Method Development For Analyzing Endogenous Metabolites In Biological Tissues Based On Fe₃O₄-Assisted LDI-MS

With the development of matrix-assisted laser desorption/ionization mass spectrometry,there are more applications on the analysis of small-molecule chemicals.In order to overcome the problem that traditional organic matrices lead to serious interference in low-mass regions,nanoparticles have been selected as an alternative matrix.Previous studies suggest that nano-Fe3O4 is an effective matrix for small-molecule analysis by laser desorption ionization mass spectrometry(LDI-MS).However,there has been no systematic study exploring the influencing factors of nano-Fe3O4 on the detection of typical metabolites,or the mechanism by which nano-Fe3O4 assists the desorption and ionization of analytes after receiving laser energy.Therefore,the content of this aspect should be explored in depth.In this study,Fe3O4 nanoparticles with different physicochemical properties(e.g.,morphologies,particle sizes,and surface hydroxyl amounts)were synthesized and characterized.Six different types of endogenous metabolites,including DL-serine,D-glucose,adenosine,arachidic acid,ceramide(d18:1/12:0),and triheptadecanoin(17:0/17:0/17:0),were used as target analytes.The method optimization was conducted based on the signal and repeatability of analytes.The results showed that,compared to other morphologies such as nano-octahedrons,nano-cubes,nano-rods,and nano-plates,the better method performance could be achieved when Fe3O4 nano-spheres were used as matrices.Under the similar surface hydroxyl amounts,the smaller particle sizes of Fe3O4 nano-spheres will obtain higher total peak intensities of analytes;Under the similar particle sizes,the higher surface hydroxyl amounts of Fe3O4 nano-spheres will obtain higher signal responses and lower standard deviations.It can be seen that Fe3O4 nanospheres(M10)with a smaller particle size and a higher surface hydroxyl content as a matrix to analyze 6 typical metabolites can improve the performance of LDI-MS methods,such as better signal strength and repeatability Therefore,we chose M10 as the optimal matrix to establish the nano-Fe3O4 assisted LDI-MS method.Compared to the traditional organic matrices such as DHB and SA,the as-obtained M10 obtained better peak intensities and repeatability,exhibiting the best overall performance for analytes in the positive ionization of LDI-MS.Good intra-or inter-spot repeatability and linearity of analytes were obtained by the optimum Fe3O4-assisted LDI-MS,indicating the reliable and potential quantitation capacity of the developed method.Based on the results of mechanism studies,the thermally driven desorption process played a vital role in LDI performance,but the chemical interactions between nano-Fe3O4 and analytes did not Last,the developed method was successfully used for the rapid analysis and localization of endogenous metabolites in biological fluids(tlapia serum,tilapia bile and human urine)and whole zebrafish tissue section samples.In addition,it also successfully detected the environmental pollutant clomiprazole,indicating that the method is practical and versatile.Accordingly,our results not only shed light on the influencing factors and mechanisms of nano-Fe3O4 for the detection of typical metabolites in LDI-MS but also propose a promising tool on chemical imaging for further elucidation of toxicological mechanisms by environmental pollutants.