Inductively Coupled Plasma Mass Spectrometry Based Hyphenated Techniques And Their Application For Elemental Analysis And Speciation In Biological System

Trace elements play important roles in the organisms. The bioactivity, bioavailability and toxicity of the elements are not only dependent on their total amount, but also highly dependent on their existing species. Therefore, trace/ultra-trace elements and their speciation analysis in biological systems are of great significance. In bioanalysis, the sample volume is always very limited, the sample matrices is always complex, and elements or their species always occur in extremely low concentration. For these reasons, trace/ultra-trace elements and their speciation analysis in biological system have become a hot and challenging topic in analytical chemistry.Inductivity coupled plasma mass spectrometry (ICP-MS) is one of the most powerful methods for trace/ultra-trace elements analysis due to its outstanding advantages including high sensitivity, high sample throughput, wide linear dynamic range, multi-element detection and isotope analysis ability. Hyphenated techniques by combining high efficient separation techniques with ICP-MS is one of the most effective and widely-used methods for elemental speciation. Sample pretreatment techniques aiming to preconcentrate the analytes and separate them from matrix can effectively improve the analytical performance. Therefore, exploring novel miniaturized, high sensitive and effective ICP-MS based hyphenated techniques is highly demanded for trace/ultra-trace elements and their speciation analysis in biological systems. Biomarcromolecules analysis by elemental labeling ICP-MS is one of the newest advances of ICP-MS, developing elemental labelling ICP-MS methods based on the interaction between the elements and the bio-molecules is extremely significance for proteomics and metallomics.The aim of this dissertation is to develop high performance liquid chromatography (HPLC)-ICP-MS hyphenated new methods for elemental speciation in biological samples; to investigate novel sample pretreatment techniques for elemental speciation in biological samples; and to develop chip-based sample pretreatment techniques for electrothermal vaporization (ETV)-ICP-MS determination of ultra-trace elements in cells and quantification of protein in biological samples. The major contents of this dissertation are described as follows:(1) A C18 column dynamically coated with zwitterionic bile acid derivative, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), was used for direct injection HPLC-UV/ICP-MS speciation of aluminum (Al) in non-spiking human serum. Small molecule Al-complex compounds of Al-citrate (Al-Cit) and large molecule Al-protein compounds of Al-transferrin (Al-Tf) were chosen as the model species and their retention behaviors on CHAPS modified C18 column were studied with UV and on-line ICP-MS detection in details. Under the optimal conditions, large-molecule Al-protein compounds and small molecule Al-complex compounds could be separated within 4 min. The detection limits of the method (LODs,3σ) were 0.74 and 0.83 ng mL-1 with the relative standard deviations (RSDs, n=7) of 2.8% and 3.0% for Al-Tf and Al-Cit, respectively. The developed method was applied to the speciation of Al in real healthy human serum and chronic hemodialysis patient serum, the experimental results show that the most of Al exists as Al-protein, and the small amount of Al exists as small molecule Al-complex compounds in human serum. Compared with the reported methods in the literature, this method has several attractive features such as simplicity, rapidness, no sample preparation required, and it provides a new strategy for studying trace amount elemental speciation in human body liquids.