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The bile salt export pump (BSEP) assay is widely used to evaluate the potential for drug-induced liver injury (DILI) early in the drug discovery process. While traditional liquid chromatography-mass spectrometry (LC-MS)-based approaches have been utilized for BSEP activity testing, they have intrinsic limitations in either throughput or the requirement for sample preparation and are difficult to scale up in order to screen drug candidates. Here we demonstrate the use of two different high-throughput MS methods based on solid-phase extraction (SPE) and desorption electrospray ionization (DESI) for high-throughput BSEP activity assessment in a label-free manner, with minimal needs for sample workup, at sampling rates of ∼11 and ∼5.5 s/sample, respectively. Both approaches were validated, compared, and successfully applied to the evaluation of 96 drug candidates for the inhibition of taurocholic acid (TCA) transport using BSEP vesicles., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

The current opioid epidemic has incentivized the discovery of new non-addictive analgesics, a process that requires the screening of opioid receptor binding, traditionally performed using radiometric assays. Here we describe a label-free alternative based on high-throughput (1 Hz) ambient mass spectrometry for screening the receptor binding of new opioid analogues.

Chemical reactions in micrometer-sized droplets can be accelerated by up to six orders of magnitude. However, this acceleration factor (ratio of rate constants relative to bulk) drops to less than 10 for millimeter-sized droplets due to the reduction in surface/volume ratio. To enhance the acceleration in millimeter-sized droplets, we use a new synthesis platform that directly doses reagent vapor onto the reaction droplet surface from a second levitated droplet. Using Katritzky transamination as a model reaction, we made quantitative measurements on size-controlled vapor-dosed droplets, revealing a 31-fold increase in reaction rate constants when examining the entire droplet contents. This enhancement is attributed to a greater reaction rate constant in the droplet surface region (estimated as 10 5 times greater than that for the bulk). The capability for substantial reaction acceleration in large droplets highlights the potential for rapid synthesis of important chemicals at useful scales. For example, we successfully prepared 23 pyridinium salts within minutes. This efficiency positions droplets as an exceptional platform for rapid, in situ catalyst synthesis. This is illustrated by the preparation of pyridinium salts as photocatalysts and their subsequent use in mediation of amine oxidation both within the same droplet., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The development and performance of two mass spectrometry (MS) workflows for the intraoperative diagnosis of isocitrate dehydrogenase (IDH) mutations in glioma is implemented by independent teams at Mayo Clinic, Jacksonville, and Huashan Hospital, Shanghai. The infiltrative nature of gliomas makes rapid diagnosis necessary to guide the extent of surgical resection of central nervous system (CNS) tumors. The combination of tissue biopsy and MS analysis used here satisfies this requirement. The key feature of both described methods is the use of tandem MS to measure the oncometabolite 2-hydroxyglutarate (2HG) relative to endogenous glutamate (Glu) to characterize the presence of mutant tumor. The experiments i) provide IDH mutation status for individual patients and ii) demonstrate a strong correlation of 2HG signals with tumor infiltration. The measured ratio of 2HG to Glu correlates with IDH-mutant (IDH-mut) glioma ( P < 0.0001) in the tumor core data of both teams. Despite using different ionization methods and different mass spectrometers, comparable performance in determining IDH mutations from core tumor biopsies was achieved with sensitivities, specificities, and accuracies all at 100%. None of the 31 patients at Mayo Clinic or the 74 patients at Huashan Hospital were misclassified when analyzing tumor core biopsies. Robustness of the methodology was evaluated by postoperative re-examination of samples. Both teams noted the presence of high concentrations of 2HG at surgical margins, supporting future use of intraoperative MS to monitor for clean surgical margins. The power of MS diagnostics is shown in resolving contradictory clinical features, e.g., in distinguishing gliosis from IDH-mut glioma., Competing Interests: Competing interests statement:Z.O. is the founder of PurSpec Technologies, which is developing miniature mass spectrometry systems.

