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Several studies have reported ionization methods to classify the chemical composition of levitated particles held in an electrodynamic balance using mass spectrometry (MS). These methods include electrospray-based paper spray (PS) ionization, plasma discharge ionization, and direct analysis in real-time (DART) ionization, with each showing advantages and disadvantages. Our recent work demonstrated that PS ionization could yield accurate data for the chemical evolution of mixed component particles undergoing evaporation. However, measurements were performed using an internal standard to account for and correct the inherent variability in the PS ionization source. Here, we explore a new electrospray-based method coupled to particle levitation-the Open Port Sampling Interface (OPSI), which provides many advantages over the PS method, with few disadvantages. In this application note we report experiments in which micron-sized particles, containing analytes such as citric acid, maleic acid, and tetraethylene glycol, were levitated and optically probed to determine their size and mass. Subsequent transfer of individual levitated particles into the OPSI allowed for the ionization and mass spectrometry analysis of these particles. We discuss the stability and reproducibility of MS measurements, demonstrate effective quantitation in both positive and negative mode, and determine the sensitivity of the OPSI to a range of analyte mass present in levitated particles. Importantly, we show stability of the OPSI over6 h without the need for normalizing signal variations with an internal standard in the sample, demonstrating robust application of the OPSI to measurements over extended periods of time.

The application of a fully automated autosampler/HPLC-ESI–MS/MS system for spatially resolved quantitative droplet-based liquid extraction surface sampling/profiling of stapled α–helical peptide ATSP-7041 in mouse whole-body thin tissue sections is reported. 20-μm-thick serial tissue sections of an ATSP-7041 dosed mouse were prepared and the absolute concentration of the targeted peptide was first determined in different organs using 2.3-mm diameter tissue punches, standard bulk tissue extraction protocols, and subsequent HPLC separation and tandem mass spectrometric analysis. The same organs/locations were then analyzed in neighboring tissue sections using the droplet-based surface sampling approach. The observed ATSP-7041 concentration using this method was always significantly lower than that measured by the tissue punch workflow at the same tissue location of a serial section. Calculated extraction efficiencies were 10.7 ± 0.5% (brain), 11.0 ± 3.2% (liver spot 1), 10.7 ± 2.6% (liver spot 2), 15.0 ± 0.6% (lung) and 12.9 ± 0.7% (blood). While these extraction efficiency values were low, they were reproducible within a given organ. This suggests that once the extraction efficiency is established for a given tissue type and drug, the reproducibility of the droplet-based approach could provide a non-labor intensive and high-throughput means to acquire spatially resolved quantitative analysis of multiple samples of the same type.

The open port interface (OPI) coupled to an atmospheric pressure ion source is used to capture, dilute, focus, and transport nanoliter volume sample droplets for high-speed mass spectrometric analysis. For typical applications, the system has been optimized to achieve 1 Hz nanoliter volume sample transfer rates while simultaneously diluting the sample >1000-fold to minimize sample matrix-induced ionization suppression. Geometric, flow, and dispensing alterations to the system presented here demonstrate that sample transfer rates for the OPI of at least 15 Hz are possible. The fluid dynamic processes that enable sampling rates of 1 Hz and greater are examined in detail by correlating computational fluid dynamics simulations, analytic calculations, experimental data, photographic footage, and reference to the fluid dynamics literature. The resulting models and experimental results provide the rationale underlying the design and tuning of the system as well as information for developing optimized analytical methods. In combination with acoustic droplet dispensing, referred to as acoustic ejection mass spectrometry (AEMS), this system can be considered to be a special case of flow injection analysis with unique features that control the peak width, symmetry, and segregation of the samples transported in a fluid while simultaneously enabling their mixing and dilution with carrier fluids. In addition, conditions are established to prevent direct contact of the sample with a surface enabling, in combination with a contact-free dispenser like acoustic ejection, a dramatic reduction in sample-to-sample carry-over.

Acoustic ejection mass spectrometry is a recently developed concept in which low nanoliter-volume sample droplets are acoustically dispensed from microtiter plate wells into a continuous fluid transfer open-port interface for subsequent ionization at atmospheric pressure. This manuscript focuses on the acoustic droplet dispensing component of a prototype system, in particular the well-to-well sampling speed, droplet volume calibration, precision, reproducibility, and various modes of operation this device enables. A new method to measure the volume of individually dispensed droplets is presented to both aid method validation and potentially assist in the tuning of acoustic dispense parameters for samples having a wide range of viscosities and surface tensions. Acoustic dispensing modes of operation discussed are high-speed, well-to-well dispensing of individual nanoliter-scale droplets from microtiter plates, including the first demonstration of 1536-well compatibility using this approach. Multiple nanoliter-volume droplets per sampling event to increase detection dynamic range is described, and a "continuous infusion" mode to provide a low sample consumption sustained steady-state signal for analyte detection optimization, improved ion statistics and signal-to-noise ratio (S/N), or time for in-depth tandem mass spectrometry of the components in a sample is presented. The concept of "bar coding" using combinations of dispensed droplet patterns to register well-plate position to specific mass spectral signals is introduced, as well as judicious well-plate sample layout to enable assay "multiplexing" as a means to maximize well-to-well sample analysis throughput, is also demonstrated.

RationaleLaser microdissection-liquid vortex capture/electrospray ionization mass spectrometry (LMD-LVC/ESI-MS) has potential for on-line classification of tissue but an investigation into what analytical conditions provide best spectral differentiation has not been conducted. The effects of solvent, ionization polarity, and spectral acquisition parameters on differentiation of mouse brain tissue regions are described.MethodsIndividual 40 x 40m microdissections from cortex, white, grey, granular, and nucleus regions of mouse brain tissue were analyzed using different capture/ESI solvents, in positive and negative ion mode ESI, using time-of-flight (TOF)-MS and sequential window acquisitions of all theoretical spectra (SWATH)-MS (a permutation of tandem-MS), and combinations thereof. Principal component analysis-linear discriminant analysis (PCA-LDA), applied to each mass spectral dataset, was used to determine the accuracy of differentiation of mouse brain tissue regions.ResultsMass spectral differences associated with capture/ESI solvent composition manifested as altered relative distributions of ions rather than the presence or absence of unique ions. In negative ion mode ESI, 80/20 (v/v) methanol/water yielded spectra with low signal/noise ratios relative to other solvents. PCA-LDA models acquired using 90/10 (v/v) methanol/chloroform differentiated tissue regions with 100% accuracy while data collected using methanol misclassified some samples. The combination of SWATH-MS and TOF-MS data improved differentiation accuracy.ConclusionsCombined TOF-MS and SWATH-MS data differentiated white, grey, granular, and nucleus mouse tissue regions with greater accuracy than when solely using TOF-MS data. Using 90/10 (v/v) methanol/chloroform, tissue regions were perfectly differentiated. These results will guide future studies looking to utilize the potential of LMD-LVC/ESI-MS for tissue and disease differentiation.

