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Biodiesel is a renewable, biodegradable, clean burning fuel, produced from vegetable oils and animal fat. It is a mixture of fatty acid alkyl esters, products that result from the catalytic transesterification of lipids. The first part of this research describes the development of a new and direct method used to rapidly and quantitatively determine the amount of free methanol in biodiesel samples. The analytical method developed is different from the current standards for methanol determination, and it is the first headspace-SPME method used to extract methanol from biodiesel as matrix. The second part of this research describes the direct analysis of acetic acid and 2uraldehyde in an aqueous mixture using headspace SPME. The direct and accurate determination and quantitation of these two analytes is very important, as they can be inhibitors or food sources for microorganisms capable of producing lipids or ethanol.

Today’s solid phase microextraction (SPME) is a well known technique that combines knowledge from different fields in an attractive, efficient, and economic way. The development of SPME has seen huge growth since its introduction as a new approach to sample preparation in the early 1990s. The applications of SPME are continuously expanding, and one of the most interesting current aspects consists of applying SPME for fast analysis of biological fluids, both in vitro and in vivo. In spite of this great potential, development of new bio-applications is considerably hindered by the lack of suitable SPME products. The goal of this study is to find SPME coatings that can be utilized for in vivo and in vitro extractions, in direct contact with a biological matrix such as blood or tissue. This thesis presents several effective ways of preparing SPME coatings based on biocompatible polymers and silica-based extractive phases, focusing on their biocompatibility as a must. After fabrication, the proposed coatings are tested for biocompatibility and analytical utility. Finally, some practical applications of the new coatings are presented, such as fast drug analysis and determination of drug plasma protein binding. Six test drugs with different physico-chemical properties are chosen for the investigation: diazepam, verapamil, lorazepam, warfarin, nordiazepam, and loperamide. It is shown that the application of these new SPME fibers for biological sample analysis greatly reduces the time required for sample preparation and limits the exposure of the analytical personnel to potentially infectious biofluids.

Today’s solid phase microextraction (SPME) is a well known technique that combines knowledge from different fields in an attractive, efficient, and economic way. The development of SPME has seen huge growth since its introduction as a new approach to sample preparation in the early 1990s. The applications of SPME are continuously expanding, and one of the most interesting current aspects consists of applying SPME for fast analysis of biological fluids, both in vitro and in vivo. In spite of this great potential, development of new bio-applications is considerably hindered by the lack of suitable SPME products. The goal of this study is to find SPME coatings that can be utilized for in vivo and in vitro extractions, in direct contact with a biological matrix such as blood or tissue. This thesis presents several effective ways of preparing SPME coatings based on biocompatible polymers and silica-based extractive phases, focusing on their biocompatibility as a must. After fabrication, the proposed coatings are tested for biocompatibility and analytical utility. Finally, some practical applications of the new coatings are presented, such as fast drug analysis and determination of drug plasma protein binding. Six test drugs with different physico-chemical properties are chosen for the investigation: diazepam, verapamil, lorazepam, warfarin, nordiazepam, and loperamide. It is shown that the application of these new SPME fibers for biological sample analysis greatly reduces the time required for sample preparation and limits the exposure of the analytical personnel to potentially infectious biofluids.