Charged microdroplets accelerate mineral disintegration.

Exploration of the unique chemical properties of interfaces can unlock new understanding. A striking example is the finding of accelerated reactions, particularly spontaneous oxidation reactions, that occur without assistance of catalysts or external oxidants at the air interface of both aqueous and organic solutions (provided they contain some water). This finding opened a new area of interfacial chemistry but also caused heated debate regarding the primary chemical species responsible for the observed oxidation. An overview of the literature covering oxidation in microdroplets with air interfaces is provided, together with a critical examination of previous findings and hypotheses. The water radical cation/radical anion pair, formed spontaneously and responsible for the electric field at or near the droplet/air interface, is suggested to constitute the primary redox species. Mechanisms of accelerated microdroplet reactions are critically discussed and it is shown that hydroxyl radical/hydrogen peroxide formation in microdroplets does not require that these species be the primary oxidant. Instead, we suggest that hydroxyl radical and hydrogen peroxide are the products of water radical cation decay in water. The importance of microdroplet chemistry in the prebiotic environment is sketched briefly and the role of partial solvation in reaction acceleration is noted., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

This brief cautionary note reports a failure in a common and useful assumption, namely, that the isotopes of the elements occur in their natural abundance ratios in commercially sourced organic compounds. Some commercial sources of tris(pentafluorophenyl)borane, B(C 6 F 5 ) 3 , show severely depleted 10 B, while materials from other suppliers display natural isotopic abundances. The depletion varies from lot-to-lot, and it was confirmed by inductively coupled plasma (ICP) mass spectrometry. The isotope 10 B is used in the nuclear power industry, as a neutron absorber in the power control rods. It is speculated that the residual 11 B generated when preparing 10 B-enriched boron carbide for control rod use, provides 11 B-rich raw material that is then used for commercial B(C 6 F 5 ) 3 synthesis.

Peptide formation from amino acids is thermodynamically unfavorable but a recent study provided evidence that the reaction occurs at the air/solution interfaces of aqueous microdroplets. Here, we show that i) the suggested amino acid complex in microdroplets undergoes dehydration to form oxazolone; ii) addition of water to oxazolone forms the dipeptide; and iii) reaction of oxazolone with other amino acids forms tripeptides. Furthermore, the chirality of the reacting amino acids is preserved in the oxazolone product, and strong chiral selectivity is observed when converting the oxazolone to tripeptide. This last fact ensures that optically impure amino acids will undergo chain extension to generate pure homochiral peptides. Peptide formation in bulk by wet-dry cycling shares a common pathway with the microdroplet reaction, both involving the oxazolone intermediate., Competing Interests: Competing interests statement:The authors declare no competing interest.

Biothreat detection has continued to gain attention. Samples suspected to fall into any of the CDC's biothreat categories require identification by processes that require specialized expertise and facilities. Recent developments in analytical instrumentation and machine learning algorithms offer rapid and accurate classification of Gram-positive and Gram-negative bacterial species. This is achieved by analyzing the negative ions generated from bacterial cell extracts with a modified linear quadrupole ion-trap mass spectrometer fitted with two-dimensional tandem mass spectrometry capabilities (2D MS/MS). The 2D MS/MS data domain of a bacterial cell extract is recorded within five s using a five-scan average after sample preparation by a simple extraction. Bacteria were classified at the species level by their lipid profiles using the random forest, k-nearest neighbor, and multilayer perceptron machine learning models. 2D MS/MS data can also be treated as image data for use with image recognition algorithms such as convolutional neural networks. The classification accuracy of all models tested was greater than 99%. Adding to previously published work on the 2D MS/MS analysis of bacterial growth and the profiling of sporulating bacteria, this study demonstrates the utility and information-rich nature of 2D MS/MS in the identification of bacterial pathogens at the species level when coupled with machine learning.

