Structure-Driven Liquid Microjunction Surface-Sampling Probe Mass Spectrometry

The rhizosphere is the narrow region of soil surrounding the roots of plants that is influenced by root exudates, root secretions, and associated microbial communities. This region is crucial to plant growth and development and plays a critical role in nutrient uptake, disease resistance, and soil transformation. Understanding the function of exogenous compounds in the rhizosphere starts with determining the spatiotemporal distribution of these molecular components. Using liquid microjunction surface-sampling probe mass spectrometry (LMJ-SSP-MS) and microfluidic devices with attached microporous membranes enables in situ, nondisruptive, and nondestructive spatiotemporal measurement of exogenous compounds from plant roots. However, long imaging times (>2 h) can negatively affect plant heath and limit temporal studies. Here, we present a novel strategy to optimize the number and location of sampling sites on these microporous membrane-covered microfluidic devices. This novel, “structure-driven” sampling workflow takes into consideration the channel structure of the microfluidic device to maximize sampling from the channels and minimize acquisition time (∼4× less time in some cases while providing similar chemical image accuracy), thus reducing stress on plants during in situ LMJ-SSP-MS analysis

Controlled-Resonant Surface Tapping-Mode Scanning Probe Electrospray Ionization Mass Spectrometry Imaging

This paper reports on the advancement of a controlled-resonant surface tapping-mode single capillary liquid junction extraction/ESI emitter for mass spectrometry imaging. The basic instrumental setup and the general operation of the system were discussed, and optimized performance metrics were presented. The ability to spot sample, lane scan, and chemically image in an automated and controlled fashion were demonstrated. Rapid, automated spot sampling was demonstrated for a variety of compound types, including the cationic dye basic blue 7, the oligosaccharide cellopentaose, and the protein equine heart cytochrome <i>c</i>. The system was used for lane scanning and chemical imaging of the cationic dye crystal violet in inked lines on glass and for lipid distributions in mouse brain thin tissue sections. Imaging of the lipids in mouse brain tissue under optimized conditions provided a spatial resolution of approximately 35 μm based on the ability to distinguish between features observed both in the optical and mass spectral chemical images. The sampling spatial resolution of this system was comparable to the best resolution that has been reported for other types of atmospheric pressure liquid extraction-based surface sampling/ionization techniques used for mass spectrometry imaging

Atomic Force Microscopy Thermally-Assisted Microsampling with Atmospheric Pressure Temperature Ramped Thermal Desorption/Ionization-Mass Spectrometry Analysis

The use of atomic force microscopy controlled nanothermal analysis probes for reproducible spatially resolved thermally assisted sampling of micrometer-sized areas (ca. 11 × 17 μm wide × 2.4 μm deep) from relatively low number-average molecular weight (<i>M</i>_<i>n</i> < 3000) polydisperse thin films of poly­(2-vinylpyridine) (P2VP) is presented. Following sampling, the nanothermal analysis probes were moved up from the surface and the probe temperature ramped to liberate the sampled materials into the gas phase for atmospheric pressure chemical ionization and mass spectrometric analysis. The procedure and mechanism for material pickup, the sampling reproducibility and sampling size are discussed, and the oligomer distribution information available from slow temperature ramps versus ballistic temperature jumps is presented. For the <i>M</i>_<i>n</i> = 970 P2VP, the <i>M</i>_<i>n</i> and polydispersity index determined from the mass spectrometric data were in line with both the label values from the sample supplier and the value calculated from the simple infusion of a solution of polymer into the commercial atmospheric pressure chemical ionization source on this mass spectrometer. With a P2VP sample of higher <i>M</i>_<i>n</i> (<i>M</i>_<i>n</i> = 2070 and 2970), intact oligomers were still observed (as high as <i>m</i>/<i>z</i> 2793 corresponding to the 26-mer), but a significant abundance of thermolysis products were also observed. In addition, the capability for confident identification of the individual oligomers by slowly ramping the probe temperature and collecting data-dependent tandem mass spectra was also demonstrated. The material type limits to the current sampling and analysis approach as well as possible improvements in nanothermal analysis probe design to enable smaller area sampling and to enable controlled temperature ramps beyond the present upper limit of about 415 °C are also discussed

Topographical and Chemical Imaging of a Phase Separated Polymer Using a Combined Atomic Force Microscopy/Infrared Spectroscopy/Mass Spectrometry Platform