The objective of the current research project relies on implementation of solvent-free, green and environmentally friendly solid phase microextraction (SPME) sample preparation alternative in the area of complex sample characterization. The advantages that the technique offers in comparison to traditional methods of sample preparation including solvent-free implementation, short sample preparation times, small sample amount requirements, advanced automation capability and minimization of matrix effects are effectively employed during ex vivo and laboratory investigations of complex samples. More important, the underlying features of the technique including miniaturized format, nonexhaustive extraction recoveries and on-site compatibility were fully exploited in order to investigate the metabolome of biological systems directly on the site. Hence, in vivo SPME extraction format was employed in direct immersion SPME sampling of biological systems, hence eliminating the crucial prerequisites associated with multiple preparative steps and incorporation of metabolism quenching that are encountered during implementation of traditional sample preparation methods in global metabolite analysis. Furthermore, in vivo sampling format was hyphenated to comprehensive two-dimensional gas chromatography – time-of-flight mass spectrometry (GCxGC-ToFMS) for high-resolution sampling of volatile and semivolatile metabolites in ‘Honeycrisp’ apples. The initial stages of the project involved evaluation of performance characteristics of commercial SPME extraction coatings in terms of extraction selectivity, extraction sensitivity and desorption efficiency by employing headspace SPME analysis of both aqueous standards spiked with representative volatile and semivolatile metabolites as well as the apple homogenate. DVB/CAR/PDMS coating was selected on the basis of optimum metabolite coverage and extraction sensitivity and was consequently employed during ex vivo and in vivo sampling assays corresponding to determination of volatile and semivolatile metabolites. The former extraction methodology incorporated appropriate sample preparation steps for quenching metabolic activity so that the relevant metabolome profile is not biased against unstable metabolites and those that are susceptible to inter-metabolite conversions which adversely impact preservation of metabolite identity. The two sample preparation assays were compared in terms of metabolite coverage and analytical precision in order to identify SPME route toward characterization of more representative metabolome and determination of instantaneous and more ‘true’ metabolism snapshoot. This is the first report illustrating the implementation of in vivo direct immersion SPME assay for non invasive determination of endogenous fruit metabolites whose profiles and contents are highly correlated to a multitude of influential fruit quality traits.

The objective of the current research project relies on implementation of solvent-free, green and environmentally friendly solid phase microextraction (SPME) sample preparation alternative in the area of complex sample characterization. The advantages that the technique offers in comparison to traditional methods of sample preparation including solvent-free implementation, short sample preparation times, small sample amount requirements, advanced automation capability and minimization of matrix effects are effectively employed during ex vivo and laboratory investigations of complex samples. More important, the underlying features of the technique including miniaturized format, nonexhaustive extraction recoveries and on-site compatibility were fully exploited in order to investigate the metabolome of biological systems directly on the site. Hence, in vivo SPME extraction format was employed in direct immersion SPME sampling of biological systems, hence eliminating the crucial prerequisites associated with multiple preparative steps and incorporation of metabolism quenching that are encountered during implementation of traditional sample preparation methods in global metabolite analysis. Furthermore, in vivo sampling format was hyphenated to comprehensive two-dimensional gas chromatography – time-of-flight mass spectrometry (GCxGC-ToFMS) for high-resolution sampling of volatile and semivolatile metabolites in ‘Honeycrisp’ apples. The initial stages of the project involved evaluation of performance characteristics of commercial SPME extraction coatings in terms of extraction selectivity, extraction sensitivity and desorption efficiency by employing headspace SPME analysis of both aqueous standards spiked with representative volatile and semivolatile metabolites as well as the apple homogenate. DVB/CAR/PDMS coating was selected on the basis of optimum metabolite coverage and extraction sensitivity and was consequently employed during ex vivo and in vivo sampling assays corresponding to determination of volatile and semivolatile metabolites. The former extraction methodology incorporated appropriate sample preparation steps for quenching metabolic activity so that the relevant metabolome profile is not biased against unstable metabolites and those that are susceptible to inter-metabolite conversions which adversely impact preservation of metabolite identity. The two sample preparation assays were compared in terms of metabolite coverage and analytical precision in order to identify SPME route toward characterization of more representative metabolome and determination of instantaneous and more ‘true’ metabolism snapshoot. This is the first report illustrating the implementation of in vivo direct immersion SPME assay for non invasive determination of endogenous fruit metabolites whose profiles and contents are highly correlated to a multitude of influential fruit quality traits.