Rationale As the spatial resolution of mass spectrometry imaging technologies has begun to reach into the nanometer regime, finding readily available or easily made resolution reference materials has become particularly challenging for molecular imaging purposes. This paper describes the fabrication, characterization and use of vertical line array polymeric spatial resolution test patterns for nano-thermal analysis/atomic force microscopy/mass spectrometry chemical imaging. Methods Test patterns of varied line width (0.7 or 1.0 μm) and spacing (0.7 or 1.0 μm) were created in an ~1-μm-thick poly(methyl methacrylate) thin film using electron beam lithography. The patterns were characterized by scanning electron microscopy, energy-dispersive X-ray spectroscopy, atomic force microscopy topography and nano-thermal analysis/mass spectrometry imaging. Results The efficacy of these polymeric test patterns for the advancement of chemical imaging technique was illustrated by their use to judge the spatial resolution improvement achieved by heating the ionization interface of the current instrument platform. The spatial resolution of the mass spectral chemical images was estimated to be 1.4 μm, based on the ability to statistically distinguish 0.7 μm-wide lines separated by 0.7 μm-wide spacings in those images when the interface cross was heated to 200 oC. Conclusions This work illustrates that e-beam lithography is a viable method to create spatial resolution test patterns in a thin film of high molecular weight polymer to allow unbiased judgment of intra-laboratory advancement and/or inter-laboratory comparison of instrument advances in nano-thermal analysis/atomic force microscopy/mass spectrometry chemical imaging.

RATIONALE An "Open Access"-like mass spectrometric platform to fully utilize the simplicity of the manual open port sampling interface for rapid characterization of unprocessed samples by liquid introduction atmospheric pressure ionization mass spectrometry has been lacking. The in-house developed integrated software with a simple, small and relatively low-cost mass spectrometry system introduced here fills this void. METHODS Software was developed to operate the mass spectrometer, to collect and process mass spectrometric data files, to build a database and to classify samples using such a database. These tasks were accomplished via the vendor-provided software libraries. Sample classification based on spectral comparison utilized the spectral contrast angle method. RESULTS Using the developed software platform near real-time sample classification is exemplified using a series of commercially available blue ink rollerball pens and vegetable oils. In the case of the inks, full scan positive and negative ion ESI mass spectra were both used for database generation and sample classification. For the vegetable oils, full scan positive ion mode APCI mass spectra were recorded. The overall accuracy of the employed spectral contrast angle statistical model was 95.3% and 98% in case of the inks and oils, respectively, using leave-one-out cross-validation. CONCLUSIONS This work illustrates that an open port sampling interface/mass spectrometer combination, with appropriate instrument control and data processing software, is a viable direct liquid extraction sampling and analysis system suitable for the non-expert user and near real-time sample classification via database matching. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA.

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-d7 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-d7 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.

Rationale Currently objective/quantitative descriptions of the quality and spatial resolution of mass spectrometry derived chemical images are not standardized. Development of these standardized metrics is required to objectively describe the chemical imaging capabilities of existing and/or new mass spectrometry imaging technologies. Such metrics would allow unbiased judgment of intra-laboratory advancement and/or inter-laboratory comparison for these technologies if used together with standardized surfaces. Methods Two image metrics, viz., "chemical image contrast" (ChemIC) based on signal-to-noise related statistical measures on chemical image pixels and "corrected resolving power factor" (cRPF) constructed from statistical analysis of mass-to-charge chronograms across features of interest in an image, were developed. These metrics, quantifying chemical image quality and spatial resolution, respectively, were used to evaluate chemical images of a model photoresist patterned surface collected using a laser ablation/liquid vortex capture mass spectrometry imaging system under different instrument operational parameters. Results The calculated ChemIC and cRPF metrics determined in an unbiased fashion the relative ranking of chemical image quality obtained with the laser ablation/liquid vortex capture mass spectrometry imaging system. These rankings were used to show that both chemical image contrast and spatial resolution deteriorated with increasing surface scan speed, increased lane spacing and decreasing size of surface features. Conclusions ChemIC and cRPF, respectively, were developed and successfully applied for the objective description of chemical image quality and spatial resolution of chemical images collected from model surfaces using a laser ablation/liquid vortex capture mass spectrometry imaging system. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA.

Rationale Laser microdissection coupled directly with mass spectrometry provides the capability of on-line analysis of substrates with high spatial resolution, high collection efficiency, and freedom on shape and size of the sampling area. Establishing the merits and capabilities of the different sampling modes that the system provides is necessary in order to select the best sampling mode for characterizing analytically challenging samples. Methods The capabilities of laser ablation spot sampling, laser ablation raster sampling, and laser 'cut and drop' sampling modes of a hybrid optical microscopy/laser ablation liquid vortex capture electrospray ionization mass spectrometry system were compared for the analysis of single cells and tissue. Results Single Chlamydomonas reinhardtii cells were monitored for their monogalactosyldiacylglycerol (MGDG) and diacylglyceryltrimethylhomo-Ser (DGTS) lipid content using the laser spot sampling mode, which was capable of ablating individual cells (~4–15 μm) even when agglomerated together. Turbid Allium Cepa cells (~150 μm) having unique shapes difficult to precisely measure using the other sampling modes could be ablated in their entirety using laser raster sampling. Intact microdissections of specific regions of a cocaine-dosed mouse brain tissue were compared using laser 'cut and drop' sampling. Since in laser 'cut and drop' sampling whole and otherwise unmodified sections are captured into the probe, 100% collection efficiencies were achieved. Laser ablation spot sampling has the highest spatial resolution of any sampling mode, while laser ablation raster sampling has the highest sampling area adaptability of the sampling modes. Conclusions Laser ablation spot sampling has the highest spatial resolution of any sampling mode, useful in this case for the analysis of single cells. Laser ablation raster sampling was best for sampling regions with unique shapes that are difficult to measure using other sampling modes. Laser 'cut and drop' sampling can be used for cases where the highest sensitivity is needed, for example, monitoring drugs present in trace amounts in tissue. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA.

