Microdialysis coupled with droplet microfluidics and mass spectrometry for determination of neurotransmitters in vivo with high temporal resolution.

Monitoring the concentration fluctuations of neurotransmitters in vivo is valuable for elucidating the chemical signals that underlie brain functions. Microdialysis sampling is a widely used tool for monitoring neurochemicals in vivo. The volume requirements of most techniques that have been coupled to microdialysis, such as HPLC, result in fraction collection times of minutes, thus limiting the temporal resolution possible. Further the time of analysis can become long for cases where many fractions are collected. Previously we have used direct analysis of dialysate by low-flow electrospray ionization-tandem mass spectrometry (ESI-MS/MS) on a triple quadrupole mass spectrometer to monitor acetylcholine, glutamate, and γ-amino-butyric acid to achieve multiplexed in vivo monitoring with temporal resolution of seconds. Here, we have expanded this approach to adenosine, dopamine, and serotonin. The method achieved limits of detection down to 2 nM, enabling basal concentrations of all these compounds, except serotonin, to be measured in vivo. Comparative analysis with LC-MS/MS showed accurate results for all compounds except for glutamate, possibly due to interference for this compound in vivo. Pairing this analysis with droplet microfluidics yields 11 s temporal resolution and can generate dialysate fractions down to 3 nL at rates up to 3 fractions per s from a microdialysis probe. The system is applied to multiplexed monitoring of neurotransmitter dynamics in response to stimulation by 100 mM K+ and amphetamine. These applications demonstrate the suitability of the droplet ESI-MS/MS method for monitoring short-term dynamics of up to six neurotransmitters simultaneously.

Simultaneous monitoring of multiple hormones from human islets of Langerhans using solid-phase extraction-mass spectrometry.

Islets of Langerhans release peptide hormones in controlled amounts and patterns to ensure proper maintenance of blood glucose levels. The overall release of the hormones is shaped by external factors and by autocrine and paracrine interactions occurring within the islets. To better understand what controls the secretion of islet-secreted peptides, and how these processes go awry in diabetes, methods to monitor the release of multiple hormones simultaneously are needed. While antibody-based assays are typically used, they are most often applied to quantification of a single hormone. Mass spectrometry (MS), on the other hand, is well suited for quantifying multiple hormones simultaneously but typically requires time-consuming separation steps with biological samples. In this report, response surface methodology was used to identify a set of optimal solid-phase extraction (SPE) conditions for the islet-secreted peptides, insulin, C-peptide, glucagon, and somatostatin. The optimized SPE method was used with multiple reaction monitoring and isotopically labeled standards to quantify secretion levels. Calibrations were linear from 0.5 to 50 nM with < 15% RSD peak area ratios. A microfluidic system was used to perfuse 30 human islets with different glucose conditions, and fractions were collected every 2 min for SPE-MS analysis. Results showed the release dynamics of the individual peptides, as well as patterns, such as positively and negatively correlated release and oscillations. This rapid SPE-MS method is expected to be useful for examining other peptide and small-molecule secretions from islets and could be applied to a number of other biological systems for investigating cellular communication.

High-Throughput Optimization of Photochemical Reactions using Segmented-Flow Nanoelectrospray-Ionization Mass Spectrometry.

Within the realm of drug discovery, high-throughput experimentation techniques enable the rapid optimization of reactions and expedited generation of drug compound libraries for biological and pharmacokinetic evaluation. Herein we report the development of a segmented flow mass spectrometry-based platform to enable the rapid exploration of photoredox reactions for early-stage drug discovery. Specifically, microwell plate-based photochemical reaction screens were reformatted to segmented flow format to enable delivery to nanoelectrospray ionization-mass spectrometry analysis. This approach was demonstrated for the late-stage modification of complex drug scaffolds, as well as the subsequent structure-activity relationship evaluation of synthesized analogs. This technology is anticipated to expand the robust capabilities of photoredox catalysis in drug discovery by enabling high-throughput library diversification.

Ion Mobility-Mass Spectrometry Coupled to Droplet Microfluidics for Rapid Protein Structure Analysis and Drug Discovery.

