Metal complexes play fundamental roles in synthetic, biological, and catalytic processes, and when doing so they are often present at very low concentrations. Determining the coordination structure of complexes at trace levels or in complicated mixtures, however, is a very difficult task. This dissertation focuses on the investigation of a new methodology, based on ion-molecule (I-M) reactions and mass spectrometry (MS), to determine the coordination structure of metal complexes sensitively. This work describes the study of the gas-phase reactivity exhibited by a variety of model transition metal complexes with different reagent ligands in a quadrupole ion trap mass spectrometer. A series of tetradentate and pentadentate ligands containing N, O, and/or S donor atoms were synthesized and complexed with late first-row transition metals. Noticeable differences in reactivity were found as the ligand field around the central metal ion was changed. The electronic structure of the metal center was also found to exert a remarkable effect on the gas-phase reactions of metal complexes, and in fact, complexes with different geometric structures could be distinguished. Angular overlap model (AOM) and density functional theory (DFT) calculations were used to explain the experimental observations. The analytical utility of the I-M reactions was also investigated. Fluctuations in reagent and buffer gas pressures and vacuum system temperature were examined as possible error sources affecting the conclusions drawn from the I-M reaction procedure. Reagent gas pressure and temperature were found to be the most important factors affecting the reproducibility of the experimental data. Because the analytical information derived from these I-M reactions relies on complexation of a reagent ligand, steric factors that might affect these reactions were also evaluated. In summary, I-M reactions can distinguish complexes with different coordinating functional groups and geometries, and these reactions have some potential for providing coordination structure information for complexes present at trace levels.

Forensic scientists attempt to solve crimes through examination of the evidence found at crime scenes. By determining the relative concentrations of certain elements present in bullets, a profile of an individual sample can be developed that will allow investigators to match ammunition from a crime scene with that from a known source. The removal of a substantial proportion of the lead matrix is required for the determination of trace elements in bullets by inductively coupled plasma mass spectrometry (ICP-MS). Several flow injection methods for the selective removal of lead from bullets have been developed. On-line solid-phase extraction with the commercially available resin Pb-Spec® removed the matrix quantitatively. Pb-Spec incorporates an immobilized crown ether, giving the resin selectivity for lead. The analytes were not retained, but passed through the column for quantification by ICP-MS. The method was validated by analyzing NIST SRMs 2415 (battery lead), 2416 (bullet lead), and 2417 (lead-based alloy). The injection of large sample volumes into a flow injection ICP-MS system produces transient peaks with flat tops. The signal at peak maximum is directly proportional to analyte concentration. Sample throughput can be improved by making replicate measurements during the residence time at peak maximum instead of requiring multiple sample injections. A successful procedure utilizing Pb-Spec for the removal of matrix lead from a bullet sample was developed, and validated by the analysis of SRMs. On-line precipitation and filtration of the matrix was examined. The bullet sample was combined with sulfuric acid, and the conditions for maximum precipitation were obtained. The resultant lead sulfate was filtered on a column packed with macerated glass wool. The formation of sulfates by the analytes was not favored, so they were detected by ICP-MS. The potential for lead preconcentration by Pb-Spec was investigated with environmental water samples, and the retention characteristics were examined in detail. The feasibility of incorporating a crown ether-containing supported liquid membrane into a flow injection manifold was investigated for the removal of high concentrations of lead from solutions. Experimental and theoretical results demonstrated that the time scale of mass transfer was too slow for flow injection applications.

