| Phospholipids (PLs) are the most abundant in lipids and theirstructures determine their amphoteric features. PLs are the mainconstituents of biological membranes and have both structural andfunctional properties. Especially, some PLs also participate in manybiological processes, like cell proliferation, differentiation, apoptosis,oxidative stress and so on. In recent years, increasing researchesdemonstrated that the metabolic disorder of PLs with the relatedmetabolites and enzymes play an important role in many malignantdiseases, including cancer of the breast, endometrium, colon, kidney andacute leukemia, malignant lymphomas, multiple myeloma. Based ondifferences in their polar head groups, the PLs are divided into severalclasses. Each class of PLs is composed of a mixture of molecular speciescontaining different fatty acids. This structural diversification leads to the analytical challenges for PLs.Selective enrichment of PLs from complex biological samples will begreat helpful for studying the change of PLs, especially for those of lowabundance species. The liquid-liquid extraction (LLE) and solid-phaseextraction (SPE) protocol are commonly used to separate PLs from totallipid extracts. The latter is a technique designed for rapid, selective samplepreparation and purification prior to chromatographic analysis, providingthe sample clean-up, high recovery and concentration essential for precisequantitative analysis. The commonly used solid-phase materials for SPEare usually porous silica or silica modified with cyanopropyl,aminopropylor2,3-dihydroxypropoxypropylgroups，etc. However, theselectivity and efficiency for PLs of these materials are not very high.It is reported that metal oxides (like ZrO2, TiO2) were proved to havehigh selectivity for phosphate group. The metal oxide can serve as lewisacid with positive charge under acidic condition and therefore it canselectively adsorbe species with phosphate group. The adsorbed specieswill desord while the metal oxide turns into lewis base under the alkalinecondition. These metal oxides have already been used in the enrichment ofphosphopeptides. For example, TiO2was used to enrich PLs from egg ordairy product. In this study, titania coated silica microspheres were synthesizedthrough water vapor-induced internal hydrolysis method and characterized.Then the composite material was employed as a novel sorbent in the SPEcolumn for specific capture of PLs prior to reversed phase liquidchromatography with evaporative light scattering detector (RPLC-ELSD)and ultra performance liquid chromatography coupled with quadrupoletime-of-flight mass spectrometry (UPLC-QTOF MS) analysis. Afterward,this specific SPE approach was introduced into the pretreatment ofbiological sample and, the experimental results showed that the titaniacoated silica microspheres have excellently selective adsorbability towardsPLs, and they are promising in phospholipid profiling andphospholipidomics study.The contents of this thesis are as follows:1．Titania coated silica microspheres (TiO2/SiO2) were synthesizedthrough water vapor-induced internal hydrolysis method and characterizedby SEM, UV-vis, XRD and BET experiment, which showed that titanialayer was uniformly distributed onto the surface of silica particles. Besides,the composite microspheres have large surface area of335m2/g anduniform distribution of pore size with average diameter of12nm, whichare suitable for the SPE sorbent. 2. The TiO2/SiO2composite microspheres were packed into the SPEcolumn, which was used in selective extraction for the PLs. The absorptiveability of the developed SPE protocol was investigated by RPLC-ELSD.Important factors, such as loading buffers, eluting buffers and elutionvolume that affect the extraction process were investigated and optimized.The experimental results obtained from the phospholipid standards revealsthat the optimized SPE approach have higher recoveries towards PLs thanpure TiO2particles, and10wt%TiO2/SiO2has the highest recoveries ofmore than85%.3. The proposed TiO2/SiO2-SPE method was then applied to extractendogenous PLs from human serum prior to UPLC-QTOF MS analysis.The responsive intensity of extracts without SPE approach (Before SPE)and SPE eluting fraction (After SPE) were compared based on metabolitesselected according to their occurrence in human serum. The results showthat662molecular feathres were observed when pure TiO2particles wereused, whereas1527molecular features were detected with the SPE columnpacked with TiO2/SiO2. The SPE method provides66.7%larger number ofunique molecular features compared with that observed without using thismethod. Also, it demonstrats that the responsive intensity of elutingfraction was higher than that of direct analysis and more PLs can be enriched and detected through this specific SPE protocol.4. The TiO2/SiO2-SPE strategy was introduced into thephospholipidomics study on Non-Hodgkin’s Lymphoma. In this part, theserum phospholipid profiling was obtained by UPLC-QTOF MS technique.Afterward, the data was firstly processed by the softare MassLynx and thenimported into the Simca-P11.5with multivariate data analysis, includingprincipal component analysis (PCA), orthogonal partial least squaresdiscriminant analysis (OPLS-DA). Based on the classification analysis ofVIP>1and the significance analysis of p<0.05, eight potential biomarkerswere screened and identified, which closely associates with non-hodgkin’slymphoma. |
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