In this paper, the use of a hybrid atomic force microscopy/infrared spectroscopy/mass spectrometry imaging platform was demonstrated for the acquisition and correlation of nanoscale sample surface topography and chemical images based on infrared spectroscopy and mass spectrometry. The infrared chemical imaging component of the system utilized photothermal expansion of the sample at the tip of the atomic force microscopy probe recorded at infrared wave numbers specific to the different surface constituents. The mass spectrometry-based chemical imaging component of the system utilized nanothermal analysis probes for thermolytic surface sampling followed by atmospheric pressure chemical ionization of the gas phase species produced with subsequent mass analysis. The basic instrumental setup, operation, and image correlation procedures are discussed, and the multimodal imaging capability and utility are demonstrated using a phase separated poly­(2-vinylpyridine)/poly­(methyl methacrylate) polymer thin film. The topography and both the infrared and mass spectral chemical images showed that the valley regions of the thin film surface were comprised primarily of poly­(2-vinylpyridine) and hill or plateau regions were primarily poly­(methyl methacrylate). The spatial resolution of the mass spectral chemical images was estimated to be 1.6 μm based on the ability to distinguish surface features in those images that were also observed in the topography and infrared images of the same surface

Combined Falling Drop/Open Port Sampling Interface System for Automated Flow Injection Mass Spectrometry

The aim of this work was to demonstrate and to evaluate the analytical performance of a combined falling drop/open port sampling interface (OPSI) system as a simple noncontact, no-carryover, automated system for flow injection analysis with mass spectrometry. The falling sample drops were introduced into the OPSI using a widely available autosampler platform utilizing low cost disposable pipet tips and conventional disposable microtiter well plates. The volume of the drops that fell onto the OPSI was in the 7–15 μL range with an injected sample volume of several hundred nanoliters. Sample drop height, positioning of the internal capillary on the sampling end of the probe, and carrier solvent flow rate were optimized for maximum signal. Sample throughput, signal reproducibility, matrix effects, and quantitative analysis capability of the system were established using the drug molecule propranolol and its isotope labeled internal standard in water, unprocessed river water and two commercially available buffer matrices. A sample-to-sample throughput of ∼45 s with a ∼4.5 s base-to-base flow injection peak profile was obtained in these experiments. In addition, quantitation with minimally processed rat plasma samples was demonstrated with three different statin drugs (atorvastatin, rosuvastatin, and fluvastatin). Direct characterization capability of unprocessed samples was demonstrated by the analysis of neat vegetable oils. Employing the autosampler system for spatially resolved liquid extraction surface sampling exemplified by the analysis of propranolol and its hydroxypropranolol glucuronide phase II metabolites from a rat thin tissue section was also illustrated

Geochemical Evidence for Rare-Earth Element Mobilization during Kaolin Diagenesis

This study investigates how saprolization influences inherent rare-earth element (REE) source rock signatures and how depositional environment(s) and diagenetic reactions ultimately impact the REE signature within sedimentary kaolin bodies. Rare-earth element geochemistry signatures are particularly useful for tracking element sources and mobility and are, therefore, powerful tools in the investigation of clay mineral formation and diagenesis. Rare-earth element and bulk chemical compositions were determined using discrete chemical analyses and chemical imaging. Saprolitic materials show an enrichment in the light and heavy REEs, compared with the parent material, with enhanced Ce/Eu anomalies. Light REEs within sedimentary kaolins are associated with phosphate mineralogy and have experienced variable degrees of diagenetic fractionation and mobilization. Cretaceous kaolins display more light REE mobility compared with Tertiary kaolins, which show very little REE fractionation. Degrees of REE fractionation are driven primarily by differences in sedimentary kaolin physical properties and the presence of organic acids in groundwater. Unfortunately, the provenance of the Georgia kaolins could not be determined based solely on the trace-element and REE compositions because fractionations during saprolization and diagenesis mask much of the inherent provenance signatures. Finally, implications for the REEs as an economic deposit and their beneficiation are discussed

Online, Absolute Quantitation of Propranolol from Spatially Distinct 20- and 40-μm Dissections of Brain, Liver, and Kidney Thin Tissue Sections by Laser Microdissection–Liquid Vortex Capture–Mass Spectrometry

Spatial resolved quantitation of chemical species in thin tissue sections by mass spectrometric methods has been constrained by the need for matrix-matched standards or other arduous calibration protocols and procedures to mitigate matrix effects (e.g., spatially varying ionization suppression). Reported here is the use of laser “cut and drop” sampling with a laser microdissection-liquid vortex capture electrospray ionization tandem mass spectrometry (LMD-LVC/ESI-MS/MS) system for online and absolute quantitation of propranolol in mouse brain, kidney, and liver thin tissue sections of mice administered with the drug at a 7.5 mg/kg dose, intravenously. In this procedure either 20 μm × 20 μm or 40 μm × 40 μm tissue microdissections were cut and dropped into the flowing solvent of the capture probe. During transport to the ESI source drug related material was completely extracted from the tissue into the solvent, which contained a known concentration of propranolol-<i>d</i>₇ as an internal standard. This allowed absolute quantitation to be achieved with an external calibration curve generated from standards containing the same fixed concentration of propranolol-<i>d</i>₇ and varied concentrations of propranolol. Average propranolol concentrations determined with the laser “cut and drop” sampling method closely agreed with concentration values obtained from 2.3 mm diameter tissue punches from serial sections that were extracted and quantified by HPLC/ESI-MS/MS measurements. In addition, the relative abundance of hydroxypropranolol glucuronide metabolites were recorded and found to be consistent with previous findings