RATIONALE: A simple method to introduce unprocessed samples into a solvent for rapid characterization by liquid introduction atmospheric pressure ionization mass spectrometry has been lacking. The continuous flow, self-cleaning open port sampling interface introduced here fills this void. METHODS: The open port sampling interface used a vertically aligned, co-axial tube arrangement enabling solvent delivery to the sampling end of the device through the tubing annulus and solvent aspiration down the center tube and into the mass spectrometer ionization source via the commercial APCI emitter probe. The solvent delivery rate to the interface was set to exceed the aspiration rate creating a continuous sampling interface along with a constant, self-cleaning spillover of solvent from the top of the probe. RESULTS: Using the open port sampling interface with positive ion mode APCI and a hybrid quadrupole time of flight mass spectrometer, rapid, direct sampling and analysis possibilities are exemplified with plastics, ballpoint and felt tip ink pens, skin, and vegetable oils. These results demonstrated that the open port sampling interface could be used as a simple, versatile and self-cleaning system to rapidly introduce multiple types of unprocessed, sometimes highly concentrated and complex, samples into a solvent flow stream for subsequent ionization andmore » analysis by mass spectrometry. The basic setup presented here could be incorporated with any self-aspirating liquid introduction ionization source (e.g., ESI, APCI, APPI, ICP, etc.) or any type of atmospheric pressure sampling ready mass spectrometer system. CONCLUSIONS: The open port sampling interface provides a means to introduce and quickly analyze unprocessed solid or liquid samples with liquid introduction atmospheric pressure ionization source without fear of sampling interface or ionization source contamination.« less

Ambient ionization mass spectrometry techniques have recently become prevalent in natural product research due to their ability to examine secondary metabolites in situ. 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 in situ. 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.

Described here are the results from the profiling of the proteins arginine vasopressin (AVP) and adrenocorticotropic hormone (ACTH) from normal human pituitary gland and pituitary adenoma tissue sections, using a fully automated droplet-based liquid-microjunction surface-sampling-HPLC-ESI-MS-MS system for spatially resolved sampling, HPLC separation, and mass spectrometric detection. Excellent correlation was found between the protein distribution data obtained with this method and data obtained with matrix-assisted laser desorption/ionization (MALDI) chemical imaging analyses of serial sections of the same tissue. The protein distributions correlated with the visible anatomic pattern of the pituitary gland. AVP was most abundant in the posterior pituitary gland region (neurohypophysis), and ATCH was dominant in the anterior pituitary gland region (adenohypophysis). The relative amounts of AVP and ACTH sampled from a series of ACTH-secreting and non-secreting pituitary adenomas correlated with histopathological evaluation. ACTH was readily detected at significantly higher levels in regions of ACTH-secreting adenomas and in normal anterior adenohypophysis compared with non-secreting adenoma and neurohypophysis. AVP was mostly detected in normal neurohypophysis, as expected. This work reveals that a fully automated droplet-based liquid-microjunction surface-sampling system coupled to HPLC-ESI-MS-MS can be readily used for spatially resolved sampling, separation, detection, and semi-quantitation of physiologically-relevant peptide and protein hormones, including AVP and ACTH, directly from human tissue. In addition, the relative simplicity, rapidity, and specificity of this method support the potential of this basic technology, with further advancement, for assisting surgical decision-making. Graphical Abstract Mass spectrometry based profiling of hormones in human pituitary gland and tumor thin tissue sections.

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.

Previous studies defined easy and difficult to hydrolyze fractions of hemicellulose that may result from bonds among cellulose, hemicellulose, and lignin. To understand how such bonds affect hydrolysis, Populus trichocarpa × Populus deltoides, holocellulose isolated from P. trichocarpa × P. deltoides and birchwood xylan were subjected to hydrothermal flow-through pretreatment. Samples were characterized by glycome profiling, HPLC, and UPLC-MS. Glycome profiling revealed steady fragmentation and removal of glycans from solids during hydrolysis. The extent of polysaccharide fragmentation, hydrolysis rate, and total xylose yield were lowest for P. trichocarpa × P. deltoides and greatest for birchwood xylan. Comparison of results from P. trichocarpa × P. deltoides and holocellulose suggested that lignin-carbohydrate complexes reduce hydrolysis rates and limit release of large xylooligomers. Smaller differences between results with holocellulose and birchwood xylan suggest xylan-cellulose hydrogen bonds limited hydrolysis, but to a lesser extent. These findings imply cell wall structure strongly influences hydrolysis.

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.

Aim: The aim of this work was to demonstrate and evaluate the analytical performance of coupling the immediate drop on demand technology to a mass spectrometer via the recently introduced open port sampling interface and ESI. Methodology & results: A maximum sample analysis throughput of 5 s per sample was demonstrated. Signal reproducibility was 10% or better as demonstrated by the quantitative analysis of propranolol and its stable isotope-labeled internal standard propranolol-d7. The ability of the system to multiply charge and analyze macromolecules was demonstrated using the protein cytochrome c. Conclusion: This immediate drop on demand technology/open port sampling interface/ESI–MS combination allowed for the quantitative analysis of relatively small mass analytes and was used for the identification of macromolecules like proteins.

The key to advancing materials is to understand and control their structure and chemistry. However, thorough chemical characterization is challenging since existing techniques characterize only a few properties of the specimen, thereby necessitating multiple measurement platforms to acquire the necessary information. The multimodal combination of atomic force microscopy (AFM) and mass spectrometry (MS) transcends existing analytical capabilities for nanometer scale spatially resolved correlation of the chemical and physical properties of a sample surface. One such hybrid system employs heated AFM cantilevers for thermal desorption (TD) sampling of molecules from a surface and subsequent gas phase ionization and detection of the liberated species by MS. Herein, we report on the use of voltage pulse trains to tailor cantilever heating such that spot sampling size was reduced and mass spectral signal was improved compared to constant voltage, static heating of the cantilever. Desorption efficiency (DE), defined as the quotient of the mass spectral signal intensity and the volume of the desorption crater, was used to judge the effectiveness of a particular tailored heating function. To guide the development and optimization of the heating functions and aid in interpreting experimental results, a 1D finite element model was developed that predicted the cantilever response to different heating functions. Three tailored heating functions that used different combinations, magnitudes, and durations of rectangular voltage pulses, were used for surface spot sampling. The resultant sampling spot size and DE were compared to the same metrics obtained with the conventional method that uses a single voltage pulse. Using a model system composed of a thin film of ink containing pigment yellow 74 as a model system, desorption craters shrunk from 2 μm, using the conventional approach, to 310 nm using the optimum tailored heating function. This same pulsed heating function produced a 381× improvement in the DE and an 8× improvement in spatial resolution compared to the conventional heating approach showing that signal/amount of material sampled was improved significantly by this new cantilever heating strategy.

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 (Mn < 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 Mn = 970 P2VP, the Mn 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 s...

