Application Of Novel In-tube Solid Phase Microextraction To The Analysis Of Volatile Metabolites In Breath Vapor

Solid-phase microextraction(SPME),a relatively efficient,solvent-free and versatile sample-preparation method,was developed by Arthur and Pawliszyn about 2 decades ago.It has the advantage of no solvent extraction,rapid,efficient,high sensitivity.SPME has been broadly applied in different fields,such as environment,food,forensics and pharmaceuticals.At present,more and more attention has been paid to the accurate determination of trace compounds in complex matrices,such as trace pollutants in atmosphere,biomarkers in biological matrices and so on.Lung cancer is one of the cancer with the highest cancer incidence and mortality around the world,its early diagnosis is important.Breath analysis is a promising approach for the non-invasive assessment of inflammatory and oxidative stress biomarkers involved in the etiopathogenesis of various respiratory conditions.EBC analysis still faces great challenges because of the complex matrix of EBC sample and extremely low content of the target analytes.Therefore,it is necessary to develop a fast,simple,efficient,stable,sensitive,and automated sample pretreatment technology to solve these problems.The traditional SPME technique involves a small-diameter fused silica rod(?1cm)coated adsorbing material that serves as the stationary phase.However,Fiber-based SPME is confronted with low sample and extraction capacity,instability and swelling in organic solvents due to the short length of the coated segment and the physical bonds between the substrate and the coatings.To solve the intractable problems existing in the traditional SPME mode,Eisert and Pawliszyn initially introduced in-tube SPME(IT-SPME)in 1997.IT-SPME is a great way to overcome problems often encountered by use of fiber-based SPME.Meanwhile,IT-SPME allows for the convenient and on-line HPLC automation of the extraction process.The key of SPME technology is the fiber coating materials,which determines the sensitivity and selectivity of SPME method.Therefore,the development of high selectivity,high efficiency and high stability of the coating and its preparation method has been a research focus,but also the key to the development of SPME Technology.In this paper,a new type of SPME coating material was prepared by electrophoretic deposition,which was combined with chromatography and mass spectrometry for the analysis and determination of small molecules in human breath metabolism and the headspace gas of lung cancer cell culture in vitro.The main study contents are listed as follows:In this work,G/PANI coating was fabricated on the internal surface of stainless-steel tube by a facile in-situ electrodeposition strategy,and a novel on-line in-tube SPME-HPLC method(IT-SPME-HPLC)was developed for the determination of trace aldehyde metabolites in human exhaled breath condensates.The morphology,microstructure and extraction performance of the resultant composites were investigated systematically.Worm-like PANI nanorods were densely packed on the hierarchical,porous and wrinkle-like surface of graphene plane.On the one hand,the embedding of polyaniline can improve the stability of the material on the substrate;on the other hand,it can avoid the overlap of the layer graphene.Graphene has been incorporated successfully into PANI polymer,which can provide more accessible adsorption sites for extraction.Some parameters related to extraction efficiency were also studied.Under the optimized conditions,the coating was used to quantitatively extract six aldehydes.Six aldehydes were determined simultaneously with low limits of detection of 0.02-0.04 nmol L-1 and good linearity(R2 ? 0.9920).The method has been applied successfully for the determination of aldehydes in human exhaled breath condensates with good recovery(70%-120%)and satisfied reproducibility(relative standard deviation:1.1%-11.9%).G/PANI coating possessed excellent mechanical and chemical stability,long lifespan and robustness.Moreover,the automated and back-flush continuous desorption mode enables rapid analysis.IT-SPME allows for the convenient automation of the extraction process by on-line coupling to HPLC.It can ensure faster analysis speed,less labor,reduced consumption of organic solvents and samples,higher accuracy and precision relative to off-line manual techniques.In this work,we introduced a novel graphene/polyaniline(G/PANI)electrodeposited coating for needle trap microextraction coupled GC/MS for the first time.The needle trap device(NTD)sampler prepared by electrodepositing G/PANI into 21-gauge stainless steel needle was evaluated for the dynamic headspace analysis of EBV and cell breath gas.The coating inside the needle exhibits enhanced mechanical stability,longer lifespan,larger specific surface area and good biocompatibility.The morphology and formation of the composite coating were confirmed by scanning electronic microscopy(SEM)and Fourier transform infrared(FTIR).The parameters for the sampling and analysis of analytes by NTD including extraction mode,sampling temperature,sampling speed,desorption parameters(temperature and time)were evaluated and optimized in the laboratory.The G/PANI coated needle was also compared with G/PANI coated fibers as well as SPME with PDMS,PDMS/CAR,PDMS/DVB,PA and DVB/CAR coated fibers.Lower limits of detection of 0.002-0.003 ?g L-1 and good linearity(R2?0.9934)are better than that of G/PANI coated fibers.The method has been applied successfully to the determination of VOCs in human exhaled breath and cell breath gas with good recovery(70%-120%)and acceptable reproducibility(relative standard deviation:Intra-day 4.8%-12.7%,Inter-day,4.2%-14.3%).It provides a new possibility to the determination of VOCs of exhaled breath both in vivo and in vitro.The specific volatile organic compounds(VOCs)exhaled by lung cancer cells in the microenvironment are the source biomarkers of lung cancer and also serve as direct evidence that the diagnosis of lung cancer by breath is possible.