Study On Rapid Mass Spectrometry Strategies For Metabolites Analysis

Metabolites are the products of the metabolism of natural life activities in the organism.As we all know,life activities are performed together by many nucleic acids,proteins,and small molecular metabolites.The functional changes of upstream nucleic acids,proteins and other macromolecules will eventually be reflected in the metabolic level.Metabolites are the embodiment of the results of life processes.Many previous studies have proved that many diseases are closely related to abnormal changes of the abundance of metabolites.Therefore,accurate and effective analysis of metabolites plays an important role in the diagnosis,prevention and treatment of diseases,the study of disease mechanism and bioenergy.However,metabolites are widely distributed with great differences in the numbers,species,abundance,polarity and other aspects.Futhermore,the biological samples matrix is complex and the interference is serious.It is a great challenge to analytical chemistry for achievement of the qualitative and quantitative analysis of metabolites quickly and accurately.At present,mass spectrometry has been widely used in various fields due to its high sensitivity,high selectivity and high efficiency in the structural analysis of compounds.In recent decades,with the rapid development of atmospheric ionization techniques,in vivo,in situ,real-time and on-line rapid mass spectrometry analysis methods have become an inevitable trend of mass spectrometry strategies.Based on the techniques of mass spectrometry,this paper is devoted to the development of fast,efficient extraction technology with good biocompatibility,and atmospheric ionization techniques with high matrix tolerance in order to realize fast,in situ structural identification and quantitative analysis of metabolites in biological samples.In the first chapter,the significance of metabolite analysis,the main analytical strategies and the mass spectrometry based rapid analytical techniques that reported in the previous literatures were summarized.The main problems of the existing mass spectrometry based rapid analytical methods and the main ideas of this paper were briefly described.In the view of the complex biological sample matrix,the atmospheric ionization mass spectrometry cannot direct quantitate the target analytes accurately in biological samples due to the serious ion suppression.In the second chapter,a fast and facile method for the determination of trace ribonucleosides in the human urine was developed by the combination of the nanocoating cellulose paper micoextraction with the nanospray ionization tandem mass spectrometry(nESI-MS/MS).Nanocoating cellulose paper was prepared through nano-precision deposition of uniform ultrathin zirconia gel film layer-by-layer on the cellulose paper using a sol-gel method.Due to the large surface area of cellulose paper and the strong affinity between zirconia and cis-diols(Lewis acid-base interaction),ribonucleoside could be selectively extracted and enriched from urine sample,thus greatly improving the detection sensitivity.Typically,the nanocoating cellulose paper was immersed into the diluted urine to extract the target analyte,and then subjected to nESI-MS/MS.The entire analytical process could be completed within 10 minutes.The method was evaluated by determining the amount of ribonucleosides(adenosine,cytidine,uridine and guanosine)in urine samples.The signal intensity of ribonucleotides extracted from nanocaoting cocellulose paper was 136-459 times higher than that from unmodified cellulose paper.The limit of detection(LODs)and limit of quantification(LOQs)were in the range of 0.014-1.26 μg·L-1,0.045-4.19 μg·L-1,respectively.The recoveries of the ribonucleotides in the urine sample were in the range of 75.64-103.49%,and the relative standard deviations(RSDs)were less than 9.36%.This method is accurate,reliable,low-cost,easy to prepare and environmentally friendly,and has a great potential in rapid and low-cost on site disease diagnosis in resource-deficient areas.In the third chapter,the stable spray could not be observed in the conventional negative-ion mode paper spray unless the spray solvent was changed into non-polar solvent owing to the electrical discharge.Inspired by ion motions driven by the opposite electrophoretic force and the steady opposite polarities of cone-jets present within AC electrospray in the previous reported literatures,we assume that serious electrical discharge in negative-ion paper spray could be alleviated by AC voltage because of the periodic relaxation of the accumulated electrons from the spray tip.Based on this point,the mathematical model of excitation inductance of high voltage AC power supply was proposed.After LTSpice simulation,a pulsed high voltage power supply with low frequency,continuously adjustable voltage,good frequency stability and small waveform distortion was successfully constructed.After AC voltage applied,detection of saturated fatty acids,phosphorylated peptides and adenosine 5’-disodium triphosphate(ATP)in negative-ion mode paper spray has been successfully achieved with high sensitivity.In addition,considering the easy availability of the application of pulsed high voltage power supply,we also designed and configured a set of fixed frequency(50 Hz)high voltage AC power supply with high adaptability and low cost.The high AC voltage was originally generated from 220 V/50 Hz AC household electric power,and then amplified by two stage transformers.The entire power supply equipment is simple,portable and low cost,which could be extended easily in various analytical environments.In the fourth chapter,because of the existence of isomers and the difficulty in locating C=C double bonds of free unsaturated fatty acids(FAs),we apply the developed AC paper spray ionization to the analysis of unsaturated FAs in human serum.Herein,the accurate in situ identification and quantification of unsaturated FAs C=C positional isomers in human serum was achieved successfully.The epoxidation reaction between the C=C bonds and the reactive oxidative species generated during the negative-ion mode AC paper spray could be rapidly and controllably performed via AC voltage.Upon collision induced dissociation(CID)tandem mass spectrometry,the epoxides of FAs were fragmented to release the characteristic diagnostic product ions,which could indicate the specific C=C positions.At the same time,the intensity of the characteristic fragment ions could be used for the quantification of the FA isomers.The LODs and LOQs of this method for unsaturated FAs were in the range of 0.018-0.051μM and 0.022-0.36 μM,respectively.Epoxidation of unsaturated fatty acids and ionization of epoxides could be achieved one-step without the need of additional sample preparation or chemical reactive reagents.Therefore,the proposed method has a promising application prospect for the point-of-care diagnosis.