RATIONALE Capture of material from a laser ablation plume into a continuous flow stream of solvent provides the means for uninterrupted sampling, transport and ionization of collected material for coupling with mass spectral analysis. Reported here is the use of vertically aligned transmission geometry laser ablation in combination with a new non-contact liquid vortex capture probe coupled with electrospray ionization for spot sampling and chemical imaging with mass spectrometry. METHODS A vertically aligned continuous flow liquid vortex capture probe was positioned directly underneath a sample surface in a transmission geometry laser ablation (355 nm, 10 Hz, 7 ns pulse width) set up to capture into solution the ablated material. The outlet of the vortex probe was coupled to the Turbo V™ ion source of an AB SCIEX TripleTOF 5600+ mass spectrometer. System operation and performance metrics were tested using inked patterns and thin tissue sections. Glass slides and slides designed especially for laser capture microdissection, viz., DIRECTOR(®) slides and PEN 1.0 (polyethylene naphthalate) membrane slides, were used as sample substrates. RESULTS The estimated capture efficiency of laser-ablated material was 24%, which was enabled by the use of a probe with large liquid surface area (~2.8 mm(2) ) and with gravity to help direct ablated material vertically down towards the probe. The swirling vortex action of the liquid surface potentially enhanced capture and dissolution not only of particulates, but also of gaseous products of the laser ablation. The use of DIRECTOR(®) slides and PEN 1.0 (polyethylene naphthalate) membrane slides as sample substrates enabled effective ablation of a wide range of sample types (basic blue 7, polypropylene glycol, insulin and cyctochrome c) without photodamage using a UV laser. Imaging resolution of about 6 µm was demonstrated for stamped ink on DIRECTOR(®) slides based on the ability to distinguish features present both in the optical and in the chemical image. This imaging resolution was 20 times better than the previous best reported results with laser ablation/liquid sample capture mass spectrometry imaging. Using thin sections of brain tissue the chemical image of a selected lipid was obtained with an estimated imaging resolution of about 50 µm. CONCLUSIONS A vertically aligned, transmission geometry laser ablation liquid vortex capture probe, electrospray ionization mass spectrometry system provides an effective means for spatially resolved spot sampling and imaging with mass spectrometry.

RATIONALE Droplet-based liquid extraction approaches for spatially resolved surface sampling coupled with high-performance liquid chromatography/mass spectrometry (HPLC/MS) provide the ability to deal with complex sample matrices and to identify isomeric compounds not distinguishable by MS methods alone. Improvements in sampling reliability, spatial resolution, spatial precision and extraction efficiency are required to further the analytical utility of such sampling systems. METHODS An autosampler capable of droplet-based liquid extraction was coupled with an HPLC/MS system. Visual inspection of the junction formation between the probe and a glass surface allowed evaluation of the liquid junction formation reliability, spatial location and size as a function of variable parameters such as solvent composition, probe-to-surface distance and droplet volume during solvent dispense and aspiration. Quantitative analysis of a component from a model surface using a weak extraction solvent was used to evaluate the effect of extraction time and number of extraction cycles on analyte extraction efficiency. RESULTS Reliable junction formation, independent of other variable parameters, was realized simply by maintaining a maximum distance of 0.4 mm between the probe and the sample surface. The smallest liquid junction diameter (1.6 mm) was observed when using a 1 μL dispensed volume and 90% aqueous extraction solvent with either methanol or acetonitrile. Good sampling precision was always achieved using an extraction solvent with at least 50% methanol or acetonitrile by volume. Quantitative sampling of rhodamine B from a magenta Sharpie ink surface using a weak extraction solvent showed that extraction efficiency could be improved by increasing the extraction time or the number of extraction cycles. CONCLUSIONS A platform employing a commercially available autosampler coupled to HPLC/MS was developed and successfully applied to investigate the effect of different sampling parameters on the reliability, spatial resolution, spatial precision and extraction efficiency of the liquid junction surface sampling process. This article is a U.S. Government work and is in the public domain in the USA.

RATIONALE Laser ablation provides for the possibility of sampling a large variety of surfaces with high spatial resolution. This type of sampling when employed in conjunction with liquid capture followed by nanoelectrospray ionization provides the opportunity for sensitive and prolonged interrogation of samples by mass spectrometry as well as the ability to analyze surfaces not amenable to direct liquid extraction. METHODS A fully automated, reflection geometry, laser ablation liquid capture spot sampling system was achieved by incorporating appropriate laser fiber optics and a focusing lens into a commercially available, liquid extraction surface analysis (LESA®)-ready Advion TriVersa NanoMate system. RESULTS Under optimized conditions about 10% of laser-ablated material could be captured in a droplet positioned vertically over the ablation region using the NanoMate robot-controlled pipette. The sampling spot size area with this laser ablation liquid capture surface analysis (LA/LCSA) mode of operation (typically about 120 µm × 160 µm) was approximately 50 times smaller than that achievable by direct liquid extraction using LESA® (ca 1 mm diameter liquid extraction spot). The setup was successfully applied for the analysis of ink on glass and paper as well as the endogenous components in Alstroemeria Yellow King flower petals. In a second mode of operation with a comparable sampling spot size, termed laser ablation/LESA®, the laser system was used to drill through, penetrate, or otherwise expose material beneath a solvent resistant surface. Once drilled, LESA® was effective in sampling soluble material exposed at that location on the surface. CONCLUSIONS Incorporating the capability for different laser ablation liquid capture spot sampling modes of operation into a LESA®-ready Advion TriVersa NanoMate enhanced the spot sampling spatial resolution of this device and broadened the surface types amenable to analysis to include absorbent and solvent-resistant materials. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA.

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 c. 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.

This paper reports on the development of a hybrid atmospheric pressure atomic force microscopy/mass spectrometry imaging system utilizing nanothermal analysis probes for thermal desorption surface sampling with subsequent atmospheric pressure chemical ionization and mass analysis. The basic instrumental setup and the general operation of the system were discussed, and optimized performance metrics were presented. The ability to correlate topographic images of a surface with atomic force microscopy and a mass spectral chemical image of the same surface, utilizing the same probe without moving the sample from the system, was demonstrated. Co-registered mass spectral chemical images and atomic force microscopy topographical images were obtained from inked patterns on paper as well as from a living bacterial colony on an agar gel. Spatial resolution of the topography images based on pixel size (0.2 μm × 0.8 μm) was better than the resolution of the mass spectral images (2.5 μm × 2.0 μm), which were limited by current mass spectral data acquisition rate and system detection levels.