Co-registered Topographical, Band Excitation Nanomechanical, and Mass Spectral Imaging Using a Combined Atomic Force Microscopy/Mass Spectrometry Platform

The advancement of a hybrid atomic force microscopy/mass spectrometry imaging platform demonstrating the co-registered topographical, band excitation nanomechanical, and mass spectral imaging of a surface using a single instrument is reported. The mass spectrometry-based chemical imaging component of the system utilized nanothermal analysis probes for pyrolytic surface sampling followed by atmospheric pressure chemical ionization of the gas-phase species produced with subsequent mass analysis. The basic instrumental setup and operation are discussed, and the multimodal imaging capability and utility are demonstrated using a phase-separated polystyrene/poly(2-vinylpyridine) polymer blend thin film. The topography and band excitation images showed that the valley and plateau regions of the thin film surface were comprised primarily of one of the two polymers in the blend with the mass spectral chemical image used to definitively identify the polymers at the different locations. Data point pixel size for the topography (390 nm × 390 nm), band excitation (781 nm × 781 nm), and mass spectrometry (690 nm × 500 nm) images was comparable and submicrometer in all three cases, but the data voxel size for each of the three images was dramatically different. The topography image was uniquely a surface measurement, whereas the band excitation image included information from an estimated 20 nm deep into the sample and the mass spectral image from 110 to 140 nm in depth. Because of this dramatic sampling depth variance, some differences in the band excitation and mass spectrometry chemical images were observed and were interpreted to indicate the presence of a buried interface in the sample. The spatial resolution of the chemical image was estimated to be between 1.5 and 2.6 μm, based on the ability to distinguish surface features in that image that were also observed in the other images

Dereplicating and Spatial Mapping of Secondary Metabolites from Fungal Cultures <i>in Situ</i>

Ambient ionization mass spectrometry techniques have recently become prevalent in natural product research due to their ability to examine secondary metabolites <i>in situ</i>. These techniques retain invaluable spatial and temporal details that are lost through traditional extraction processes. However, most ambient ionization techniques do not collect mutually supportive data, such as chromatographic retention times and/or UV/vis spectra, and this can limit the ability to identify certain metabolites, such as differentiating isomers. To overcome this, the droplet–liquid microjunction–surface sampling probe (droplet–LMJ–SSP) was coupled with UPLC–PDA–HRMS–MS/MS, thus providing separation, retention times, MS data, and UV/vis data used in traditional dereplication protocols. By capturing these mutually supportive data, the identity of secondary metabolites can be confidently and rapidly assigned <i>in situ</i>. Using the droplet–LMJ–SSP, a protocol was constructed to analyze the secondary metabolite profile of fungal cultures without any sample preparation. The results demonstrate that fungal cultures can be dereplicated from the Petri dish, thus identifying secondary metabolites, including isomers, and confirming them against reference standards. Furthermore, heat maps, similar to mass spectrometry imaging, can be used to ascertain the location and relative concentration of secondary metabolites directly on the surface and/or surroundings of a fungal culture

Dereplicating and Spatial Mapping of Secondary Metabolites from Fungal Cultures <i>in Situ</i>

Ambient ionization mass spectrometry techniques have recently become prevalent in natural product research due to their ability to examine secondary metabolites <i>in situ</i>. These techniques retain invaluable spatial and temporal details that are lost through traditional extraction processes. However, most ambient ionization techniques do not collect mutually supportive data, such as chromatographic retention times and/or UV/vis spectra, and this can limit the ability to identify certain metabolites, such as differentiating isomers. To overcome this, the droplet–liquid microjunction–surface sampling probe (droplet–LMJ–SSP) was coupled with UPLC–PDA–HRMS–MS/MS, thus providing separation, retention times, MS data, and UV/vis data used in traditional dereplication protocols. By capturing these mutually supportive data, the identity of secondary metabolites can be confidently and rapidly assigned <i>in situ</i>. Using the droplet–LMJ–SSP, a protocol was constructed to analyze the secondary metabolite profile of fungal cultures without any sample preparation. The results demonstrate that fungal cultures can be dereplicated from the Petri dish, thus identifying secondary metabolites, including isomers, and confirming them against reference standards. Furthermore, heat maps, similar to mass spectrometry imaging, can be used to ascertain the location and relative concentration of secondary metabolites directly on the surface and/or surroundings of a fungal culture