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.

Continuous and automated slug flow nanoextraction for rapid partition coefficient measurement.

Octanol-water partition coefficients (log Kow) are widely used in pharmaceutical and environmental chemistry to assess the lipophilicity of compounds. Traditionally log Kow is determined using a shake-flask method that uses milliliters of sample and solvent and requires hours for preparation, extraction, and analysis. Here, we report an automated system for rapid log Kow determination for an array of compounds using slug flow nanoextraction (SFNE) enabled by a microfluidic chip. In the method, an autosampler is used to introduce 1 µL of sample into a microfluidic device that segments the injected volume into a series of 4 nL slugs that are each paired to an adjacent octanol slug. Each octanol-water phase pair is compartmentalized by an immiscible fluorous carrier fluid. During flow, rapid extraction occurs at each octanol-water interface. The resulting linear array of slugs flows into an online UV absorbance detector that is used to determine concentrations in the phases, allowing the log Kow to be measured. The microfluidic device allows toggling between two-phase "aqueous plug" generation (aqueous sample separated by fluorous carrier fluid) and three-phase "phase pair" generation. In this way, online calibration for detection in the aqueous phase can be achieved. The method is applied to determining log Kow for a panel of seven pharmaceutical compounds, including complete calibration curves, at three different pHs in under 2 h using 5 µL of extraction standard and 2.9 µL of octanol per extraction standard analyzed.

High-Throughput Liquid-Liquid Extractions with Nanoliter Volumes.

Current methods for liquid-liquid extractions generally require microliter to milliliter volumes of solvents and sample, long equilibration times, and manual procedures. Extraction methods for samples in microfluidic systems have been limited as this tool is difficult to implement on the nanoliter or smaller scale. We have developed slug-flow nanoextraction (SFNE), a method based on droplet microfluidics that allows multiple liquid-liquid extractions to be performed simultaneously in a capillary tube, using only 5 nL of sample and extraction solvent per extraction. Each two-phase slug is segmented from the others by immiscible carrier fluid. We found rapid extractions (<5 s), partition coefficients matching those achieved at larger scale extractions, and potential to preconcentrate samples through volume manipulation. This method was used to accurately and rapidly determine octanol-water partition coefficients (Kow), determining identical Kow as the shake-flask method for acetaminophen, Kow = 2.48 ± 0.02. The measurement, along with calibration and 12 replicates, was complete within 5 min, consuming under 150 nL of solvent and sample. The method was also applied to extract analytes from complex biological samples prior to electrospray ionization-tandem mass spectrometry (ESI-MS/MS) at a rate of 6 s per sample, allowing for simultaneous determination of five different drugs spiked into human plasma, synthetic urine (SU), and artificial cerebral spinal fluid (aCSF) using ethyl acetate as the extraction phase. The signal-to-noise (S/N) for analytes improved up to 19-fold compared to direct ESI-MS of single-phase droplets (aqueous plugs segmented by carrier fluid), with limits of detection down to 7 nM (35 amol).

CE-MS with electrokinetic supercharging and application to determination of neurotransmitters.

Electrokinetic supercharging (EKS) is known as one of the most effective online electrophoretic preconcentration techniques, though pairing with it with mass spectrometry has presented challenges. Here, EKS is successfully paired with ESI-MS/MS to provide a sensitive and robust method for analysis of biogenic amines in biological samples. Injection parameters including electric field strength and the buffer compositions used for the separation and focusing were investigated to achieve suitable resolution, high sensitivity, and compatibility with ESI-MS. Using EKS, the sensitivity of the method was improved 5000-fold compared to a conventional hydrodynamic injection with CZE. The separation allowed for baseline resolution of several neurotransmitters within 16 min with LODs down to 10 pM. This method was applied to targeted analysis of seven biogenic amines from rat brain stem and whole Drosophila tissue. This is the first method to use EKS with CE-ESI-MS/MS to analyze biological samples.

Metal cation control of electroosmotic flow magnitude in phospholipid-coated capillaries.

CZE has become widespread for the separation and analysis of biomolecules such as proteins and peptides, due to factors such as, the speed of the separations, low sample volume, and high resolution associated with the technique. However, the separation of biomolecules by CZE does present a significant challenge due to the electrostatic attraction and adsorption of cationic, or cation containing, biomolecules to the capillary surface. To that end numerous methods have been developed to passivate, or protect the surface, in order to prevent the adsorption of analytes. Yet, in the process of protecting the capillary surface, the potential for further modification of the EOF, a factor crucial to effective analyte resolution, is greatly diminished. In seeking to overcome this limitation we have explored the potential of incorporating a range of metal cations into a phospholipid bilayer capillary coating. It has previously been established that the inclusion of calcium into the separation buffer with a phospholipid coating will reverse the EOF in the capillary. Here, we present our investigation of a broader range of metal cations included in the separation buffer (Ca(2+) , Mg(2+) , Co(2+) , Ni(2+) , Sr(2+) , Ba(2+) , and Ce(3+) ) revealing that the choice of metal cation can drastically influence the EOF, with observed values between -3.80 × 10(-4) and -5.74 × 10(-5) cm(2) /V·s.