This study uses a rapid tandem mass-spectrometry method to determine water content in complex organic solutions. Emphasis is placed on trace-water analysis by a fast and accurate alternative to the Karl-Fischer method. In this new method, water is captured by a charge-labeled molecular probe. Water binds strongly with high specificity to the strongly electrophilic aldehyde site in a charge-labelled molecule (N-methylpyridinium); competitive binding by other analytes is effectively discriminated against in the mass-measurement step. Quantitative determinations are made over a wide concentration range, 0.001 % (10 ppm) to 99 %, with better than 10 % relative standard deviation, along with short (1 min) analysis times using small sample volumes (several μL). Applications include water measurement in simple organic solvents, for example, deuterated solvents, as well as in complex mixtures, for example, organic reaction mixtures. Additionally, this method allows for water monitoring in levitated droplets. Mechanistic investigations into the impact of water on important chemical processes in organic synthesis and environmental science are reported., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The low-temperature plasma (LTP) probe is a common plasma-based source used for ambient desorption-ionization mass spectrometry (MS). While the LTP probe has been characterized in detail with MS, relatively few studies have used optical spectroscopy. In this paper, two-dimensional (2D) imaging at selected wavelengths is used to visualize important species in the LTP plasma jet. First, 2D steady-state images of the LTP plume for N 2 + (391.2 nm), He I (706.5 nm), and N 2 (337.1 nm) emissions were recorded under selected plasma conditions. Second, time-resolved 2D emission maps of radiative species in the LTP plasma jet were recorded through the use of a 200 ns detection gate and varying gate delays with respect to the LTP trigger pulse. Emission from He I, N 2 + , and N 2 in the plasma jet region was found to show a transient behavior (often referred to as plasma bullets) lasting only a few microseconds. The N 2 + and He I maps were highly correlated in spatial and temporal structure. Further, emission from N 2 showed two maxima in time, one before and one after the maximum emission for N 2 + and He I, due to an initial electronic excitation wave and ion-electron recombination, respectively. Third, the interaction of the LTP probe with a sample substrate and an electrically grounded metallic needle was studied. Emission from a fluorophore on the sample substrate showed an initial photon-induced excitation from plasma-generated photons followed by electronic excitation by other plasma species. The presence of a grounded needle near the plasma jet significantly extended the plasma jet lifetime and also generated a long-lived corona discharge on the needle. The effect of LTP operating parameters on emission spectra was correlated with mass-spectral results including reagent-ion signals. Lastly, five movies provide a side-by-side comparison of the temporal behavior of emitting species and insights into the interactions of the emission clouds with a sample surface as well as an external needle. Temporally and spatially resolved imaging provided insights into important processes in the LTP plasma jet, which will help improve analyte ion sampling in LTP-MS.

It is argued that each of the three key steps in drug discovery, (i) reaction screening to find successful routes to desired drug candidates, (ii) scale up of the synthesis to produce amounts adequate for testing, and (iii) bioactivity assessment of the candidate compounds, can all be performed using mass spectrometry (MS) in a sequential fashion. The particular ionization method of choice, desorption electrospray ionization (DESI), is both an analytical technique and a procedure for small-scale synthesis. It is also highly compatible with automation, providing for high throughput in both synthesis and analysis. Moreover, because accelerated reactions take place in the secondary DESI microdroplets generated from individual reaction mixtures, this allows either online analysis by MS or collection of the synthetic products by droplet deposition. DESI also has the unique advantage, amongst spray-based MS ionization methods, that complex buffered biological solutions can be analyzed directly, without concern for capillary blockage. Here, all these capabilities are illustrated, the unique chemistry at droplet interfaces is presented, and the possible future implementation of DESI-MS based drug discovery is discussed.

