SU-8-Based Microchips for Capillary Electrophoresis and Electrospray Ionization Mass Spectrometry

Miniaturization of analytical instrumentation is attracting growing interest in response to the explosive demand for rapid, yet sensitive analytical methods and low-cost, highly automated instruments for pharmaceutical and bioanalyses and environmental monitoring. Microfabrication technology in particular, has enabled fabrication of low-cost microdevices with a high degree of integrated functions, such as sample preparation, chemical reaction, separation, and detection, on a single microchip. These miniaturized total chemical analysis systems (microTAS or lab-on-a-chip) can also be arrayed for parallel analyses in order to accelerate the sample throughput. Other motivations include reduced sample consumption and waste production as well as increased speed of analysis. One of the most promising hyphenated techniques in analytical chemistry is the combination of a microfluidic separation chip and mass spectrometer (MS). In this work, the emerging polymer microfabrication techniques, ultraviolet lithography in particular, were exploited to develop a capillary electrophoresis (CE) separation chip which incorporates a monolithically integrated electrospray ionization (ESI) emitter for efficient coupling with MS. An epoxy photoresist SU-8 was adopted as structural material and characterized with respect to its physicochemical properties relevant to chip-based CE and ESI/MS, namely surface charge, surface interactions, heat transfer, and solvent compatibility. As a result, SU-8 was found to be a favorable material to substitute for the more commonly used glass and silicon in microfluidic applications. In addition, an infrared (IR) thermography was introduced as direct, non-intrusive method to examine the heat transfer and thermal gradients during microchip-CE. The IR data was validated through numerical modeling. The analytical performance of SU-8-based microchips was established for qualitative and quantitative CE-ESI/MS analysis of small drug compounds, peptides, and proteins. The CE separation efficiency was found to be similar to that of commercial glass microchips and conventional CE systems. Typical analysis times were only 30-90 s per sample indicating feasibility for high-throughput analysis. Moreover, a mass detection limit at the low-attomole level, as low as 10E+5 molecules, was achieved utilizing MS detection. The SU-8 microchips developed in this work could also be mass produced at low cost and with nearly identical performance from chip to chip. Until this work, the attempts to combine CE separation with ESI in a chip-based system, amenable to batch fabrication and capable of high, reproducible analytical performance, have not been successful. Thus, the CE-ESI chip developed in this work is a substantial step toward lab-on-a-chip technology.

Heated Nebulizer Microchips for Mass Spectrometry

Miniaturized analytical devices, such as heated nebulizer (HN) microchips studied in this work, are of increasing interest owing to benefits like faster operation, better performance, and lower cost relative to conventional systems. HN microchips are microfabricated devices that vaporize liquid and mix it with gas. They are used with low liquid flow rates, typically a few µL/min, and have previously been utilized as ion sources for mass spectrometry (MS). Conventional ion sources are seldom feasible at such low flow rates. In this work HN chips were developed further and new applications were introduced. First, a new method for thermal and fluidic characterization of the HN microchips was developed and used to study the chips. Thermal behavior of the chips was also studied by temperature measurements and infrared imaging. An HN chip was applied to the analysis of crude oil – an extremely complex sample – by microchip atmospheric pressure photoionization (APPI) high resolution mass spectrometry. With the chip, the sample flow rate could be reduced significantly without loss of performance and with greatly reduced contamination of the MS instrument. Thanks to its suitability to high temperature, microchip APPI provided efficient vaporization of nonvolatile compounds in crude oil. The first microchip version of sonic spray ionization (SSI) was presented. Ionization was achieved by applying only high (sonic) speed nebulizer gas to an HN microchip. SSI significantly broadens the range of analytes ionizable with the HN chips, from small stable molecules to labile biomolecules. The analytical performance of the microchip SSI source was confirmed to be acceptable. The HN microchips were also used to connect gas chromatography (GC) and capillary liquid chromatography (LC) to MS, using APPI for ionization. Microchip APPI allows efficient ionization of both polar and nonpolar compounds whereas with the most popular electrospray ionization (ESI) only polar and ionic molecules are ionized efficiently. The combination of GC with MS showed that, with HN microchips, GCs can easily be used with MS instruments designed for LC-MS. The presented analytical methods showed good performance. The first integrated LC–HN microchip was developed and presented. In a single microdevice, there were structures for a packed LC column and a heated nebulizer. Nonpolar and polar analytes were efficiently ionized by APPI. Ionization of nonpolar and polar analytes is not possible with previously presented chips for LC–MS since they rely on ESI. Preliminary quantitative performance of the new chip was evaluated and the chip was also demonstrated with optical detection. A new ambient ionization technique for mass spectrometry, desorption atmospheric pressure photoionization (DAPPI), was presented. The DAPPI technique is based on an HN microchip providing desorption of analytes from a surface. Photons from a photoionization lamp ionize the analytes via gas-phase chemical reactions, and the ions are directed into an MS. Rapid analysis of pharmaceuticals from tablets was successfully demonstrated as an application of DAPPI.