Transmission geometry laser ablation directly into a formed liquid microjunction of a continuous flow liquid microjunction surface sampling probe/electrospray ionization emitter was utilized for molecular and elemental detection and mass spectrometry imaging. The ability to efficiently capture and ionize ablated material was demonstrated by the detection of various small soluble n-mers of polyaniline and silver ion solvent clusters formed from laser ablation of electropolymerized polyaniline and silver thin films, respectively. In addition, analysis of surfaces that contain soluble components was accomplished by coating or laminating the sample with an insoluble film to enable liquid junction formation without directly extracting material from the surface. The ability to perform mass spectrometry imaging at a spatial resolution of about 50 μm was illustrated by using laminated inked patterns on a microscope slide. In general, these data demonstrate at least an order of magnitude signal enhancement compared to the noncontact, laser ablation droplet capture-based surface sampling/ionization approaches that have been previously presented.

This paper describes the coupling of ambient laser ablation surface sampling, accomplished using a laser capture microdissection system, with atmospheric pressure chemical ionization mass spectrometry for high spatial resolution multimodal imaging. A commercial laser capture microdissection system was placed in close proximity to a modified ion source of a mass spectrometer designed to allow for sampling of laser ablated material via a transfer tube directly into the ionization region. Rhodamine 6G dye of red sharpie ink in a laser etched pattern as well as cholesterol and phosphatidylcholine in a cerebellum mouse brain thin tissue section were identified and imaged from full scan mass spectra. A minimal spot diameter of 8 m was achieved using the 10X microscope cutting objective with a lateral oversampling pixel resolution of about 3.7 m. Distinguishing between features approximately 13 m apart in a cerebellum mouse brain thin tissue section was demonstrated in a multimodal fashion including co-registered optical and mass spectral chemical images.

Rationale A continuous-flow liquid microjunction surface sampling probe extracts soluble material from surfaces for direct ionization and detection by mass spectrometry. Demonstrated here is the on-line coupling of such a probe with high-performance liquid chromatography/mass spectrometry (HPLC/MS) enabling extraction, separation and detection of small molecules and proteins from surfaces in a spatially resolved (~0.5 mm diameter spots) manner. Methods A continuous-flow liquid microjunction surface sampling probe was connected to a six-port, two-position valve for extract collection and injection to an HPLC column. A QTRAP® 5500 hybrid triple quadrupole linear ion trap equipped with a Turbo V™ ion source operated in positive electrospray ionization (ESI) mode was used for all experiments. The system operation was tested with the extraction, separation and detection of propranolol and associated metabolites from drug dosed tissues, caffeine from a coffee bean, cocaine from paper currency, and proteins from dried sheep blood spots on paper. Results Confirmed in the tissue were the parent drug and two different hydroxypropranolol glucuronides. The mass spectrometric response for these compounds from different locations in the liver showed an increase with increasing extraction time (5, 20 and 40 s). For on-line separation and detection/identification of extracted proteins from dried sheep blood spots, two major protein peaks dominated the chromatogram and could be correlated with the expected masses for the hemoglobin α and β chains. Conclusions Spatially resolved sampling, separation, and detection of small molecules and proteins from surfaces can be accomplished using a continuous-flow liquid microjunction surface sampling probe coupled on-line with HPLC/MS detection.

As the spatial resolution of mass spectrometry imaging technologies has begun to reach into the nanometer regime, finding readily available or easily made resolution reference materials has become particularly challenging for molecular imaging purposes. This paper describes the fabrication, characterization and use of vertical line array polymeric spatial resolution test patterns for nano-thermal analysis/atomic force microscopy/mass spectrometry chemical imaging.Test patterns of varied line width (0.7 or 1.0 μm) and spacing (0.7 or 1.0 μm) were created in an ~1-μm-thick poly(methyl methacrylate) thin film using electron beam lithography. The patterns were characterized by scanning electron microscopy, energy-dispersive X-ray spectroscopy, atomic force microscopy topography and nano-thermal analysis/mass spectrometry imaging.The efficacy of these polymeric test patterns for the advancement of chemical imaging techniques was illustrated by their use to judge the spatial resolution improvement achieved by heating the ionization interface of the current instrument platform. The spatial resolution of the mass spectral chemical images was estimated to be 1.4 μm, based on the ability to statistically distinguish 0.7-μm-wide lines separated by 0.7-μm-wide spacings in those images when the interface cross was heated to 200°C.This work illustrates that e-beam lithography is a viable method to create spatial resolution test patterns in a thin film of high molecular weight polymer to allow unbiased judgment of intra-laboratory advancement and/or inter-laboratory comparison of instrument advances in nano-thermal analysis/atomic force microscopy/mass spectrometry chemical imaging. Published in 2017. This article is a U.S. Government work and is in the public domain in the USA.

An "Open Access"-like mass spectrometric platform to fully utilize the simplicity of the manual open port sampling interface for rapid characterization of unprocessed samples by liquid introduction atmospheric pressure ionization mass spectrometry has been lacking. The in-house developed integrated software with a simple, small and relatively low-cost mass spectrometry system introduced here fills this void.Software was developed to operate the mass spectrometer, to collect and process mass spectrometric data files, to build a database and to classify samples using such a database. These tasks were accomplished via the vendor-provided software libraries. Sample classification based on spectral comparison utilized the spectral contrast angle method.Using the developed software platform near real-time sample classification is exemplified using a series of commercially available blue ink rollerball pens and vegetable oils. In the case of the inks, full scan positive and negative ion ESI mass spectra were both used for database generation and sample classification. For the vegetable oils, full scan positive ion mode APCI mass spectra were recorded. The overall accuracy of the employed spectral contrast angle statistical model was 95.3% and 98% in case of the inks and oils, respectively, using leave-one-out cross-validation.This work illustrates that an open port sampling interface/mass spectrometer combination, with appropriate instrument control and data processing software, is a viable direct liquid extraction sampling and analysis system suitable for the non-expert user and near real-time sample classification via database matching. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA.

Rationale Currently, the absolute quantitation aspects of droplet-based surface sampling for tissue analysis using a fully automated autosampler/high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) system have not been fully evaluated. Knowledge of extraction efficiency and its reproducibility is required to judge the potential of the method for absolute quantitation of analytes from tissue sections. Methods Adjacent tissue sections of propranolol-dosed mouse brain (10-μm-thick), kidney (10-μm-thick) and liver (8-, 10-, 16- and 24-μm-thick) were obtained. The absolute concentration of propranolol was determined in tissue punches from serial sections using standard bulk tissue extraction protocols and subsequent HPLC separations and MS/MS analysis. These values were used to determine propranolol extraction efficiency from the tissues with the droplet-based surface sampling approach. Results Extraction efficiency of propranolol using 10-μm-thick brain, kidney and liver tissues using droplet-based surface sampling varied between ~45 and 63%. The extraction efficiency decreased from ~65% to ~36% with liver thickness increasing from 8 μm to 24 μm. Selecting half of the samples as standards, the precision and accuracy of propranolol concentrations were determined for the other half of the samples that were employed as a quality control data set. The resulting precision (±15%) and accuracy (±3%) were within acceptable limits. Conclusions Quantitation of adjacent mouse tissue sections of different organs and of various thicknesses by droplet-based surface sampling in comparison with bulk extraction of tissue punches showed that extraction efficiency was incomplete using the former method, and that it depended on the organ and tissue thickness. However, once extraction efficiency was determined and applied, the droplet-based approach provided satisfactory quantitation accuracy and precision for assay validations. Thus, once the extraction efficiency was calibrated for a given tissue type, tissue thickness and drug, the droplet-based approach provides a non-labour-intensive and high-throughput means to acquire spatially resolved quantitative analysis of multiple samples of the same type. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA.