Although microdialysis (MD) and solid-phase microextraction (SPME) are widely used sampling techniques, a comparison study has not been performed to date. The goal of the research presented was not only to address this issue but also to develop new analytical methods that were more suitable for in vivo study using MD and SPME. A new calibration method called kinetic microdialysis was developed for in vivo sampling. Two MD probes with different flow rates were simultaneously inserted into the symmetric parts of sampling system. A simple empirical equation was proposed to calculate the analyte concentrations in the sample matrix using two different dialysate concentrations. Several factors that influenced the correction factor in this equation were discussed. An excellent correlation was observed between the calculated and theoretical value. This method was subsequently applied for in vivo sampling, for the measurement of pesticide allocation in the different leaves of a jade plant (Crassula ovata). Compared to the other reported MD calibration methods, this novel approach offers several advantages including simplicity, speed, robustness, and increased accuracy. The on-fiber standardization technique for solid-coated SPME was studied and a theoretical model is proposed for the isotropic behavior of adsorption and desorption, based on Fick’s law of diffusion and the Langmuir model. The isotropy of the adsorption and desorption of analytes onto and from the surface of porous solid SPME fiber was validated with the use of a commercially available fiber, a 50 um carbowax/templated resin (CW/TPR) for carbamate pesticide analysis in various in vitro sample matrices. Time constants were comparable for the adsorption and desorption processes. Equilibrium constants and fiber capacities were calculated with the Langmuir Isotherm Model. A kinetic method was developed to calibrate adsorption using desorption. This calibration corrected for the sample matrix effects and minimized displacement effects as a pre-equilibrium extraction. The technique was successfully applied to the analysis of pesticides in river water and white wine. This developed method could be potentially applied for in vivo study. A new kinetic calibration was developed using dominant pre-equilibrium desorption by SPME. The calibration was based on isotropism between absorption and desorption, which was proved theoretically and experimentally in an aqueous solution and semi-solid matrix. This approach allows for the calibration of absorption using desorption to compensate for matrix effects. Moreover, concentration profiles are initially proposed to verify isotropism between the absorption and desorption, while providing a linear approach to obtain time constants for the purpose of quantitative analysis. This linear approach is more convenient, robust and accurate than the non-linear version with the previously used time profiles. Furthermore, the target analytes are used as the internal standards, thus radioactive or deuterated internal standards are not necessary. In addition, dominant pre-equilibrium desorption utilizes the pre-equilibrium approach and offers a shorter sample preparation time, which is typically suitable for in vivo sampling. This kinetic calibration method was successfully applied to prepare samples of polycyclic aromatic hydrocarbons (PAHs) in a flow-through system and in vivo pesticide sampling in a jade plant (Crassula ovata). Previous field studies utilizing SPME predominantly focused on volatile compounds in air or water. Earlier in vivo sampling studies utilizing SPME were limited to liquid matrices, namely blood. In this study, SPME was developed for in vivo laboratory and field sampling of pharmaceuticals in fish muscle. Pre-equilibrium extraction was used to shorten in vivo sampling time. The use of pre-equilibrium desorption rates are proposed as a means to calibrate pre-equilibrium extractions. Excellent linearity was found between the free concentrations determined by SPME from the muscle of living fish and the waterborne concentrations of several pharmaceuticals. It is also firstly proposed a simple SPME method to determine free and total concentrations simultaneously in a living tissue using the known protein binding value. The utility of in vivo SPME sampling under field conditions was evaluated in wild fish collected from a number of different river locations under varying degrees of influence from municipal wastewater effluents. Diphenhydramine and diltiazem were detected in the muscle of fish downstream of a local wastewater treatment plant. Based on this study, SPME technique has demonstrated several important advantages for laboratory and field in vivo sampling. The development of a rapid, robust, easy to deploy technique which combines sampling, extraction and concentration into one step is a potentially important tool for use in vivo field-based sampling. MD and SPME methods have been developed and compared through in vitro and in vivo study. For in vitro study (juice, milk and orange jelly), both methods offered accurate and precise results (recovery: 88-105% with RSD < 15%) for complex sample matrices by standard addition method. The limits of quantification (LOQs) of the two methods developed were below the tolerance levels in milk set by the United Nations Food and Agriculture Organization (FAO). Compared to MD, the fully automated SPME procedure offered several advantages including high-throughput and more efficient sampling, less labor intensity, and capability for batch analysis. For in vivo study, kinetic calibrations were performed using retrodialysis and in-fiber standardization techniques for MD and SPME, respectively. Quantitative analysis was performed to measure pesticide concentrations in living tissue, i.e., the leaves of a living jade plant (Crassula ovata). Although both techniques provided sampling with minimal perturbation to the system under study, SPME was more sensitive, precise and accurate, suitable for field sampling and had a wider application than MD. It demonstrated that SPME has the potential to replace MD for in vivo study.

