Improving Sensitivity and Throughput for Bioanalytical Measurements with Mass Spectrometry using Microfluidics and Online Sample Preparation

Abstract

Mass spectrometry (MS) has advantages as an analytical technique including label-free detection, high degrees of specificity and selectivity, capability for simultaneous monitoring/determination of numerous analytes, and rapid ionization/analysis, though throughput and sensitivity improvements are regularly sought. As a result, MS is commonly interfaced with different technologies including separations. Capillary electrophoresis (CE) is often selected over liquid chromatography(LC) for advantages including low volume requirements and faster separations, though online preconcentration is often necessary to improve limits of detection (LODs). In this work, a powerful preconcentration technique is paired with CE-MS to obtain LODs down to 10 pM, addressing the sensitivity limitation. Preconcentration here was shown to improve LODs by 5000-fold compared to a typical hydrodynamic injection for CE-MS. This method was applied for simultaneous determination of seven neurochemicals in biological samples with an excellent linear dynamic range (pM-µM). To improve throughputs, microfluidic sample introduction, especially droplet microfluidics, has shown promise for use with electrospray ionization (ESI)-MS, demonstrating sample introduction rates up to 30 Hz and droplet sizes down to 65 pL.1,2 In this dissertation, several droplet microfluidic assays are developed and paired with nanoelectrospray ionization (nESI) or ESI (droplet-nESI) for high throughput analyses. In one method, 5 nL droplets are generated at the end of a microdialysis probe to achieve temporal resolution near 10 s, substantially better than a standard LC-MS analysis with 5–20 min temporal resolution with microdialysis, offering much deeper insight into neurochemical dynamics. Using low flow nESI with MS/MS to overcome ionization suppression, we achieve low nM LODs (down to 2 nM) for simultaneous monitoring of seven neurochemicals, including trace neurotransmitters. This was applied to monitoring neurochemical dynamics in mouse brains in response to multiple drugs. In another method, droplet-nESI is used for high throughput screening in a directed enzyme evolution workflow. Directed evolution can provide much higher reaction yields, but screening can take weeks. Here, droplets are generated from well-plate reactions and infused into a sensitive nESI-MS/MS method to measure reaction yields with product LODs down to 12 nM. Over 1700 reactions (> 550 in triplicate) are screened in a single day, and over 1700 reactions (in triplicate) are screened in total. Seven enzymes with higher enzymatic activity are identified while achieving 10-fold higher throughput than LC-MS. Finally, a new mode of liquid-liquid extraction is developed called slug flow nanoextraction (SFNE) that uses only 5 nL of each phase per extraction, and partitioning equilibrium is reached within seconds while flowing in-line with a detector. This was applied for online sample clean-up of pharmaceuticals from biofluids in-line with MS/MS analysis, demonstrating nearly 20-fold-improvements in detection sensitivity with up to 60 extractions performed during a single infusion. Furthermore, an entirely online and automated system is developed for SFNE. This system is applied to screen 21 octanol-water partition coefficients (Kow) within 2 hr. Kows are an important physiochemical characteristic for understanding drug bioavailability, though current measurements are low-throughput. In a comparison, the developed method offers 10-fold higher throughput and 40-fold lower volume requirements than a typical workflow. The work described in this dissertation, though diverse, pushes forward what can be performed with mass spectrometry and bioanalysis. Each chapter introduces an analytically novel or innovative approach for the analysis of small molecules that advances important fields of research including physiochemical characterization, high throughput screening with mass spectrometry, and sensitive bioanalysis.