The development of nephrocalcinosis, the progressive formation and deposition of oxalate crystals in the kidneys, in premature infants is a relatively common and potentially serious condition. Although hyperoxaluria and hypercalciurea are both associated with the pathogenesis of nephrocalcinosis, it is believed that hyperoxaluria may be more important in enhancing the risk of calcium oxalate precipitation. Therefore, dietary measures to decrease urinary oxalate excretion in preterm infants may lead to decreased incidence of nephrocalcinosis. Total parenteral nutrition (TPN) solutions can act as a direct source of oxalate introduction into premature infants since they contain ascorbic acid (vitamin C) which is known to oxidize to oxalate in-vitro. Since increased urinary oxalate excretion has been reported in infants receiving TPN is was of clinical interest to be able to determine whether the infusion of TPN solutions into premature infants resulted in an unnecessary oxalate burden. A robust ion chromatographic method with suppressed conductivity detection was developed for the reliable determination of oxalate at part-per-million (ppm) levels in TPN solutions. An eluent system based on sodium carbonate and sodium bicarbonate was developed, permitting the separation and selective detection of oxalate from the major anionic components of the TPN matrix. The interference effect of method induced oxalate formation from ascorbate, which is known to hinder the determination of oxalate in TPN by currently available methodologies, was shown to be absent. The application of uncertainty in measurement was applied to ensure that a low level of experimental uncertainty was carried through to the obtained data. The effect of non-linear analyte response that is commonly reported when using carbonate and bicarbonate eluents with suppressed conductivity was evaluated. The deviation of calibration data from a linear response was found to be only very slight over the oxalate concentrations studied (0.2 ppm–30 ppm). The application of the developed method to the preliminary study of oxalate formation in TPN subjected to phototherapy conditions was successfully demonstrated.

A method was developed for the determination of total arsenic in rice grain by microwave-assisted digestion inductively coupled plasma mass spectrometry. Standard calibration solutions were matrix-matched with respect to acid concentration and carbon content post-digest. The importance of eliminating the drying step during sample preparation procedures was investigated. The method was validated with spikes containing standard arsenate solutions into the rice matrix, and with certified reference material SRM1568a (rice flour) from NIST. The method was successfully applied to a commercially available rice sample. Four arsenic species [arsenate (As(V)), arsenite (As(III)), dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA)] were extracted from rice grains by microwave-assisted extraction and separated with high performance liquid chromatography inductively coupled plasma mass spectrometry. The method includes a novel sample clean-up step involving a dialysis procedure to decrease the amount of large starch molecules in the injection solution, in order to minimize poor resolution of chromatographic peaks and maximize column life. The method was validated with spikes of standard arsenic solutions, added to the rice matrix before the extraction procedure. Literature reference values for arsenic species quantification in SRM1568a (rice flour) were also compared. This method was successfully applied to a commercially available rice sample. A study into improvements in reverse phase-HPLC separations of arsenic species was conducted. For the first time, a Sunfire C8 column from Waters (Milford, CT) was employed for the separation of arsenic species in rice extracts. This column was compared to a Symmetry C8 column with respect to total elution time, detection limits, interference effects, and column life, and evaluated with respect to peak resolution, shifts in retention times, and peak symmetry.

Studies of the interaction of the relevant metal, metalloid or nanoparticulate species with biological systems are underpinned by the provision of reliable information about chemical composition of the relevant materials. Often, no methods of chemical analysis are available. The work described in this dissertation centers on developing methods to help with studies for a variety of analytes and samples. A method was developed for the determination of 11 trace elements (As, Cd, Co, Cr, Cu, Fe, Mg, Mn, Pb, Ti, and Zn) in human breast milk and infant formulas by inductively coupled plasma optical emission spectrometry (ICP-OES) following microwave-assisted digestion. A method was established for the determination of trace elements, with an emphasis on titanium as titanium dioxide, in snack foods and consumer products. The interactions of some dissolved metals, including rare earth elements, and metallo-nanoparticles (silver, gold, titanium dioxide, aluminum oxide, and iron) with aquatic plants were studied. After exposure in a variety of mesocosms, the partitioning of the elemental species between various compartments was quantified by ICP-MS and ICP-OES following microwave-assisted digestion. An ICP mass spectrometry (MS) method has also been developed to quantify the uptake of gold and silver nanoparticles by C. elegans. Uptake of gold nanoparticles was size dependent, suggesting increased ingestion efficiency with increased particle diameter. The feasibility of discriminating between suspended TiO2 nanoparticles and dissolved titanium by the analysis of the rapid transient signal events obtained from the ICP-MS instrument operated in a rapid response mode was also developed. Data handling parameters were established that allowed a distinction in the signals for nanoparticulate and standard solutions. Spikes in the signal were defined by distinct parameters using the mean and standard deviation, where a spike in the signal was defined as a signal > x¯ + ks (k =3). This approach however did lead to a statistical difference in the spike signal events for solutions and nanosuspensions.

