At many times during this project I found myself asking the question,"Why am I doing this?" In my attempt to answer, I would always seem to recount my positive experiences with the analytical sciences. Thus, I feel compelled to give thanks to those who were integral to my education in the analytical sciences and inspirations to my professional development. First, I thank the University of Maryland for encouraging me to pursue an education in the sciences. Second, I thank the graduate program at the University of Florida for providing me an opportunity to focus in the analytical sciences and teaching me how to formulate question and thought. Third, I thank Bristol-Myers Squibb for balancing my hunger for the application of analytical sciences with the need to experience collaboration, interaction, and growth. To each of the above mentioned institutions, I am grateful for the support and continued source of inspiration. To all the people at the above mentioned institutions, I will hold dear the friendships, relationships, and memories that are the result of success and failure. And finally, I wish to thank my loving wife and family for their continual encouragement and support for everything I do. For this, I am truly blessed. xii ACKNOWLEDGMENTS CHAPTER 1 1

Nanospray experiments were performed on an ensemble of drug molecules and their commonly known metabolites to compare performance with conventional electrospray ionization (ESI) and to evaluate equimolar response capabilities. Codeine, dextromethorphan, tolbutamide, phenobarbital, cocaine, and morphine were analyzed along with their well-known metabolites that were formed via hydroxylation, dealkylation, hydrolysis, and glucuronidation. Nanospray exhibited a distinct trend toward equimolar response when flow rate was reduced from 25 nL/min to less than 10 nL/min. A more uniform response between the parent drug and the corresponding metabolites was obtained at flow rates of 10 nL/min or lower. The largest discrepancy was within +/-50% for plasma samples. Nanospray was used as a calibrator for conventional ESI liquid chromatography/tandem mass spectrometry (LC/MS/MS) and normalization factors were applied to the quantitation of an acyl-glucuronide metabolite of a proprietary compound in rat plasma. A nanospray calibration method was developed with the standard curve of the parent drug to generate quantitative results for drug metabolites within +/-20% of that obtained with reference standards and conventional ESI. The nanospray method provides a practical solution for the quantitative estimation of drug metabolites in drug discovery when reference standards are not available.

Droplets or plugs within multiphase microfluidic systems have rapidly gained interest as a way to manipulate samples and chemical reactions on the femtoliter to microliter scale. Chemical analysis of the plugs remains a challenge. We have discovered that nanoliter plugs of sample separated by air or oil can be analyzed by electrospray ionization mass spectrometry when pumped directly into a fused silica nanospray emitter tip. Using leu-enkephalin in methanol and 1% acetic acid in water (50:50 v:v) as a model sample, we found carry-over between plugs was < 0.1% and relative standard deviation of signal for a series of plugs was 3%. Detection limits were 1 nM. Sample analysis rates of 0.8 Hz were achieved by pumping 13 nL samples separated by 3 mm long air gaps in a 75 μm inner diameter tube. Analysis rates were limited by the scan time of the ion trap mass spectrometer. The system provides a robust, rapid, and information-rich method for chemical analysis of sample in segmented flow systems. SirMultiphase flow in capillary or microfluidic systems has generated considerable interest as a way to partition and process many discrete samples or synthetic reactions in confined spaces. 1-4 A common arrangement is a series of aqueous plugs or droplets separated by gas or immiscible liquid such that each plug can act as a small, individual vial or reaction vessel. 4,5 Methods for formation and manipulation of plugs on the femtoliter to nanoliter scale have recently been developed. [2][3][4][6][7][8][9][10][11] The sophistication of these methods has rapidly increased so that it is now possible to perform many common laboratory functions such as sampling, 12 splitting, 13-15 reagent addition, 16, 17 concentration,18 , 19 and dilution 20 on plugs in microfluidic systems. A frequent emphasis is that such manipulations can be performed automatically at high-throughput. These miniaturized multiphase flow systems have roots in the popular technique of continuous flow analysis (also known as segmented flow analysis) which uses air-segmentation of samples for high-throughput assays in clinical, industrial and environmental applications. 21,22 A limiting factor in using and studying multiphase flows is the paucity of methods to chemically analyze the contents of plugs. Optical methods such as colorimetry 22 and fluorescence are most commonly used. 20 Systems for electrophoretic analysis of segmented flows have recently been developed. 23,24 Drawbacks of these methods are that they require that the analytes be Mass spectrometry (MS) is an attractive analytical technique for analysis of segmented flows because it has the sensitivity and speed to be practically useful for low volume samples analyzed at high-throughput. Mass spectrometry has been coupled to segmented flow by collecting samples onto a plate for MALDI-MS26 or a moving belt interface for electron impact ionization-MS.27 ICP-MS of air-segmented samples has been demonstrated on a relatively large sample format (0.2 mL samples).28 MS analysis of acoustically levitat...

Low-flow electrospray ionization is typically a purely electrostatic method, used without supporting sheath-gas nebulization. Complex spray morphology results from a large number of possible spray emission modes. Spray morphology may assume the optimal Taylor cone-jet spray mode under equilibrium conditions. When coupling to nanobore gradient elution chromatography, however, stability of the Taylor cone-jet spray mode is compromised by the gradient of mobile phase physiochemical properties. The common spray modes for aqueous/ organic mobile phases were characterized using orthogonal (strobed illumination) transmitted light and (continuous illumination) scattered light imaging. Correlation of image sets from these complementary illumination methods provides the basis for spray mode identification using qualitative and quantitative image analysis. An automated feedback-controlled electrospray source was developed on a computer capable of controlling electrospray potential using an image-processing based algorithm for spray mode identification. The implementation of the feedback loop results in a system that is both self-starting and self-tuning for a specific spray mode or modes. Thus, changes in mobile phase composition and/or flow rate are compensated in real-time and the source is maintained in the cone-jet or pulsed cone-jet spray modes. [7]. The principal means of increasing the operable ESI flow rate was with the addition of either coaxial or cross flow sheath gas to aid in the droplet formation suitable for ion generation and mass analysis [8 -10].While much research and development effort was aimed at increasing electrospray's operable flow rate, a number of groups conducted studies at lower flow rates [11][12][13][14]. Early observations by Gale and Smith [11] showed that the flow rate could be reduced to 200 nL/min without reducing the signal-to-noise (S/N) ratio. Wilm and Mann [12,15] demonstrated that flow rates could be reduced another order of magnitude, to the 10 to 20 nL/min level, with no significant reduction in S/N. At approximately the same time, Emmet and Caprioli [13] demonstrated exceptionally high sensitivity for peptide analysis by directly coupling nanobore (50 -100 m inside diameter) LC columns to low-flow (100 -200 nL/min) ESI. Ultra-low flow rates of less than 1 nL/min have been shown to yield significant ion current suitable for MS [14,16] and enable the direct coupling of small bore (Ϸ5 m) capillary electrophoresis, with sub-attomole sensitivity [17]. Collectively, these various nanoliter-per-minute ESI-MS methods have become known as nanospray.Recent experiments suggest that operation at nanospray flow rates effect ionization on a fundamental level. Ionization effects have been observed for both off-line [18,19] and on-line [20] nanospray methods. It is important to note that electrostatic attraction between mobile phase and counter-electrode is typically the sole source of mobile phase flow for off-line nanospray [12,

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