Late-stage diversification of drug molecules is an important strategy in drug discovery that can be facilitated by reaction screening using high-throughput experimentation. Here we present a rapid method for functionalizing bioactive molecules based on accelerated reactions in microdroplets. Reaction mixtures are nebulized at throughputs better than 1 reaction/second and the accelerated reactions occurring in the microdroplets are followed by desorption electrospray ionization mass spectrometry (DESI-MS). Because the accelerated reactions occur on the millisecond timescale, they allow an overall screening throughput of 1 Hz working at the low nanogram scale. Using this approach, an opioid agonist (PZM21) and an antagonist (naloxone) were diversified using three reactions important in medicinal chemistry: sulfur fluoride exchange (SuFEx) click reactions, imine formation reactions, and ene-type click reactions. Some 269 functionalized analogs of naloxone and PZM21 were generated and characterized by tandem mass spectrometry (MS/MS) after screening over 500 reactions., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

We demonstrate proof-of-concept for point-of-care assessment of long-term alcohol consumption by measuring phosphatidylethanol in blood/dried blood spots with nano-electrospray ionization and MS/MS using a miniature mass spectrometer. 'Abstinence', 'moderate', and 'chronic' consumption could be distinguished rapidly for both sample types, and quantitative performance was obtained with blood (LoQ-100 ng mL -1 ).

The sulfur fluoride exchange (SuFEx) reaction is significant in drug discovery, materials science, and chemical biology. Conventionally, it involves installation of SO 2 F followed by fluoride exchange by a catalyst. We report catalyst-free Aza-Michael addition to install SO 2 F and then SuFEx reaction with amines, both occurring in concert, in microdroplets under ambient conditions. The microdroplet reaction is accelerated by a factor of ∼10 4 relative to the corresponding bulk reaction. We suggest that the superacidic microdroplet surface assists SuFEx reaction by protonating fluorine to create a good leaving group. The reaction scope was established by performing individual reactions in microdroplets of 18 amines in four solvents and confirmed using high-throughput desorption electrospray ionization experiments. The study demonstrates the value of microdroplet-assisted accelerated reactions in combination with high-throughput experimentation for characterization of reaction scope., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Lipidomic and metabolomic profiles of sporulated and vegetative Bacillus subtilis and Bacillus thuringiensis from irradiated lysates were recorded using a quadrupole ion trap mass spectrometer modified to perform two-dimensional tandem mass spectrometry (2D MS/MS). The 2D MS/MS data domains, acquired using a 1.2 s scan of negative ions generated by nanoelectrospray ionization of microwave irradiated spores, showed the presence of dipicolinic acid (DPA) as well as various lipids. Aside from microwave radiation to extract DPA and lipids from spores, sample preparation was minimal. Characteristic lipid and metabolic profiles were observed using 10 7 ─10 8 cells of the two Bacillus species. Major features of the lipid profiles observed for the vegetative states included sets of phosphatidylglycerol (PG) lipids. Product ion spectra were extracted from the 2D MS/MS data, and they provided structural information on the fatty acid components of the PG lipids. The study demonstrates the flexibility, speed, and informative power of metabolomic and lipidomic fingerprinting for identifying the presence of spore-forming biological agents using 2D MS/MS as a rapid profiling screening method.

Tissue microarrays (TMAs) are commonly used for the rapid analysis of large numbers of tissue samples, often in morphological assessments but increasingly in spectroscopic analysis, where specific molecular markers are targeted via immunostaining. Here we report the use of an automated high-throughput system based on desorption electrospray ionization (DESI) mass spectrometry (MS) for the rapid generation and online analysis of high-density (6144 samples/array) TMAs, at rates better than 1 sample/second. Direct open-air analysis of tissue samples (hundreds of nanograms) not subjected to prior preparation, plus the ability to provide molecular characterization by tandem mass spectrometry (MS/MS), make this experiment versatile and applicable to both targeted and untargeted analysis in a label-free manner. These capabilities are demonstrated in a proof-of-concept study of frozen brain tissue biopsies where we showcase (i) a targeted MS/MS application aimed at identification of isocitrate dehydrogenase mutation in glioma samples and (ii) an untargeted MS tissue type classification using lipid profiles and correlation with tumor cell percentage estimates from histopathology. The small sample sizes and large sample numbers accessible with this methodology make for a powerful analytical system that facilitates the identification of molecular markers for later use in intraoperative applications to guide precision surgeries and ultimately improve patient outcomes., (© 2022. The Author(s).)