RATIONALE Ascorbic acid (AA) and folic acid (FA) are water-soluble vitamins and are usually fortified in food and dietary supplements. For the safety of human health, proper intake of these vitamins is recommended. Improvement in the analysis time required for the quantitative determination of these vitamins in food and nutritional formulations is desired. METHODS A simple and fast (~5 min) in-tube sample preparation was performed, independently for FA and AA, by mixing extraction solvent with a powdered sample aliquot followed by agitation, centrifugation, and filtration to recover an extract for analysis. Quantitative detection was achieved by flow-injection (1 μL injection volume) electrospray ionization tandem mass spectrometry (ESI-MS/MS) in negative ion mode using the method of standard addition. RESULTS The method of standard addition was employed for the quantitative estimation of each vitamin in a sample extract. At least 2 spiked and 1 non-spiked sample extract were injected in triplicate for each quantitative analysis. Given an injection-to-injection interval of approximately 2 min, about 18 min was required to complete the quantitative estimation of each vitamin. The concentration values obtained for the respective vitamins in the standard reference material (SRM) 3280 using this approach were within the statistical range of the certified values provided in the NIST Certificate of Analysis. The estimated limit of detections of FA and AA were 13 and 5.9 ng/g, respectively. CONCLUSIONS Flow-injection ESI-MS/MS was successfully applied for the rapid quantitation of FA and AA in SRM 3280 multivitamin/multielement tablets. Published 2012. This article is a US Government work and is in the public domain in the USA.

The rapid and direct analysis of the amount and spatial distribution of exogenous chloroquine (CHQ) and CHQ metabolites from tissue sections by liquid extraction surface sampling analysis coupled with tandem mass spectrometry (LESA-MS/MS) was demonstrated. LESA-MS/MS results compared well with previously published CHQ quantification data collected by organ excision, extraction and fluorescent detection. The ability to directly sample and analyze spatially resolved exogenous molecules from tissue sections with minimal sample preparation and analytical method development has the potential to facilitate the assessment of target tissue penetration of pharmaceutical compounds, to establish pharmacokinetic/pharmacodynamic relationships, and to complement established pharmacokinetic methods used in the drug discovery process during tissue distribution assessment.

The use of flow-injection electrospray ionization tandem mass spectrometry for rapid and high-throughput mass spectral analysis of selected B vitamins, viz., B1, B2, B3, B5, and B6, in nutritional formulations was demonstrated. A simple and rapid (~5 min) in-tube sample preparation was performed. Automated flow injection introduced 1 μL of the extracts directly into the mass spectrometer ion source without chromatographic separation. Sample-to-sample analysis time was 60 s. Quantitative capabilities of the flow-injection analysis were tested using the method of standard additions and SRM 3280. The quantity determined for each B vitamin in SRM 3280 was within the statistical range provided for the respective certified values. This approach was also applied to two different commercial vitamin supplement tablets and proved to be successful in the quantification of the selected B vitamins, as evidenced by an agreement with the label values and the results obtained using isotope dilution liquid chromatography/mass spectrometry.

A commercially available chip-based infusion nanoelectrospray ionization system was used to ionize metallo alkylporphyrins for mass spectrometric detection and structure elucidation by mass spectrometry. Different ionic forms of model compounds (nickel (II), vanadyl (II), copper (II), and cobalt (II) octaethylporphyrin) were created by using two different types of conductive pipette tips supplied with the device. These pipette tips provide the conductive contact to solution at which the electrolysis process inherent to electrospray takes places in the device. The original unmodified, bare carbon-impregnated plastic pipette tips were exploited to intentionally electrochemically oxidize (ionize) the porphyrins to form molecular radical cations for detection. Use of modified pipette tips, with a surface coating devised to inhibit analyte mass transport to the surface or slow the kinetics of the analyte electrochemical reactions, was shown to limit the ionic species observed in the mass spectra of these porphyrins largely, but not exclusively, to the protonated molecule. Under the conditions of these experiments, the effective upper potential limit for oxidation with the uncoated pipette tip was 1.1 V or less, and the coated pipette tips effectively prevented the oxidation of analytes with redox potentials greater than about 0.25 V. Product ion spectra of either molecular ionic species could be used to determine the alkyl chain length on the porphyrin macrocycle. The utility of this electrochemical ionization approach for the analysis of naturally occurring samples was demonstrated using nickel geoporphyrin fractions isolated from Gilsonite bitumen. Acquiring neutral loss spectra as a means to improve the specificity of detection in these complex natural samples was also illustrated.

A blotting method that transfers analytes separated on wettable high-performance thin-layer chromatography (HPTLC) plates to a hydrophobic reversed-phase C8 HPLTC plate suitable for analysis with a liquid microjunction surface sampling probe electrospray ionization mass spectrometry system was described and demonstrated. The simple blotting procedure transfers the analyte from the wettable plate to the topmost surface of a rigidly backed, easy-to-mount hydrophobic substrate that already has been proven viable for analysis by this sampling probe/mass spectrometry system. The utility of the approach was demonstrated by the analysis of a four-component peptide mixture originally separated on a ProteoChrom® HPTLC cellulose sheet and then blotted onto the reversed-phase HPTLC plate.

This paper describes the coupling of ambient pressure transmission geometry laser ablation with a liquid-phase sample collection into a continuous flow surface sampling probe/electrospray emitter for mass spectrometry based chemical imaging. The flow probe/emitter device was placed in close proximity to the surface to collect the sample plume produced by laser ablation. The sample collected was immediately aspirated into the probe and onto the electrospray emitter, ionized and detected with the mass spectrometer. Freehand drawn ink lines and letters and an inked fingerprint on microscope slides were analyzed. The circular laser ablation area was about 210 µm in diameter and under the conditions used in these experiments the spatial resolution, as determined by the size of the surface features distinguished in the chemical images, was about 100 µm. Published in 2011 by John Wiley & Sons, Ltd.