Native mass spectrometry coupled to ion mobility (IM-MS) has become an important tool for the investigation of protein structure and dynamics upon ligand binding. Additionally, collisional activation or collision induced unfolding (CIU) can further probe conformational changes induced by ligand binding; however, larger scale screens have not been implemented due to limitations associated with throughput and sample introduction. In this work we explore the high-throughput capabilities of CIU fingerprinting. Fingerprint collection times were reduced 10-fold over traditional data collections through the use of improved smoothing and interpolation algorithms. Fast-CIU was then coupled to a droplet sample introduction approach using 40 nL droplet sample volumes and 2 s dwell times at each collision voltage. This workflow, which increased throughput by ∼16-fold over conventional nanospray CIU methods, was applied to a 96-compound screen against Sirtuin-5, a protein target of clinical interest. Over 20 novel Sirtuin-5 binders were identified, and it was found that Sirtuin-5 inhibitors will stabilize specific Sirtuin-5 gas-phase conformations. This work demonstrates that droplet-CIU can be implemented as a high-throughput biophysical characterization approach. Future work will focus on improving the throughput of this workflow and on automating data acquisition and analysis.

Small molecules released from islets of Langerhans determined by liquid chromatography - mass spectrometry.

Islets of Langerhans are the endocrine tissue within the pancreas that secrete hormones for maintenance of blood glucose homeostasis. A variety of small molecules including classical neurotransmitters are also released from islets. While the roles of most of these small molecules are unknown, some have been hypothesized to play a critical role in islet physiology. To better understand their role on islet function, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to separate and quantify 39 small molecules released from islets. Benzoyl chloride derivatization of analyte molecules was used to impart retention and facilitate electrospray ionization efficiency. Separation was achieved on a 2.1 × 150 mm column packed with 2.7 µm core-shell C18 particles. Calibration curves showed excellent linearity between the concentration and analyte response, with relative standard deviations of the analyte responses below 15% and limits of detection from 0.01-40 nM. The method was applied to examine small molecules released from murine and human islets of Langerhans after static incubation and perfusion with glucose. Results showed a decrease in secretion rates with increasing glucose concentration for most of the analytes. Secretion rates were found to be higher in human islets compared to their murine counterpart. This method will be useful in understanding the roles of small molecules in biological systems.

Rapid liquid chromatography-mass spectrometry quantitation of glucose-regulating hormones from human islets of Langerhans.

Glucose homeostasis is maintained through the secretion of peptide hormones, such as insulin, somatostatin, and glucagon, from islets of Langerhans, clusters of endocrine cells found in the pancreas. This report describes an LC-MS method using multiple reaction monitoring for quantitation of insulin, C-peptide, glucagon, and somatostatin secretion from human islet populations. For rapid analysis, a 5 min separation was achieved using a 2.1 × 30 mm (i.d. x length) C18 column with 2.7 µm diameter core shell particles. A sacrificial protein hydrolysate was used with the sample and found to improve signal magnitude, repeatability, and to reduce carryover between runs. At optimized gradient conditions, the gradient run time was 4.55 min producing an average peak width of 0.3 min, a minimum resolution of 1.2, and a peak capacity of 20. As a proof of concept, the method was used to measure secretions from static incubations of human islets from 2 donors. Insulin and C-peptide were quantified and matched well with literature values of these hormones. We expect that this antibody-free quantitation of multiple hormones secreted from islets will provide insights into the temporal relationships of these peptides in the future.

A droplet microfluidic platform for high-throughput photochemical reaction discovery.

The implementation of continuous flow technology is critical towards enhancing the application of photochemical reactions for industrial process development. However, there are significant time and resource constraints associated with translating discovery scale vial-based batch reactions to continuous flow scale-up conditions. Herein we report the development of a droplet microfluidic platform, which enables high-throughput reaction discovery in flow to generate pharmaceutically relevant compound libraries. This platform allows for enhanced material efficiency, as reactions can be performed on picomole scale. Furthermore, high-throughput data collection via on-line ESI mass spectrometry facilitates the rapid analysis of individual, nanoliter-sized reaction droplets at acquisition rates of 0.3 samples/s. We envision this high-throughput screening platform to expand upon the robust capabilities and impact of photochemical reactions in drug discovery and development.

High-Throughput Nanoelectrospray Ionization-Mass Spectrometry Analysis of Microfluidic Droplet Samples.

Droplet microfluidics enables high-throughput manipulation of fL-µL volume samples. Methods implemented for the chemical analysis of microfluidic droplets have been limited in scope, leaving some applications of droplet microfluidics difficult to perform or out of reach entirely. Nanoelectrospray ionization-mass spectrometry (nESI-MS) is an attractive approach for droplet analysis, because it allows rapid, label-free, information-rich analysis with high mass sensitivity and resistance to matrix effects. Previous proof-of-concept systems for the nESI-MS analysis of droplets have been limited by the microfluidics used so that stable, long-term operation needed for high-throughput applications has not been demonstrated. We describe a platform for the stable analysis of microfluidic droplet samples by nESI-MS. Continuous infusion of droplets to an nESI emitter was demonstrated for as long as 2.5 h, corresponding to analysis of over 20 000 samples. Stable signal was observed for droplets as small as 65 pL and for throughputs as high as 10 droplets/s. A linear-concentration-based response and sample-to-sample carryover of <3% were also shown. The system is demonstrated for measuring products of in-droplet enzymatic reactions.