Amide bond formation, the essential condensation reaction underlying peptide synthesis, is hindered in aqueous systems by the thermodynamic constraints associated with dehydration. This represents a key difficulty for the widely held view that prebiotic chemical evolution leading to the formation of the first biomolecules occurred in an oceanic environment. Recent evidence for the acceleration of chemical reactions at droplet interfaces led us to explore aqueous amino acid droplet chemistry. We report the formation of dipeptide isomer ions from free glycine or L-alanine at the air-water interface of aqueous microdroplets emanating from a single spray source (with or without applied potential) during their flight toward the inlet of a mass spectrometer. The proposed isomeric dipeptide ion is an oxazolidinone that takes fully covalent and ion-neutral complex forms. This structure is consistent with observed fragmentation patterns and its conversion to authentic dipeptide ions upon gentle collisions and for its formation from authentic dipeptides at ultra-low concentrations. It also rationalizes the results of droplet fusion experiments that show that the dipeptide isomer facilitates additional amide bond formation events, yielding authentic tri- through hexapeptides. We propose that the interface of aqueous microdroplets serves as a drying surface that shifts the equilibrium between free amino acids in favor of dehydration via stabilization of the dipeptide isomers. These findings offer a possible solution to the water paradox of biopolymer synthesis in prebiotic chemistry.

Microdroplets show unique chemistry, especially in their intrinsic redox properties, and to this we here add a case of simultaneous and spontaneous oxidation and reduction. We report the concurrent conversions of several phosphonates to phosphonic acids by reduction (R-P → H-P) and to pentavalent phosphoric acids by oxidation. The experimental results suggest that the active reagent is the water radical cation/anion pair. The water radical cation is observed directly as the ionized water dimer while the water radical anion is only seen indirectly though the spontaneous reduction of carbon dioxide to formate. The coexistence of oxidative and reductive species in turn supports the proposal of a double-layer structure at the microdroplet surface, where the water radical cation and radical anion are separated and accumulated., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Electrospray deposition of copper salt-containing microdroplets onto the liquid surface of an electrically grounded reaction mixture leads to the formation of Cu nanoclusters, which then catalyze the azide-alkyne cycloaddition (AAC) reaction to form triazoles. This method of in situ nanocatalyst preparation provided 17 times higher catalytic activity compared to that in the conventional catalytic reaction. The gentle landing of the Cu-containing droplets onto the liquid surface forms a thin film of catalyst which promotes the heterogeneous AAC reaction while showing diffusion-controlled kinetics. UV-vis spectral characterization confirms that the catalyst is comprised of Cu nanoclusters. This unique catalytic strategy was validated using several substrates and the corresponding products were confirmed by tandem mass spectrometry (MS/MS) analysis., (© 2022 The Authors. ChemPlusChem published by Wiley-VCH GmbH.)

Reactions in microdroplets can be accelerated and can present unique chemistry compared to reactions in bulk solution. Here, we report the accelerated oxidation of aromatic sulfones to sulfonic acids in microdroplets under ambient conditions without the addition of acid, base, or catalyst. The experimental data suggest that the water radical cation, (H 2 O) +• , derived from traces of water in the solvent, is the oxidant. The substrate scope of the reaction indicates the need for a strong electron-donating group (e.g., p -hydroxyl) in the aromatic ring. An analogous oxidation is observed in an aromatic ketone with benzoic acid production. The shared mechanism is suggested to involve field-assisted ionization of water at the droplet/air interface, its reaction with the sulfone (M) to form the radical cation adduct, (M + H 2 O) +• , followed by 1,2-aryl migration and C-O cleavage. A remarkably high reaction rate acceleration (∼10 3 ) and regioselectivity (∼100-fold) characterize the reaction.

