Assessment Of Schiff Base Compounds As Matrices For The Analyses Of Small Molecules By Negative Ion MALDI Mass Spectrometry

Matrix assisted laser desorption/ionization mass spectrometry(MALDI-MS),with characteristics of soft ionization and the ability to obtain a complete molecular ion information,is often used for the analyses of macromolecules such as proteins,nucleic acids and synthetic polymers.Furthermore,it has the irreplaceable advantages in simple sample preparation procedure,high sensitivity,less sample consumption and good tolerance to salt for the analyses of small molecules including animal and plant metabolites,pesticide residues etc.In the MALDI-MS analysis,matrixes are often required,however,traditional matrix ions appeared interferes in the range of the low molecular mass when detected the small molecules.Also unequal cocrystallization of matrix and analyte influences the reproducibility of analyte signals,which is not conducive to quantitative analysis.Therefore,in this thesis,we synthesized some new Schiff base compounds and used them as MALDI matrices for detection of small molecule analytes in the negative ion mode by MALDI-MS,the experimental content mainly include the following three aspects:(1)Six kinds of Schiff base compounds(S1,S2,S3,S4,S5 and S6)were synthesized through dehydration condensation reaction of anilines and aldehydes.The synthesized compounds were then used and evaluated as MALDI matrices for the analyses of saturated and unsaturated fatty acids in the negative ion mode.The method of ultraviolet absorption and quantum chemistry calculations were used to explore rationality of Schiff bases as matrixes.Finally,S1 was selected as an optimal MALDI matrix for testing standard fatty acids and free fatty acids in the serum.The liquid-liquid extraction was used to crude extract of free fatty acids in serum,the ―seeds‖ method was used for sample dropping and an internal standard(C17:1)was added.As results,reproducible signals and quantitative analysis were achieved for the analyses of free fatty acids in human serum.Our results demonstrated that the Schiff base S1 as MALDI matrix showed advantages of no matrix interference,high sensitivity,simple separation step and less sample cost etc.(2)The application of the Schiff base S1 as MALDI matrix was further examined in the negative ion mode by testing of a series of acidic and alkanol molecules,including 6 amino acids(tryptophan,lysine,histidine,threonine,leucine and glutamic acid),two kinds of phospholipids(1,2-2oleoyl-sn-glycerol-3phospholipids and sphingomyelin),four small molecular drugs(ethinylestradiol,artemisinin,adriamycin and dexamethasone)and tannic acid molecule.As a result,all the tested analytes can be detected in the mass spectra.Deprotonated amino acids ions were detected with various signal-to-noise ratios(S/N).According to quantum chemistry calculations,we found that the amino acid which held the lower ionization energy showed higher signal-to-noise ratio.In the analyses of phospholipids,high signal-to-noise ratio was achievd and without the matrix interference.The formation pathways of phospholipids fragments were proposed and discussed.The Schiff base S1 has been demonstrated as an effective MALDI matrix for wide application in the dectection of small molecule including acids,lipids and alcohols in the negative ion mode by MALDI-MS.(3)Previously,compounds 9AA,DMAN and DPN have been used as MALDI matrixes in the negative ion mode for the detection of small molecule analytes and showed the advantage of less matrix interference.Here,the Schiff base S1 are compared with these alkaline matrices to show advantages and disadvantages for analyses of small molecules on the basis of following experiments: 1)the mole ratio effect of matrix to sample on analyte abundances;2)stability evaluation of the matrix under vacuum condition;3)the effect of matrix concentration on the matrix peak interferences;4)solid ultraviolet experiments of matrix crystals;5)quantum chemical calculation of carboxyl H-O bond length in the gas and liquid phase,and the proton affinity(PA),the proton affinity of deprotonated molecules(PA[M-H]-),ionization energy(IE)and electron affinity(EA)of these alkaline matrices in the gas phase for those alkaline matrices and S1 matrix.Results showed that the Schiff base matrix S1 had the advantages of vacuum stability,high sensitivity and potential use for quantitative analysis.In summary,a new Schiff base matrix S1 was developed and evaluated for the analyses of a series of small molecules,which showed advantages of reducing matrix interference in low m/z region,and enabling simple,quick and accurate quantitative analyses of fatty acids in serum.At the same time,quantum chemical methods were used to explain the mechanism of matrix performance.We hope that the presentation in this thesis may provide sufficient information for people to choose reasonable matrix under the theory guidance for the analyses of small molecule by MALDI-MS.