This thesis presents enhanced analytical methods developed for complex aqueous sample analysis based on solid phase microextraction (SPME). First, the laboratory evaluation of the kinetic calibration approach in aqueous sample analysis using SPME is discussed. A modified SPME device, Polydimethylsiloxane (PDMS) rod passive sampler, was developed and the kinetic calibration method based on the standard preloaded in the extraction phase was applied to determine the time-weighted average (TWA) concentration of organic pollutants in water. Later, the SPME technique was used to investigate the complex interactions between the organic pollutants and humic organic matter (HOM) present in the aqueous samples. The kinetics of the SPME approach in complex aqueous samples was studied. The concentration of freely dissolved analytes and the total concentration of the target analytes in the sample matrix were determined by SPME sampling. The usefulness of the SPME approach for binding studies was further demonstrated by determining the sorption coefficient, a useful parameter for studying the bioavailability of the organic pollutants in the environment. In addition, the commercial Computational Fluid Dynamics (CFD) software COMSOL Multiphysics was used to predict the kinetics of analyte extraction and flow pattern under different experimental conditions using the SPME technique. A good agreement between the prediction and the experimental data confirms the advantages of the CFD application for experimental optimization thus minimizing the need of extensive experiments.

Solid phase microextraction (SPME) has gained much popularity for in vivo applications recently. Thus far, there are two types of pre-equilibrium kinetic calibration that have been applied to in vivo SPME: on-fibre standardization and dominant pre-equilibrium desorption. Both of these techniques have their own advantages and disadvantages. To address the limitations presented by these two techniques, a third pre-equilibrium kinetic calibration method, the diffusion-based interface model, was investigated. The diffusion-based interface model had been successfully applied to air and water samples but was never utilized for in vivo SPME studies. For the first part of the research, on-fibre standardization, dominant pre-equilibrium desorption, and diffusion-based interface model were compared in terms of accuracy, precision, and experimental procedures, by using a flow-through system. These three kinetic calibrations were further validated by equilibrium SPME extraction and protein-plasma precipitation, a current state-of-the-art sampling method. The potential of diffusion-based interface model was yet again demonstrated in the second part of the research project. This calibration method was applied to comparative pharmacokinetic studies of two drugs, fenoterol and methoxyfenoterol, on 5 rats. To provide a constant sampling rate as required for diffusion-based interface model, a SPME animal sampling autosampler, AccuSampler®, was utilized. It custom-written program allowed the entire SPME sampling procedure excluding insertion and removal of SPME probes to be automated. Furthermore, to validate the results obtained by SPME, the AccuSampler® was programmed to withdraw blood after each SPME sampling time point for conventional method analysis using protein-plasma precipitation. The well correlated data obtained by SPME sampling and the conventional method illustrated the potential of diffusion-based interface model as an excellent choice for future in vivo SPME applications.