The sulfotransferase (SULT) 2B1b, which catalyzes the sulfonation of 3β-hydroxysteroids, has been identified as a potential target for prostate cancer treatment. However, a major limitation for SULT2B1b-targeted drug discovery is the lack of robust assays compatible with high-throughput screening and inconsistency in reported kinetic data. For this reason, we developed a novel label-free assay based on high-throughput (>1 Hz) desorption electrospray ionization mass spectrometry (DESI-MS) for the direct quantitation of the sulfoconjugated product (CV<10 %; <1 ng analyte). The performance of this DESI-based assay was compared against a new fluorometric coupled-enzyme method that we also developed. Both methodologies provided consistent kinetic data for the reaction of SULT2B1b with its major substrates, indicating the affinity trend pregnenolone>DHEA>cholesterol, for both the phospho-mimetic and wild-type SULT2B1b forms. The novel DESI-MS assay developed here is likely generalizable to other drug discovery efforts and is particularly promising for identification of SULT2B1b inhibitors with potential as prostate cancer therapeutics., (© 2022 Wiley-VCH GmbH.)

HIV prevention and treatment with injectable cabotegravir and/or rilpivirine administered once every 4 to 8 weeks is an attractive alternative to daily therapy. Prescribed dosage and drug concentrations in plasma are based on patient data collected in clinical trials, but actual patients are expected to exhibit more variability in drug concentrations, which is important to quantify. Here, we demonstrate the first quantitative point-of-care assay with a miniature mass spectrometer to assess these drug concentrations in whole blood. Quantitative performance is obtained using paper spray ionization in combination with tandem mass spectrometry (MS/MS) in the clinically relevant concentration range of both drugs. Limits of quantitation (LoQs) of cabotegravir and rilpivirine are measured to be 750 ng/mL and 20 ng/mL, respectively. The assay turnaround time is < 4 min, and strong linear relationships are established between MS/MS responses and concentration, with percentage of relative standard deviations (RSDs) that are <15% at concentrations above the LoQs. The speed, portability, low power consumption, and specificity offered by the miniature instrument should make it an appropriate platform for measuring drug concentrations in a walk-in clinic using small volumes of patient blood., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

Spontaneous oxidation of compounds containing diverse X=Y moieties (e.g., sulfonamides, ketones, esters, sulfones) occurs readily in organic-solvent microdroplets. This surprising phenomenon is proposed to be driven by the generation of an intermediate species [M+H 2 O] +· : a covalent adduct of water radical cation (H 2 O +· ) with the reactant molecule (M). The adduct is observed in the positive ion mass spectrum while its formation in the interfacial region of the microdroplet (i.e., at the air-droplet interface) is indicated by the strong dependence of the oxidation product formation on the spray distance (which reflects the droplet size and consequently the surface-to-volume ratio) and the solvent composition. Importantly, based on the screening of a ca. 21,000-compound library and the detailed consideration of six functional groups, the formation of a molecular adduct with the water radical cation is a significant route to ionization in positive ion mode electrospray, where it is favored in those compounds with X=Y moieties which lack basic groups. A set of model monofunctional systems was studied and in one case, benzyl benzoate, evidence was found for oxidation driven by hydroxyl radical adduct formation followed by protonation in addition to the dominant water radical cation addition process. Significant implications of molecular ionization by water radical cations for oxidation processes in atmospheric aerosols, analytical mass spectrometry and small-scale synthesis are noted., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Qiu, Morato, Huang and Cooks.)

The growth of the bacterium E. coli was monitored by targeting the phospholipid constituents through desorption electrospray ionization and characterizing individual sets of isomers by recording the full 2D MS/MS data domain in a single scan of a modified quadrupole ion trap mass spectrometer. The experiments tested the applicability of the new instrumental capabilities which include sample interrogation at the molecular level for multiple components at speeds of <10 seconds/sample. The major lipids observed were phosphatidylethanolamines and phosphatidylglycerols and the growth experiment showed fatty acid chain modification from alkene to cyclopropyl groups over time. Notably, these novel MS scans were also performed using desorption electrospray ionization (DESI) to quickly sample complex mixtures without pre-separation. This demonstration experiment has implications for the value of ambient ionization mass spectrometry for monitoring biological systems on physiologically relevant timescales.