In Vivo Chemical Monitoring at High Spatiotemporal Resolution Using Microfabricated Sampling Probes and Droplet-Based Microfluidics Coupled to Mass Spectrometry.

An essential approach for in vivo chemical monitoring is to use sampling probes coupled with analytical methods; however, this method traditionally has limited spatial and temporal resolution. To address this problem, we developed an analytical system that combines microfabricated push-pull sampling probes with droplet-based microfluidics. The microfabricated probe provides spatial resolution approximately 1000-fold better than that of common microdialysis probes. Microfabrication also facilitated integration of an extra channel into the probe for microinjection. We created microfluidic devices and interfaces that allowed manipulation of nanoliter droplet samples collected from the microfabricated probe at intervals of a few seconds. Use of droplet-based microfluidics prevented broadening of collected zones, yielding 6 s temporal resolution at 100 nL/min perfusion rates. Resulting droplets were analyzed by direct infusion nanoelectrospray ionization (nESI) mass spectrometry for simultaneous determination of glutamine, glutamate, γ-aminobutyric acid, and acetylcholine. Use of low infusion rates that enabled nESI (50 nL/min) was critical to allowing detection in the complex samples. Addition of 13C-labeled internal standards to the droplet samples was used for improved quantification. Utility of the overall system was demonstrated by monitoring dynamic chemical changes evoked by microinjection of high potassium concentrations into the brain of live rats. The results showed stimulated neurochemical release with rise times of 15 s. This work demonstrates the potential of coupling microfabricated sampling probes to droplet-based mass spectrometric assays for studying chemical dynamics in a complex microenvironment at high spatiotemporal resolution.

Subsecond electrophoretic separations from droplet samples for screening of enzyme modulators.

High-throughput screening (HTS) using multiwell plates and fluorescence plate readers is a powerful tool for drug discovery and evaluation by allowing tens of thousands of assays to be completed in 1 day. Although this method has been successful, electrophoresis-based methods for screening are also of interest to avoid difficulties associated fluorescence assays such as requirements to engineer fluorogenic reactions and false positives. We have developed a method using droplet microfluidics to couple multiwell plate-based assays to microchip electrophoresis (MCE) to screen enzyme modulators. Samples contained in multiwell plates are reformatted in to plugs with a sample volume of 8 nL segmented by an immiscible oil. The segmented flow sample streams are coupled to a hybrid polydimethylsiloxane-glass microfluidic device capable of selectively extracting the aqueous samples from the droplet stream and rapidly analyzing by MCE with laser-induced fluorescence detection. This system was demonstrated by screening a test library of 140 compounds against using protein kinase A. For each sample in the screen, two droplets are generated, allowing approximately 6 MCE injections per sample. Using a 1 s separation at 2000 V/cm, we are able to analyze 96 samples in 12 min. Separation resolution between the internal standard, substrate, and product is 1.2 and average separation efficiency is 16,000 plates/s using real samples. Twenty-five compounds were identified as modulators during primary screening and verified using dose-response curves.

Evaluating the complexation behavior and regeneration of boron selective glucaminium-based ionic liquids when used as extraction solvents.

Glucaminium-based ionic liquids are a new class of solvents capable of extracting boron-species from water with high efficiency. The complexation behavior of these ILs with borate was thoroughly studied using (11)B NMR. Two different complexes, namely, monochelate complex and bischelate complex, were observed. (11)B NMR was used extensively to determine the formation constants for monochelate and bischelate complexes. The IL concentration was observed to have a significant effect on the IL-borate complexes. Using an in situ dispersive liquid-liquid microextraction (in situ DLLME) method, the extraction efficiency for boron species was increased dramatically when lithium bis[(trifluoromethyl)sulfonyl]imide (LiNTf(2)) was used as the metathesis salt in an aqueous solution containing 0.1M sodium chloride. IL regeneration after extraction was achieved using 0.1M hydrochloric acid. The extraction efficiency of boron species was consistent when the IL was employed after three regeneration cycles. The selectivity of the IL for boron species in synthetic seawater samples was similar to performing the same extraction from Milli-Q water samples.