The direct separation of isomeric glucuronide metabolites from propranolol dosed tissue extracts by differential mobility spectrometry-mass spectrometry (DMS-MS) with the use of the polar gas-phase chemical modifier acetonitrile was demonstrated. The DMS gas-phase separation was able to resolve the isomeric metabolites with separation times on the order of milliseconds instead of minutes which is typically required when using pre-ionization chromatographic separation methods. Direct separation of isomeric metabolites from the complex tissue extract was confirmed by implementing a high-performance liquid chromatography (HPLC) separation prior to the DMS-MS analysis to pre-separate the species of interest. The ability to separate isomeric exogenous metabolites directly from a complex tissue extract is expected to facilitate the drug development process by increasing analytical throughput without the requirement for pre-ionization cleanup or separation strategies.

Direct liquid extraction based surface sampling, a technique previously demonstrated with continuous flow and autonomous pipette liquid microjunction surface sampling probes, has recently been implemented as a liquid extraction surface analysis (LESA) mode on a commercially available chip-based infusion nanoelectrospray ionization (nanoESI) system. In the present paper, the LESA mode was applied to the analysis of 96-well format custom-made solid-phase extraction (SPE) cards, with each well consisting of either a 1 or a 2mm diameter monolithic hydrophobic stationary phase. These substrate wells were conditioned, loaded with either single or multi-component aqueous mixtures, and read out using the commercial nanoESI system coupled to a hybrid triple quadrupole/linear ion trap mass spectrometer or a linear ion trap mass spectrometer. The extraction conditions, including extraction/nanoESI solvent composition, volume, and dwell times, were optimized in the analysis of targeted compounds. Limit of detection and quantitation as well as analysis reproducibility figures of merit were measured. Calibration data was obtained for propranolol using a deuterated internal standard which demonstrated linearity and reproducibility. A 10� increase in signal and cleanup of micromolar angiotensin II from a concentrated salt solution was demonstrated. In addition, a multicomponent herbicide mixture at ppb concentration levels was analyzed using MS 3 spectra for compound identification in the presence of isobaric interferences. Published in 2011 by John Wiley & Sons, Ltd.

Nanometer- scale proximal probe thermal desorption/electrospray ionization mass spectrometry (TD/ESI-MS) was demonstrated for molecular surface sampling of caffeine from a thin film using a 30 nm diameter nanothermal analysis (nano-TA) probe tip in an atomic force microscope (AFM) coupled via a vapor transfer line and ESI interface to a MS detection platform. Using a probe temperature of 350 °C and a spot sampling time of 30 s, conical desorption craters 250 nm in diameter and 100 nm deep were created as shown through subsequent topographical imaging of the surface within the same system. Automated sampling of a 5 × 2 array of spots, with 2 μm spacing between spots, and real time selective detection of the desorbed caffeine using tandem mass spectrometry was also demonstrated. Estimated from the crater volume (∼2 × 10(6) nm(3)), only about 10 amol (2 fg) of caffeine was liberated from each thermal desorption crater in the thin film. These results illustrate a relatively simple experimental setup and means to acquire in an automated fashion submicrometer scale spatial sampling resolution and mass spectral detection of materials amenable to TD. The ability to achieve MS-based chemical imaging with 250 nm scale spatial resolution with this system is anticipated.

Analyte electrolysis using a repetitively pulsed high voltage ion source was investigated and compared to that using a regular, continuously operating direct current high voltage ion source in electrospray ionization mass spectrometry. The extent of analyte electrolysis was explored as a function of the length and frequency of the high voltage pulse using the model compound reserpine in positive ion mode. Using +5 kV as the maximum high voltage amplitude, reserpine was oxidized to its 2, 4, 6 and 8-electron oxidation products when direct current high voltage was employed. In contrast, when using a pulsed high voltage, oxidation of reserpine was eliminated by employing the appropriate high voltage pulse length and frequency. This effect was caused by inefficient mass transport of the analyte to the electrode surface during the duration of the high voltage pulse and the subsequent relaxation of the emitter electrode/electrolyte interface during the time period when the high voltage was turned off. This mode of ESI source operation allows for analyte electrolysis to be quickly and simply switched on or off electronically via a change in voltage pulse variables.

This letter describes the coupling of ambient pressure transmission geometry laser ablation with a liquid phase sample collection method for surface sampling and ionization with subsequent mass spectral analysis. A commercially available autosampler was adapted to produce a liquid droplet at the end of the syringe injection needle while in close proximity to the surface to collect the sample plume produced by laser ablation. The sample collection was followed by either flow injection or a high-performance liquid chromatography (HPLC) separation of the extracted components and detection with electrospray ionization mass spectrometry (ESI-MS). To illustrate the analytical utility of this coupling, thin films of a commercial ink sample containing rhodamine 6G and of mixed isobaric rhodamine B and 6G dyes on glass microscope slides were analyzed. The flow injection and HPLC-ESI-MS analysis revealed successful laser ablation, capture, and with HPLC, the separation of the two compounds. The ablated circular area was about 70 μm in diameter for these experiments. The spatial sampling resolution afforded by the laser ablation, as well as the ability to use sample processing methods like HPLC between the sample collection and ionization steps, makes this combined surface sampling/ionization technique a highly versatile analytical tool.

The electrolysis inherent to the operation of the electrospray ionization (ESI) source used with mass spectrometry (MS) is a well-known attendant effect of generating unipolar spray droplets and may affect the analysis of the analyte of interest. Undesirable electrolysis of an analyte may be prevented by limiting the emitter electrode current and/or the mass transport characteristics of the system. However, these ways to avoid analyte electrolysis may not be applcable in all ESI-MS experiments. For example, in the case of specific nanospray systems (e.g. the wire-in-a-capillary bulk-loaded or chip-based tip-loaded nanospray configurations), the solution flow rate is fixed in the 50-500 nL/min range and the electrode surface to volume ratio is large presenting a very effcient analyte to electrode mass transport configuration. In these situations, control over the interfacial potential of the working electrode via homogeneous or traditional heterogeneous (sacrificial metal) redox buffering is a possible way to prevent analyte electrolysis. However, byproducts of these redox buffering approaches can appear in the mass spectra and/or they can chemically alter the analyte. For example, the main reason for using hydroquinone as a homogeneous redox buffer, in addition to its relatively low oxidation potential, is that neither the original compound nor itsmore » oxidation product benzoquinone can be detected directly by ESI-MS. However, benzoquinone can alter analytes with thiol functional groups by reacting with those groups via a 1,4-Michael addition.« less

