest from human plasma and/or other biological fluids (urine and saliva). The extraction module will be designed to process very small sample volumes (micro-SPE) in short time through the assistance of magnetic stirring. Several parameters will be optimized in order to maximize recoveries, for example: adsorption and desorption time; stirring speed; type, volume and fractions of the elution solvent; volume, pH and ionic strength of the sample. In particular, the effects of an acid treatment with nitric acid will be studied to modulate oxidation grade of BP and, consequently, its affinity for analytes with different polarity.
At the present moment, BP has still been little explored within the framework of sample preparation, but its peculiar characteristics make it a promising material for the development of original extraction devices designable in several shapes, sizes and realization modes.
Buckypaper (BP) is a relatively new material and so far almost unused for extraction purposes [1-4]. Nevertheless it has all peculiarities to act as an adsorbing membrane: high porosity, high specific adsorption capacity, good permeability and potential reuse. It can be designed in several shapes, sizes and realization modes. Moreover, BP handling is safe because there is no dispersion of free CNTs into the atmosphere. Finally, BP intrinsic affinity towards non polar, moderately polar and aromatic organic compounds [5] can be extended to highly polar analytes by introducing hydroxyl, carbonyl and carboxyl groups on the CNTs sidewalls, as a result of strong oxidizing treatment.
For example, isoprostanes (IsoPs) are compounds of great clinical interest, because they are biomarkers of oxidative stress (BOSs), generated for non-enzymatic peroxidation of arachidonic acid [9,10]. Depending on the functionalities on the prostanoid ring, different classes of IsoPs have identified: F2-IsoPs, E2-IsoPs, D2-IsoPs, A2-IsoPs, J2-IsoPs. Among these, F2-IsoPs are the golden standard because chemically stable and present in all fluids and tissues. Depending on the position of free radical attack on the linear chains, 4 series of regioisomers can be formed (referred to as 5-series, 12-series, 8-series and 15-series), each of them counting 8 diastereoisomers for a total of 64 compounds. Among these, the regioisomers of the 15-series and 5-series occur at higher concentrations in vivo because less prone to further oxidation [9,10]. At large, the analysis of these substances is complicated by the variety of compounds and by the great number of isomers. Enzyme immunoassay (EIA) and radioimmunoassay (RIA) are high-throughput methods that do not require expensive instrumentation; nevertheless, the polyclonal antibodies used for binding the IsoPs exhibit cross-reactivity with prostaglandins, causing the overestimation of IsoPs [11]. GC coupled to mass spectrometry (MS) involves a preliminary derivatization step which makes it unfit for routine analysis [12-15]. LC-MS has the advantage of sensitivity, selectivity and the direct analysis. In the literature, almost all of the GC-MS and LC-MS methods deal with the dosage of a single marker (especially 15-F2t-IsoP) [10,12,16] or, at most, a pair of markers (15-F2t -IsoP and 5-F2t-IsoP) [16,18]. Only a couple of publications report the simultaneous LC-MS determination of six IsoPs [17,18]. They are multi-analyte methods based on long extraction procedures that include a preliminary chemical hydrolysis step and a clean-up on SPE C18 cartridge. IsoPs, having logP 4 and pKa 4.5, seem to be the ideal candidates for retention on BP. In fact, in our previous work [4], we found that adsorption on BP is favoured for analytes with logP > 1.5 and pKa differing from 7.5. Therefore, the study of a novel extraction procedure based on the development of a BP stirring micro-SPE device, alternative to the classic on-cartridge SPE clean-up, represents a desirable innovation with respect to the state-of-the-art.
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