In the last two decades, protein drugs have enjoyed a rapid growth and achieved a tremendous success in treating human diseases. However, the presence of physiological barriers greatly impedes the efficient delivery of such unconventional large molecule drugs, and therefore limits their clinical utility. An elegant way to address this challenge takes advantage of certain endogenous transporter proteins, such as human transferrin (Tf), whose ability to traverse physiological barriers has been extensively exploited. However, methods to investigate Tf-based drug delivery remained insufficient and unsatisfactory until recent development of quantitative mass spectrometry (MS). Hereby, MS-based methods have been developed and validated for quantitation of exogenous Tf in biological fluids. Particularly, different O18-labeling based approaches have been evaluated, modified and developed in this work, in order to achieve the most reliable quantitation. Alternatively, a novel approach based on indium labeling and inductively coupled plasma mass spectrometry (ICP-MS) detection has been developed for sensitive quantitation of Tf in biological fluids. The second aspect of this dissertation work focuses on the application of MS-based methods for characterization of protein drugs at different levels, ranging from protein identification, covalent structure, conformation, and interaction with physiological partners. Particularly, an O18-labeling assisted approach has been developed to identification of protein deamidation products. This new approach can readily distinguish between the two deamidated isomers. Also, an LC-MS based method has been developed for ranking the susceptibility of protein disulfide bonds to reduction, which could be applied to several disulfide bond-related analyses. Finally, a recently designed growth hormone transferrin fusion protein was studied using MS-based methods, and the molecular basis for its successful oral delivery was revealed.

Amperometric and coulometric reticulated vitreous carbon (RVC) detectors were constructed and evaluated for detection in flow injection analysis. These detectors offer a wide range of fraction conversion up to 100%. The flow rate dependency of the current was studied and compared with porous electrode theory. The precision, analytical range, band-broadening, and detection limits are comparable to that of other glassy carbon amperometric detectors. Pulsed flow electrolysis with the RVC electrode was developed for amperometry and voltammetry using lock-in amplification to measure the a.c. response which is independent of the large background current. Linearity and detection limits were determined and factors affecting the detection limits were investigated. The RVC electrode was shown to have rapid diffusion layer relaxation due to high conversion and thin layer electrolysis. Computer calculations of thin layer electrolysis were used to predict the mass transfer properties of the electrode and the results were compared with those of other electrode geometries. A new on-line system for studying the secretion of catecholamines from stimulated bovine adrenal cells in culture allows greater precision in kinetic measurements of the onset of secretion and the desensitization. The low-volume system uses flow injection analysis with which variable amounts of stimulant injected into the flowline pass through a packed bed of microbeads to which the cells are attached and the secreted catecholamines are electrochemically detected downstream. The low dispersion of the system provides low distortion of the secretory response and good detection limits. The desensitization in the presence of long-term stimulation with acetylcholine at room temperature was first order. Dramatic differences in the amount secreted and the recovery time of the cells were found at physiological temperature. Studies of secretion in the presence of nucleotides and varying concentrations of calcium revealed a depression of secretion in the presence of ATP and a minimum rate of desensitization in 1 mM calcium. More catecholamine was secreted in the absence of sodium than in its presence. The versatility of the technique for investigating desensitization and secretion due to one affector in the presence of another was demonstrated.

The work presented is divided into three sections. The first describes the study of ac voltammetric methods in flowing streams. The objective was to determine the dependence of the ac current on concentration, frequency, ac amplitude and dc scan rate for static and flowing conditions. The goal was to show that the ac current would be independent of the flow rate under the proper experimental conditions. The ac voltammetric experiment was shown to be independent of the flow rate over the entire range of flow rates studied. The second section describes the design and development of an ac electrochemical instrument that uses a lock-in amplifier with phase sensitive detection. A phase sensitive detector was designed that utilizes digital counting techniques and precision phase shifting circuitry to optimize the measurement of the faradaic signal in the presence of the capacitive background. The goal was to improve the detection limits for ac electrochemical measurements ten to one-hundred fold. Electrical measurements were made to evaluate performance characteristics, such as minimum detectable signal, dynamic range, dynamic reserve and phase angle resolution. The results from the electrical characterization data showed that the lock-in amplifier with digital phase sensitive detection performed very closely to design specifications. The final section describes a study made to evaluate the performance of the new ac electrochemical instrument in making actual ac electrochemical measurements. AC linear scan and amperometric experiments were performed on a reversible redox system, the ferri-ferrocyanide couple, at a carbon paste electrode. The object was to determine detection limits for these experiments with the new instrumentation. The detection limits obtained for the ac electrochemical experiments are 10 to 50 times better than previously reported detection limits for ac methods.