(2) A new method based on room-temperature ionic liquids (RTILs) improved reversed-phase (RP)-HPLC-ICP-MS for selenium speciation has been developed. The different parameters affecting the retention behaviors of six target selenium species especially the effect of RTILs as mobile phase additives have been studied, it was found that the mobile phase consisting of 0.4%(v/v) 1-Butyl-3-methylimidazolium chloride ([BMIM]Cl),0.4% (v/v) 1-Butyl-2,3-dimethylimidazolium tetrafluroborate ([BMMIM]BF4) and 99.2%(v/v) water has effectively improved the peak profile and six target selenium species including Na2Se03 (Se(IV)), Na2Se04 (Se(VI)), L-Selenocystine (SeCys2), DL-Selenomethionine (SeMet), Se-methylseleno-L-Cysteine (MeSeCys), Seleno-D, L-Ethionine (SeEt) were separated within 8 min. The developed method was validated and successfully applied to the speciation of selenium in Se-enriched yeasts and clover. For fresh Se-enriched yeast cells, it was found that the spiked SeCys2 in living yeast cells could transform to SeMet. Compared with other RP- ion-pair (IP)-HPLC-ICP-MS approaches for selenium speciation, the proposed method possessed the advantages including ability to regulate the retention time of the target selenium species by selecting the suitable RTILs and adjusting their concentration, simplicity, rapidness and low injection volume, thus providing wide potential applications for elemental speciation in biological systems.(3) A new sorbent of poly[styrene-co-(sodium styrene sulfonate)-co-divinylbenzene] coated Fe3O4 magnetic nanoparticles (Fe3O4@PSS MNPs) has been synthesized by one step emulsion polymerization. Based on the cation exchange interaction between the sulfonate group on the Fe3O4@PSS MNPs and the target seleno-amino acids, a new method of magnetic solid phase extraction (MSPE)-HPLC-ICP-MS for seleno-amino acids speciation in Se-enriched yeast cells has been developed. Taking SeCys2, MeSeCys, L-y-glutamyl-Se-methyl-L-selenocysteine (GluMeSeCys), SeMet and SeEt as target selenium species, a series of factors that influence the MSPE were investigated in details. The conditions for subsequent HPLC-ICP-MS determination were optimized as well. Under the optimal conditions, the LODs (3σ) for target seleno-amino acids were in the range of 21.5-89.9 ng L-1 with the RSDs (n=7) ranging in 6.7-12%, the enrichment factors were varied from 10 to 92-fold, and the linear ranges were over three orders of magnitudes (R2>0.99). To validate the accuracy of this method, certified reference materials SELM-1 was analyzed, and the determined values were in good agreement with the certified values. The proposed method was also successfully applied for the target seleno-amino acids speciation in Se-enriched yeast cells, and the recoveries for the spiked samples were in the range of 78.8-106%. The proposed MSPE-HPLC-ICP-MS method is characterized with low cost, fast separation, high enrichment factor and low LODs.(4) A novel method based on in-tube hollow fiber-solid phase microextraction (in-tube HF-SPME) on-line coupled with RP-IP-HPLC-ICP-MS was developed for arsenic speciation in biological samples. Partial sulfonated poly(styrene) (PSP) and mixed-sol of 3- mercapto propyltrimethoxysilane (yMPTS) and N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AAPTS) were prepared and coated on the inner surface of polypropylene HF. The prepared yMPTS-AAPTS/PSP coated HF was characterized by FT-IR spectroscopy and scanning electron microscope (SEM). With arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMA), dimethylarsenic acid (DMA), arsenobetaine (AsB) and arsenocholine (AsC) as target arsenic species, a series of factors that influence the in-tube HF-SPME were investigated in details, such as pH value, sample volume and flow rate, elution conditions and interference of co-existing ions. Under the optimal conditions, the sample frequency was 6.5 h-1, the LODs (3σ) for target arsenic species were in the range of 0.017-0.053μg L-1 with RSDs (n=7) ranging in 1.5-5.2%, and the enrichment factors were varied from 4 to 19-fold. To validate the accuracy of this method, certified reference materials DORM-2 (dogfish) and CRM No.18 (human urine) were analyzed, and the determined values were in good agreement with the certified values. The proposed method was also successfully applied for arsenic speciation in healthy human urine samples, and the recoveries for the spiked samples were in the range of 92.6-107%. The proposed in-tube HF-SPME-HPLC-ICP-MS method is low cost, time- and labor-saving, and suitable for simultaneously organic and inorganic arsenic speciation in biological samples.(5) Quantitative analysis of trace levels of heavy metals in human cells is critical to environmental and toxicological research. A new strategy for determining ultratrace levels of cadmium (Cd), mercury (Hg), and lead (Pb) in cultured cells was described. It involves the integration of cell sample introduction and magnetic solid phase microextraction (MSPME) on a microfluidic chip, combined with ETV-ICP-MS detection.