Although microdialysis (MD) and solid-phase microextraction (SPME) are widely used sampling techniques, a comparison study has not been performed to date. The goal of the research presented was not only to address this issue but also to develop new analytical methods that were more suitable for in vivo study using MD and SPME. A new calibration method called kinetic microdialysis was developed for in vivo sampling. Two MD probes with different flow rates were simultaneously inserted into the symmetric parts of sampling system. A simple empirical equation was proposed to calculate the analyte concentrations in the sample matrix using two different dialysate concentrations. Several factors that influenced the correction factor in this equation were discussed. An excellent correlation was observed between the calculated and theoretical value. This method was subsequently applied for in vivo sampling, for the measurement of pesticide allocation in the different leaves of a jade plant (Crassula ovata). Compared to the other reported MD calibration methods, this novel approach offers several advantages including simplicity, speed, robustness, and increased accuracy. The on-fiber standardization technique for solid-coated SPME was studied and a theoretical model is proposed for the isotropic behavior of adsorption and desorption, based on Fick’s law of diffusion and the Langmuir model. The isotropy of the adsorption and desorption of analytes onto and from the surface of porous solid SPME fiber was validated with the use of a commercially available fiber, a 50 um carbowax/templated resin (CW/TPR) for carbamate pesticide analysis in various in vitro sample matrices. Time constants were comparable for the adsorption and desorption processes. Equilibrium constants and fiber capacities were calculated with the Langmuir Isotherm Model. A kinetic method was developed to calibrate adsorption using desorption. This calibration corrected for the sample matrix effects and minimized displacement effects as a pre-equilibrium extraction. The technique was successfully applied to the analysis of pesticides in river water and white wine. This developed method could be potentially applied for in vivo study. A new kinetic calibration was developed using dominant pre-equilibrium desorption by SPME. The calibration was based on isotropism between absorption and desorption, which was proved theoretically and experimentally in an aqueous solution and semi-solid matrix. This approach allows for the calibration of absorption using desorption to compensate for matrix effects. Moreover, concentration profiles are initially proposed to verify isotropism between the absorption and desorption, while providing a linear approach to obtain time constants for the purpose of quantitative analysis. This linear approach is more convenient, robust and accurate than the non-linear version with the previously used time profiles. Furthermore, the target analytes are used as the internal standards, thus radioactive or deuterated internal standards are not necessary. In addition, dominant pre-equilibrium desorption utilizes the pre-equilibrium approach and offers a shorter sample preparation time, which is typically suitable for in vivo sampling. This kinetic calibration method was successfully applied to prepare samples of polycyclic aromatic hydrocarbons (PAHs) in a flow-through system and in vivo pesticide sampling in a jade plant (Crassula ovata). Previous field studies utilizing SPME predominantly focused on volatile compounds in air or water. Earlier in vivo sampling studies utilizing SPME were limited to liquid matrices, namely blood. In this study, SPME was developed for in vivo laboratory and field sampling of pharmaceuticals in fish muscle. Pre-equilibrium extraction was used to shorten in vivo sampling time. The use of pre-equilibrium desorption rates are proposed as a means to calibrate pre-equilibrium extractions. Excellent linearity was found between the free concentrations determined by SPME from the muscle of living fish and the waterborne concentrations of several pharmaceuticals. It is also firstly proposed a simple SPME method to determine free and total concentrations simultaneously in a living tissue using the known protein binding value. The utility of in vivo SPME sampling under field conditions was evaluated in wild fish collected from a number of different river locations under varying degrees of influence from municipal wastewater effluents. Diphenhydramine and diltiazem were detected in the muscle of fish downstream of a local wastewater treatment plant. Based on this study, SPME technique has demonstrated several important advantages for laboratory and field in vivo sampling. The development of a rapid, robust, easy to deploy technique which combines sampling, extraction and concentration into one step is a potentially important tool for use in vivo field-based sampling. MD and SPME methods have been developed and compared through in vitro and in vivo study. For in vitro study (juice, milk and orange jelly), both methods offered accurate and precise results (recovery: 88-105% with RSD < 15%) for complex sample matrices by standard addition method. The limits of quantification (LOQs) of the two methods developed were below the tolerance levels in milk set by the United Nations Food and Agriculture Organization (FAO). Compared to MD, the fully automated SPME procedure offered several advantages including high-throughput and more efficient sampling, less labor intensity, and capability for batch analysis. For in vivo study, kinetic calibrations were performed using retrodialysis and in-fiber standardization techniques for MD and SPME, respectively. Quantitative analysis was performed to measure pesticide concentrations in living tissue, i.e., the leaves of a living jade plant (Crassula ovata). Although both techniques provided sampling with minimal perturbation to the system under study, SPME was more sensitive, precise and accurate, suitable for field sampling and had a wider application than MD. It demonstrated that SPME has the potential to replace MD for in vivo study.