Palladium-catalyzed Suzuki-Miyaura (SM) coupling is widely utilized in the construction of carbon-carbon bonds. In this study, nanoelectrospray ionization mass spectrometry (nanoESI-MS) is applied to simultaneously monitor precatalysts, catalytic intermediates, reagents, and products of the SM cross-coupling reaction of 3-Br-5-Ph-pyridine and phenylboronic acid. A set of Pd cluster ions related to the monoligated Pd (0) active catalyst is detected, and its deconvoluted isotopic distribution reveals contributions from two neutral molecules. One is assigned to the generally accepted Pd(0) active catalyst, seen in MS as the protonated molecule, while the other is tentatively assigned to an oxidized catalyst which was found to increase as the reaction proceeds. Oxidative stress testing of a synthetic model catalyst 1,5-cyclooctadiene Pd XPhos (COD-Pd-XPhos) performed using FeCl 3 supported this assignment. The formation and conversion of the oxidative addition intermediate during the catalytic cycle was monitored to provide information on the progress of the transmetalation step., (© 2022 Wiley-VCH GmbH.)

Late-stage functionalization (LSF) of drug molecules is an approach to generate modified molecules that retain functional groups present in the active drugs. Here, we report a study that seeks to characterize the potential value of high-throughput desorption electrospray ionization mass spectrometry (HT DESI-MS) for small-scale rapid LSF. In conventional route screening, HT-based DESI-MS provides contactless, rapid analysis, reliable and reproducible data, minimal sample requirement, and exceptional tolerance to high salt concentrations. Ezetimibe (E), an established hypertension drug, is targeted for modification by LSF. C-H alkenylation and azo-click reactions are utilized to explore this approach to synthesis and analytical characterization. The effect of choice of reactant, stoichiometry, catalyst, and solvent are studied for both reactions using high throughput DESI-MS experiments. Optimum conditions for the formation of LSF products are established with identification by tandem mass spectrometry (MS/MS)., (© 2021 Wiley-VCH GmbH.)

Spore lysis of Bacillus species is achieved by brief (1 min) microwave irradiation while tandem mass spectrometry (MS/MS) allows identification of the characteristic spore marker, dipicolinic acid. This rapid measurement, made on 10 5 -10 8 spores, has significant implications for biothreat recognition.

Advances in ambient ionization techniques have facilitated the direct analysis of complex mixtures without sample preparation. Significant attention has been given to innovating ionization methods so that multiple options are now available, allowing for ready selection of the best methods for particular analyte classes. These ambient techniques are commonly implemented on benchtop systems, but their potential application with miniature mass spectrometers for in situ measurements is even more powerful. These applications require that attention be paid to tailoring the mass spectrometric methodology for the on-site operation. In this study, combinations of scan modes are employed to efficiently determine what tandem mass spectrometry (MS/MS) operations are most useful for detecting sulfonamides using a miniature ion trap after ionization. First, mixtures of representative sulfonamide antibiotics were interrogated using a 2D MS/MS scan on a benchtop ion trap in order to determine which class-specific fragments (ionic or neutral) are shared between the sulfonamides and thus have diagnostic value. Then, three less-used combination scans were recorded: (i) a simultaneous precursor ion scan was used to detect both analytes and an internal standard in a single ion injection event to optimize quantitative performance; (ii) a simultaneous precursor/neutral loss scan was used to improve detection limits; and finally, (iii) the simultaneous precursor/neutral loss scan was implemented in a miniature mass spectrometer and representative sulfonamides were detected at concentrations as low as 100 ng/mL by nano-electrospray and 0.5 ng absolute by paper spray ionization, although improvements in the stability of the home-built instrumentation are needed to further optimize performance.