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.

Methods for the determination of trace elements in interfering matrices were developed. The methods involve two strategies for the reduction of interferences: the use of new pretreatment methods for use with existing instrumentation, and the development of new instrumentation. A comparison was made of inductively coupled plasma-mass spectrometry (ICP-MS) detection limits and interference reduction for arsenic by hydride generation, flow injection, continuous aspiration, and mixed gas methods. Practical considerations in implementing these common techniques, as well as in the general use and maintenance of an ELAN 5000 ICP-MS instrument are presented for the benefit of new users. Two approaches for the elimination of interferences in the determination of arsenic in gold by ICP-MS. The main challenges in this analysis were the arsenic signal suppression by gold and the polyatomic interference due to chloride. A method was developed for the batch mode reductive precipitation removal of interfering gold matrix from the sample prior to hydride generation and detection of arsenic. This method has great potential value as the basis for other simple methods for the removal of transition metals that interfere with hydride generation. An on-line anion exchange method for the removal of interfering gold prior to hydride generation was subsequently developed to improve the speed of analysis. The batch-mode reduction method for the removal of gold resulted in a better detection limit than the anion exchange method. However, the automated anion exchange gold removal method required less bench chemistry, was faster, consumed less reagents, and created less mess than the off-line method. On-line solid phase extraction methods are a valuable and increasingly popular method for the removal of matrix interferences. The automation of sample pretreatment as well as the small sample size required when the technique is combined with flow injection make it ideal for use in the analysis of hazardous materials. The severe signal suppression of uranium on lithium, beryllium, magnesium, and aluminum was overcome by the on-line flow injection-solid phase extraction removal of uranium using a column packed with Tru-Spec resin. The column was regenerated on-line using ammonium oxalate. The development of an internal combustion engine as a novel source for atomic emission spectrometry was begun. This preliminary stage of development focused on the identification of important experimental parameters to be considered in future work. A one cylinder four stroke engine was modified to allow optical access through the cylinder head. Combustion radiation was monitored with a photomultiplier tube and the signal was stored using LabView software. It is hoped that this work will be the basis for the development of an inexpensive, portable, rugged instrument that will accept a variety of fuels.

Advances in knowledge of toxicology, clinical chemistry, and nutrition have made the mere determination of an element in a sample insufficient. Both the amount of an element and the forms in which it is present need to be determined. Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful element selective detection method for elemental speciation studies, and was employed as a detection method for high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) separations. An ICP-MS ion pair HPLC method for the separation of selenoamino acid standards was developed. The method was used to identify and quantify selenoamino acids present in water extracts of selenium enriched allium and yeast samples which have been shown to have cancer preventive effects in animal or human studies. Commercially available selenium nutritional supplements were also evaluated. Selenium speciation was shown to vary with level of selenium enrichment and among samples. The method was shown to be compatible with standard ICP-MS operating conditions and electrospray ionization mass spectrometry (ESI-MS). The developed method was also shown to be applicable for sulfur and tellurium speciation studies. The oxidation and reduction of selenoamino acids was investigated by HPLC-ICP-MS. Selenoamino acids were shown to be readily oxidized, and the formation of oxidation products were monitored with time. The presence of oxidized or reduced selenoamino acids in extracts of selenium enriched samples was investigated. The reversibility of the oxidation of some selenoamino acids was demonstrated, and identification of the oxidation products was attempted by ESI-MS. A preliminary evaluation of a CE-ICP-MS interface for metalloprotein speciation was carried out using model proteins and human serum, and its effects on the separation characterized. The adsorption of lead to CE capillaries was investigated. The use of commercially coated and dynamically coated capillaries resulted in reduced lead adsorption.

Flow injection hydride generation atomic absorption spectrometry is the practice of derivatizing the analyte to the volatile hydride by flow injection and introducing it to the spectrometer in the gas phase. Although hydride generation is well developed, there are still aspects and nuances which require investigation to improve the understanding of this method of sample introduction. The suggestion that the addition of various surfactants to the reagents in the hydride forming reaction has beneficial effects has been investigated. Although, in previous reports, an increase in sensitivity had been reported in the presence of surfactants, such an improvement was not observed for the flow injection procedure used in the work described in this dissertation. The effect of dissolved salts on the hydride forming reaction has been investigated. An increase in sensitivity in the presence of high concentrations of dissolved salts has been observed. This has been attributed to an increase in the proportion of the volatile hydride which is removed from the reagents in the on-line procedure. In the hydride trapping procedure, the hydrides are trapped on the interior of an electrothermal atomizer prior to atomization. Although previous reports have indicated that one may improve the limit of detection simply by increasing sample size, a practical and theoretical investigation concluded that the relationship is not linear, and that the limit of detection is limited by impurities in the reagents. Methods for the determination of lead by hydride generation with quartz tube and electrothermal atomization have been developed. The use of an oxidizing agent makes this method a little more complicated than for other elements. The determination of total selenium in urine has been attempted using an on-line digestion procedure in which bromine is generated in-situ. Whilst this method has been demonstrated to be effective in an off-line procedure, the development of a method with on-line digestion was not successful. The use of an alternative oxidizing agent and energy supply mechanism would be appear to be necessary.

The area of atomic spectrometry has long suffered from inefficient sample preparation and introduction systems. Microwave heating can be a powerful technique for improving these systems but has tending not to be well understood and used in analytical chemistry. The purpose of this work has been to use microwave energy in order to improve sample preparation and introduction for plasma source spectrometry. An objective of this work has been to develop an on-line pressurized microwave digestion system. This system was developed to be capable of digesting difficulty soluble organic materials with nitric acid alone rapidly while reducing possibilities for contamination and providing the possibility for automation. The use of microwave energy has been considered as a technique for desolvating an aqueous aerosol prior to solvent removal using a Nafion dryer. The purpose of this system was to increase the efficiency of sample introduction to a plasma source mass spectrometer. In order to accomplish this some fundamental studies of microwave interaction with water droplets were performed and a theoretical background was developed.