The objective of this study was to develop new methods for the determination and speciation of arsenic in soils. Ion-pairing chromatography was used for the separation of 4 toxicologically important arsenic species (As(III), As(V), monomethylarsonic acid (MMAA), and dimethylarsinic acid (DMAA)). Hydride generation atomic absorption spectrometry (HG-AAS) or inductively coupled plasma mass spectrometry (ICP-MS) was used for detection. Extraction of arsenic species from soils was achieved using various techniques such as shaking, sonication, and microwave-assisted extraction. Even though As(V), DMAA and MMAA were quantitatively extracted from spiked soils, the recoveries for As(III) were low due to its irreversible adsorption into the soil. In order to determine acid-extractable arsenic in soils, a procedure based on flow injection and slurry sampling with on-line microwave extraction (SS-MW-HG-AAS) was developed. The on-line microwave extraction increases the recovery of the adsorbed arsenic from 30 to 70%. The manifold was also successfully coupled to an ICP-MS for the multielemental analyses of soils. In order to eliminate the use of liquid nitrogen for the speciation of arsenic using cryo trapping, a thermal desorption instrument equipped with a Peltier cooler was utilized. The arsines from the HG manifold were directed into the U-tube of the instrument, which is kept at −30°C. At this temperature, arsine eluted with little retention but the organo-arsines were trapped and then released after arsine completely eluted. A new nebulizer design that provides better sensitivity than commercially available nebulizers are described. This nebulizer is constructed by inserting one fused silica capillary into another and then inserting this double capillary system into a PEEK tube (Double Capillary Nebulizer-DCN). Unlike the conventional concentric nebulizers where the argon delivery is from the outer area, the uniqueness of this nebulizer was the delivery of the argon from the inner and outer parts of the liquid flow to increase the efficiency of the interaction between the liquid and the gas. The performance of the DCN was comparable to the Meinhard nebulizer and some of the DCNs prepared were found to perform even better. The cost of a DCN is approximately 15% of the price of a Meinhard nebulizer.

The determination of the total element content in a biological sample or system has become outdated in recent years. Trace element speciation has become increasingly important in environmental, nutritional, biological and medical sciences. Speciation of an element includes the identification of the compound to which the element is bound and the quantification of the element associated with the particular compound. Various separation techniques coupled with elemental detection techniques have been used for the speciation of trace elements. Capillary electrophoresis (CE) has been under development for more than a decade. It offers rapid separation with high separation efficiency and resolution, and it also requires very small sample size which makes it especially attractive for biological sample analysis. Currently, inductively coupled plasma mass spectrometry (ICP-MS) is one of the most sensitive elemental detection techniques, with detection limits down to part per trillion levels. Research on interfacing CE and ICP-MS has been in progress during the past five years. Different nebulizers have been used for the CE-ICP-MS interfaces. In this research project, two nebulizers, a concentric and an ultrasonic (USN), are used as CE-ICP-MS interfaces. Metallothioneins (MTs) are used to characterize and optimize the interfaces. Metallothionein isoforms are separated and determined by the CE-ICP-MS. With the concentric nebulizer interface, negative pressure had to be applied at the inlet of the CE capillary in order to maintain the separation resolution. No suction occurred in the USN interface. Signal intensity was improved with the USN interface compared to the concentric nebulizer; however, signal-to-noise ratio was only marginally improved owing to the higher noise level. Protein-bound zinc in human serum albumin and $\alpha\sb2$-macroglobulin was determined with the USN interface. In addition to being a model protein for characterization of the interfaces, metallothionein is also one of the major zinc-binding proteins that is of interest in this project. Separation and determination of metal-contents of MTs in different buffers by CE-ICP-MS was examined with on-line photodiode array detector. Analysis of serum proteins by CE-ICP-MS was investigated. The possibility of ICP-MS detection of protein-bound zinc in albumin and $\alpha\sb2$-macroglobulin is demonstrated. Zinc isotope ratio measurement of these proteins is also attempted, however, the signal sensitivity available from current instrumentation and interface does not ensure precise and accurate measurement. Further improvement of the interface and instrumentation is necessary.