In this work, a commercially available autosampler was adapted to perform direct liquid microjunction (LMJ) surface sampling followed by a high-pressure liquid chromatography (HPLC) separation of the extract components and detection with electrospray ionization mass spectrometry (ESI-MS). To illustrate the utility of coupling a separation with this direct liquid extraction based surface sampling approach, four different organs (brain, lung, kidney, and liver) from whole-body thin tissue sections of propranolol dosed and control mice were examined. The parent drug was observed in the chromatograms of the surface sampling extracts from all the organs of the dosed mouse examined. In addition, two isomeric phase II metabolites of propranolol (an aliphatic and an aromatic hydroxypropranolol glucuronide) were observed in the chromatograms of the extracts from lung, kidney, and liver. Confirming the presence of one or the other or both of these glucuronides in the extract from the various organs was not possible without the separation. These drug and metabolite data obtained using the LMJ surface sampling/HPLC-MS method and the results achieved by analyzing similar samples by conventional extraction of the tissues and subsequent HPLC-MS analysis were consistent. The ability to directly and efficiently sample from thin tissue sections via a liquid extraction and then perform a subsequent liquid phase separation increases the utility of this liquid extraction surface sampling approach.

Planar separations, which include thin layer chromatography and gel electrophoresis, are in widespread use as important and powerful tools for conducting separations of complex mixtures. To increase the utility of planar separations, new methods are needed that allow in situ characterization of the individual components of the separated mixtures. A large number of atmospheric pressure surface sampling and ionization techniques for use with mass spectrometry have emerged in the past several years, and several have been investigated as a means for mass spectrometric read-out of planar separations. In this article, we review the atmospheric pressure surface sampling and ionization techniques that have been used for the read-out of planar separation media. For each technique, we briefly explain the operational basics and discuss the analyte type for which it is appropriate and some specific applications from the literature.

An atmospheric pressure proximal probe thermal desorption sampling method coupled with secondary ionization by electrospray or atmospheric pressure chemical ionization was demonstrated for the mass spectrometric analysis of a diverse set of compounds (dyestuffs, pharmaceuticals, explosives and pesticides) separated on various high-performance thin-layer chromatography plates. Line scans along or through development lanes on the plates were carried out by moving the plate relative to a stationary heated probe positioned close to or just touching the stationary phase surface. Vapors of the compounds thermally desorbed from the surface were drawn into the ionization region of a combined electrospray ionization/atmospheric pressure chemical ionization source where they merged with reagent ions and/or charged droplets from a corona discharge or an electrospray emitter and were ionized. The ionized components were then drawn through the atmospheric pressure sampling orifice into the vacuum region of a triple quadrupole mass spectrometer and detected using full scan, single ion monitoring, or selected reaction monitoring mode. Studies of variable parameters and performance metrics including the proximal probe temperature, gas flow rate into the ionization region, surface scan speed, read-out resolution, detection limits, and surface type are discussed.

A fully automated liquid extraction-based surface sampling device utilizing an Advion NanoMate chip-based infusion nanoelectrospray ionization system is reported. Analyses were enabled for discrete spot sampling by using the Advanced User Interface of the current commercial control software. This software interface provided the parameter control necessary for the NanoMate robotic pipettor to both form and withdraw a liquid microjunction for sampling from a surface. The system was tested with three types of analytically important sample surface types, viz., spotted sample arrays on a MALDI plate, dried blood spots on paper, and whole-body thin tissue sections from drug dosed mice. The qualitative and quantitative data were consistent with previous studies employing other liquid extraction-based surface sampling techniques.

A simple and automated spot sampling operation mode for a liquid microjunction surface sampling probe/electrospray ionization mass spectrometry (LMJ-SSP/ESI-MS) system is reported. Prior manual and automated spot sampling methods with this probe relied on a careful, relatively slow alignment of the probe and surface distance (20 microm spacing) to form the probe-to-surface liquid microjunction critical to successful surface sampling. Moreover, sampling multiple spots required retraction of the surface from the probe and a repeat of this careful probe-to-surface distance alignment at the next sampling position. With the method described here, the probe was not positioned as close to the surface, the exact probe-to-surface positioning was found to be less critical (spanning distances from about 100-300 microm), and this distance was not altered during the sampling of an entire array of sample spots. With the probe positioned within the appropriate distance from the surface, the liquid microjunction was formed by letting the liquid from the sampling end of the probe extend out from the probe to the surface. This was accomplished by reducing the self-aspiration liquid flow rate of the probe to a value less than the volume flow rate pumped into the probe. When the self-aspiration rate of the probe was subsequently increased, analytes on the surface that dissolved at the liquid microjunction were aspirated back into the probe with the liquid that created the liquid microjunction and electrosprayed. Presented here are the basics of this new sampling mode, as well as data that illustrate the potential analytical capabilities of the device to conduct high-throughput quantitative analysis.

This paper reports on the conversion of a liquid microjunction surface sampling probe (LMJ-SSP) into a two-electrode electrochemical cell using a conductive sample surface and the probe as the two electrodes with an appropriate battery powered circuit. With this LMJ-SSP, two-electrode cell arrangement, tagging of analyte thiol functionalities (in this case peptide cysteine residues) with hydroquinone tags was initiated electrochemically using a hydroquinone-doped solution when the analyte either was initially in solution or was sampled from a surface. Efficient tagging (∼90%), at flow rates of 5–10 µL/min, could be achieved for up to at least two cysteines on a peptide. The high tagging efficiency observed was explained with a simple kinetic model. In general, the incorporation of a two-electrode electrochemical cell, or other multiple electrode arrangement, into the LMJ-SSP is expected to add to the versatility of this approach for surface sampling and ionization coupled with mass spectrometric detection. Published in 2009 by John Wiley & Sons, Ltd.

Desorption electrospray ionization mass spectrometry (DESI-MS) was demonstrated as a method to detect and identify peptides from two-dimensional separations of cytochrome c and myoglobin tryptic digests on ProteoChrom HPTLC Cellulose sheets. Data-dependent tandem mass spectra were acquired during lane scans across the TLC plates. Peptides and the corresponding proteins were identified using a protein database search software. Two-dimensional distributions of identified peptides were mapped for each separated protein digest. Sequence coverages for cytochrome c and myoglobin were 81 and 74%, respectively. These compared well with those determined using the more standard HPLC/ESI-MS/MS approach (89 and 84%, respectively). Preliminary results show that use of more sensitive instrumentation has the potential for improved detection of peptides with low Rf values and improvement in sequence coverage. However, less multiple charging and more sodiation were seen in HPTLC/DESI-MS spectra relative to HPLC/ESI-MS spectra, which can affect peptide identification by MS/MS. Methods to increase multiple charging and reduce the extent of sodiation are currently under investigation. Published in 2008 by John Wiley & Sons, Ltd.