Spectroscopy of tissue triglyceride composition : In vitro and in vivo studies

Lipid analysis is commonly performed by gas chromatography (GC) in laboratory conditions. Spectroscopic techniques, however, are non-destructive and can be implemented noninvasively in vivo. Excess fat (triglycerides) in visceral adipose tissue and liver is known predispose to metabolic abnormalities, collectively known as the metabolic syndrome. Insulin resistance is the likely cause with diets high in saturated fat known to impair insulin sensitivity. Tissue triglyceride composition has been used as marker of dietary intake but it can also be influenced by tissue specific handling of fatty acids. Recent studies have shown that adipocyte insulin sensitivity correlates positively with their saturated fat content, contradicting the common view of dietary effects. A better understanding of factors affecting tissue triglyceride composition is needed to provide further insights into tissue function in lipid metabolism. In this thesis two spectroscopic techniques were developed for in vitro and in vivo analysis of tissue triglyceride composition. In vitro studies (Study I) used infrared spectroscopy (FTIR), a fast and cost effective analytical technique well suited for multivariate analysis. Infrared spectra are characterized by peak overlap leading to poorly resolved absorbances and limited analytical performance. In vivo studies (Studies II, III and IV) used proton magnetic resonance spectroscopy (1H-MRS), an established non-invasive clinical method for measuring metabolites in vivo. 1H-MRS has been limited in its ability to analyze triglyceride composition due to poorly resolved resonances. Using an attenuated total reflection accessory, we were able to obtain pure triglyceride infrared spectra from adipose tissue biopsies. Using multivariate curve resolution (MCR), we were able to resolve the overlapping double bond absorbances of monounsaturated fat and polyunsaturated fat. MCR also resolved the isolated trans double bond and conjugated linoleic acids from an overlapping background absorbance. Using oil phantoms to study the effects of different fatty acid compositions on the echo time behaviour of triglycerides, it was concluded that the use of long echo times improved peak separation with T2 weighting having a negligible impact. It was also discovered that the echo time behaviour of the methyl resonance of omega-3 fats differed from other fats due to characteristic J-coupling. This novel insight could be used to detect omega-3 fats in human adipose tissue in vivo at very long echo times (TE = 470 and 540 ms). A comparison of 1H-MRS of adipose tissue in vivo and GC of adipose tissue biopsies in humans showed that long TE spectra resulted in improved peak fitting and better correlations with GC data. The study also showed that calculation of fatty acid fractions from 1H-MRS data is unreliable and should not be used. Omega-3 fatty acid content derived from long TE in vivo spectra (TE = 540 ms) correlated with total omega-3 fatty acid concentration measured by GC. The long TE protocol used for adipose tissue studies was subsequently extended to the analysis of liver fat composition. Respiratory triggering and long TE resulted in spectra with the olefinic and tissue water resonances resolved. Conversion of the derived unsaturation to double bond content per fatty acid showed that the results were in accordance with previously published gas chromatography data on liver fat composition. In patients with metabolic syndrome, liver fat was found to be more saturated than subcutaneous or visceral adipose tissue. The higher saturation observed in liver fat may be a result of a higher rate of de-novo-lipogenesis in liver than in adipose tissue. This thesis has introduced the first non-invasive method for determining adipose tissue omega-3 fatty acid content in humans in vivo. The methods introduced here have also shown that liver fat is more saturated than adipose tissue fat.