γ-mercaptopropyl-trimethoxysilane (yMPTS) modified silica-coated magnetic nanoparticles were synthesized and employed as the extraction material for microextraction of Cd, Pb, and Hg. Under an external magnetic field, these magnetic nanoparticles were self-assembled in microchannels to form a solid phase packed column. The formation mechanism of the on-chip magnetic solid phase packed column and the main factors influencing the packing and analytical performance were investigated. Under the optimized conditions, MSPME enabled enrichment of Cd, Hg, and Pb by a factor of>40, and resulted in improved LODs (3σ) for Cd (0.72 ng L-1), Hg (0.86 ng L-1), and Pb (1.12 ng L-1) using ETV-ICP-MS. Quantitative analysis of trace Cd, Hg and Pb in HepG2 cells were achieved by applying the microfluidic system that integrated cell rupture, mixing, magnetic extraction and elution into one device, followed by ultrasensitive detection of ETV-ICP-MS, which required only microliter of samples. Analysis of approximately 5000 cells revealed that the average amounts of Cd, Hg and Pb in a single HepG2 cell were on the order of a few femtograms, and that the cellular levels increased with increasing concentrations of these metals in cell cultures. The concentrations of Cd, Hg, and Pb detectable in HepG2 cells were several orders of magnitude lower than their IC50 values, suggesting that the technique is potentially useful for measuring these heavy metals in studies of chronic metal toxicity.(6) A sensitive and selective method of magnetic immunoassay on microfluidic chip based on a sandwich-type immunoreaction with PbS nanoparticle (NPs) labels combined with ETV-ICP-MS was proposed for the determination of carcinoembryonic antigen (CEA). For this purpose, a microfluidic chip for magnetic immunoassay was designed and fabricated, the prepared iminodiacetic acid (IDA) modified silica coated magnetic nanoparticles (MNPs) was packed in its central microchannel to form a solid phase column by self-assembly under the magnetic field. After a complete immunoreaction among primary antibody, CEA and secondary antibody labeled with PbS NPs on magnetic solid phase packed-column, the concentration of CEA was quantified by ETV-ICP-MS determination of Pb released from captured PbS NPs with an acid-dissolution step. By optimizing a series conditions for immunoassay including blocking, incubation and elution, the established method presented a LOD (3σ) of 0.058μg L-1 for CEA based on the Pb signal, with the RSD (n=7) of 6.7%. The response of the method for CEA was linear over a dynamic range from 0.2 to 50μg L-1, and the enrichment factor of 2-folds (from 60μL sample to 30μL elution) was obtained. The proposed method was successfully applied for CEA determination in real human serum, and the analytical results are in good agreement with that obtained by the currently used clinical analytical method of chemiluminescent immunoassay. The developed method represents various advantages such as fast speed, sensitivity, selectivity, low sample/reagents consumption, versatility, and can be easily extended to other biological and medical assays.(7) A new method based on cloud point extraction (CPE)-ETV-ICP-MS has been proposed for the speciation of inorganic selenium in environmental waters. When the temperature of the system is higher than the cloud point temperature (CPT) of the selected surfactant Triton X-114, the complex of Se(IV) with ammonium pyrrolidine dithiocarbamate (APDC) can be extracted into the surfactant-rich phase, whereas the Se(VI) remains in aqueous solutions. Thus, an in situ separation of Se(IV) and Se(VI) and the preconcentration of Se(IV) could be realized. The surfactant-rich phase was diluted to 200μL with methanol and 10μL of them was introduced into the ETV-ICP-MS for subsequently determination. For Se(VI) determination, Se(VI) was reduced to Se(IV) prior to CPE, and its assay was based on subtracting Se(IV) from total selenium. The main factors affecting the CPE and the vaporization behavior of the analyte were investigated in details. Under the optimized experimental conditions, the LOD for Se(IV) was 8.0 ng L-1 with an enhancement factor of 39 when 10 mL of sample solution was preconcentrated to 0.2 mL. The RSD (n=7) was found to be 3.9%. The proposed method was validated and applied to the speciation of inorganic selenium in different environmental water samples with the recoveries for the spiked samples in the range of 82-102%.