Breath Testing by Fourier Transform Infrared Spectroscopy for Solvent Intoxication Diagnostics

Technical or contaminated ethanol products are sometimes ingested either accidentally or on purpose. Typical misused products are black-market liquor and automotive products, e.g., windshield washer fluids. In addition to less toxic solvents, these liquids may contain the deadly methanol. Symptoms of even lethal solvent poisoning are often non-specific at the early stage. The present series of studies was carried out to develop a method for solvent intoxication breath diagnostics to speed up the diagnosis procedure conventionally based on blood tests. Especially in the case of methanol ingestion, the analysis method should be sufficiently sensitive and accurate to determine the presence of even small amounts of methanol from the mixture of ethanol and other less-toxic components. In addition to the studies on the FT-IR method, the Dräger 7110 evidential breath analyzer was examined to determine its ability to reveal a coexisting toxic solvent. An industrial Fourier transform infrared analyzer was modified for breath testing. The sample cell fittings were widened and the cell size reduced in order to get an alveolar sample directly from a single exhalation. The performance and the feasibility of the Gasmet FT-IR analyzer were tested in clinical settings and in the laboratory. Actual human breath screening studies were carried out with healthy volunteers, inebriated homeless men, emergency room patients and methanol-intoxicated patients. A number of the breath analysis results were compared to blood test results in order to approximate the blood-breath relationship. In the laboratory experiments, the analytical performance of the Gasmet FT-IR analyzer and Dräger 7110 evidential breath analyzer was evaluated by means of artificial samples resembling exhaled breath. The investigations demonstrated that a successful breath ethanol analysis by Dräger 7110 evidential breath analyzer could exclude any significant methanol intoxication. In contrast, the device did not detect very high levels of acetone, 1-propanol and 2-propanol in simulated breath. The Dräger 7110 evidential breath ethanol analyzer was not equipped to recognize the interfering component. According to the studies the Gasmet FT-IR analyzer was adequately sensitive, selective and accurate for solvent intoxication diagnostics. In addition to diagnostics, the fast breath solvent analysis proved feasible for controlling the ethanol and methanol concentration during haemodialysis treatment. Because of the simplicity of the sampling and analysis procedure, non-laboratory personnel, such as police officers or social workers, could also operate the analyzer for screening purposes.

Spectroscopy of tissue triglyceride composition : In vitro and in vivo studies

Lipid analysis is commonly performed by gas chromatography (GC) in laboratory conditions. Spectroscopic techniques, however, are non-destructive and can be implemented noninvasively in vivo. Excess fat (triglycerides) in visceral adipose tissue and liver is known predispose to metabolic abnormalities, collectively known as the metabolic syndrome. Insulin resistance is the likely cause with diets high in saturated fat known to impair insulin sensitivity. Tissue triglyceride composition has been used as marker of dietary intake but it can also be influenced by tissue specific handling of fatty acids. Recent studies have shown that adipocyte insulin sensitivity correlates positively with their saturated fat content, contradicting the common view of dietary effects. A better understanding of factors affecting tissue triglyceride composition is needed to provide further insights into tissue function in lipid metabolism. In this thesis two spectroscopic techniques were developed for in vitro and in vivo analysis of tissue triglyceride composition. In vitro studies (Study I) used infrared spectroscopy (FTIR), a fast and cost effective analytical technique well suited for multivariate analysis. Infrared spectra are characterized by peak overlap leading to poorly resolved absorbances and limited analytical performance. In vivo studies (Studies II, III and IV) used proton magnetic resonance spectroscopy (1H-MRS), an established non-invasive clinical method for measuring metabolites in vivo. 1H-MRS has been limited in its ability to analyze triglyceride composition due to poorly resolved resonances. Using an attenuated total reflection accessory, we were able to obtain pure triglyceride infrared spectra from adipose tissue biopsies. Using multivariate curve resolution (MCR), we were able to resolve the overlapping double bond absorbances of monounsaturated fat and polyunsaturated fat. MCR also resolved the isolated trans double bond and conjugated linoleic acids from an overlapping background absorbance. Using oil phantoms to study the effects of different fatty acid compositions on the echo time behaviour of triglycerides, it was concluded that the use of long echo times improved peak separation with T2 weighting having a negligible impact. It was also discovered that the echo time behaviour of the methyl resonance of omega-3 fats differed from other fats due to characteristic J-coupling. This novel insight could be used to detect omega-3 fats in human adipose tissue in vivo at very long echo times (TE = 470 and 540 ms). A comparison of 1H-MRS of adipose tissue in vivo and GC of adipose tissue biopsies in humans showed that long TE spectra resulted in improved peak fitting and better correlations with GC data. The study also showed that calculation of fatty acid fractions from 1H-MRS data is unreliable and should not be used. Omega-3 fatty acid content derived from long TE in vivo spectra (TE = 540 ms) correlated with total omega-3 fatty acid concentration measured by GC. The long TE protocol used for adipose tissue studies was subsequently extended to the analysis of liver fat composition. Respiratory triggering and long TE resulted in spectra with the olefinic and tissue water resonances resolved. Conversion of the derived unsaturation to double bond content per fatty acid showed that the results were in accordance with previously published gas chromatography data on liver fat composition. In patients with metabolic syndrome, liver fat was found to be more saturated than subcutaneous or visceral adipose tissue. The higher saturation observed in liver fat may be a result of a higher rate of de-novo-lipogenesis in liver than in adipose tissue. This thesis has introduced the first non-invasive method for determining adipose tissue omega-3 fatty acid content in humans in vivo. The methods introduced here have also shown that liver fat is more saturated than adipose tissue fat.

Process Analytical Technology Approach on Fluid Bed Granulation and Drying : Identifying Critical Relationships and Constructing the Design Space

Fluid bed granulation is a key pharmaceutical process which improves many of the powder properties for tablet compression. Dry mixing, wetting and drying phases are included in the fluid bed granulation process. Granules of high quality can be obtained by understanding and controlling the critical process parameters by timely measurements. Physical process measurements and particle size data of a fluid bed granulator that are analysed in an integrated manner are included in process analytical technologies (PAT). Recent regulatory guidelines strongly encourage the pharmaceutical industry to apply scientific and risk management approaches to the development of a product and its manufacturing process. The aim of this study was to utilise PAT tools to increase the process understanding of fluid bed granulation and drying. Inlet air humidity levels and granulation liquid feed affect powder moisture during fluid bed granulation. Moisture influences on many process, granule and tablet qualities. The approach in this thesis was to identify sources of variation that are mainly related to moisture. The aim was to determine correlations and relationships, and utilise the PAT and design space concepts for the fluid bed granulation and drying. Monitoring the material behaviour in a fluidised bed has traditionally relied on the observational ability and experience of an operator. There has been a lack of good criteria for characterising material behaviour during spraying and drying phases, even though the entire performance of a process and end product quality are dependent on it. The granules were produced in an instrumented bench-scale Glatt WSG5 fluid bed granulator. The effect of inlet air humidity and granulation liquid feed on the temperature measurements at different locations of a fluid bed granulator system were determined. This revealed dynamic changes in the measurements and enabled finding the most optimal sites for process control. The moisture originating from the granulation liquid and inlet air affected the temperature of the mass and pressure difference over granules. Moreover, the effects of inlet air humidity and granulation liquid feed rate on granule size were evaluated and compensatory techniques used to optimize particle size. Various end-point indication techniques of drying were compared. The ∆T method, which is based on thermodynamic principles, eliminated the effects of humidity variations and resulted in the most precise estimation of the drying end-point. The influence of fluidisation behaviour on drying end-point detection was determined. The feasibility of the ∆T method and thus the similarities of end-point moisture contents were found to be dependent on the variation in fluidisation between manufacturing batches. A novel parameter that describes behaviour of material in a fluid bed was developed. Flow rate of the process air and turbine fan speed were used to calculate this parameter and it was compared to the fluidisation behaviour and the particle size results. The design space process trajectories for smooth fluidisation based on the fluidisation parameters were determined. With this design space it is possible to avoid excessive fluidisation and improper fluidisation and bed collapse. Furthermore, various process phenomena and failure modes were observed with the in-line particle size analyser. Both rapid increase and a decrease in granule size could be monitored in a timely manner. The fluidisation parameter and the pressure difference over filters were also discovered to express particle size when the granules had been formed. The various physical parameters evaluated in this thesis give valuable information of fluid bed process performance and increase the process understanding.