Ambient Ionization Research Articles

High-throughput direct profiling of natural products in plant leaves using ambient ionization high-resolution mass spectrometry.

The distribution of Danqi tongmai tablet in rats at different timeframe by UHPLC-LTQ-orbitrap mass spectrometry and nanospray desorption electrospray ionization mass spectrometry imaging.

Desorption Electrospray Ionization (DESI) is a unique ambient ionization technique in mass spectrometry that operates under atmospheric pressure without the need for vacuum systems, chemical matrices, or extensive sample preparation. These characteristics distinguish DESI from conventional ionization methods and enable quasi on-demand molecular analysis directly from native surfaces and tissues, an analytical capability otherwise impractical for many time-sensitive or spatially constrained applications. Since its development by Cooks et al. in 2004, DESI-MS has become an indispensable method for both qualitative and quantitative applications. It operates by propelling charged microdroplets (typically <10 μm in diameter) onto sample surfaces, producing gas-phase ions through soft ionization. This review presents a critical evaluation of DESI performance across a range of uses, including surface profiling, molecular imaging, and in situ diagnostics. DESI-MS allows analysis rates exceeding 2 samples per second in formats such as 96-well plates, with detection limits in the low nanogram range. Imaging applications have demonstrated spatial resolutions below 200 μm, with scan speeds reaching 100 μm s-1, enabling detailed molecular mapping in biological tissues. Technological advancements, such as infrared laser-assisted DESI (IR-LADESI), enclosed DESI modules, and microdroplet-driven reaction screening, have expanded DESI's applicability across pharmaceutical, clinical, forensic, and environmental fields. This review synthesizes developments from 2020 to 2025, emphasizing technical principles, instrumentation progress, and analytical performance, and positions DESI-MS as a leading tool in modern mass spectrometry.

Rapid evaporative ionization mass spectrometry (REIMS) is an ambient ionization mass spectrometry technique that enables real-time evaluation of several complex traits from a single measurement. The objectives of this study were: (1) to investigate the capability of REIMS to accurately identify and predict sheep carcass characteristics and flavor based on consumer response utilizing data acquired by I-Knife, and (2) to compare the ability of 2 electrodes (Meat Probe vs. I-Knife) to differentiate carcass characteristics and cooked meat flavor. For objective 1, 200 sheep carcasses were used to generate I-Knife REIMS data from the external fat and the surface lean of the Biceps femoris muscle (45 min postmortem) as well as meat patties (7 d postmortem). These patties were further used to evaluate consumer preferences using sensory analysis. Objective 2 was achieved by comparing the predictive performance of I-Knife and Meat Probe REIMS data collected from meat patties. The results demonstrated that REIMS analysis of raw meat samples can be used to accurately predict and classify cooked sheep meat flavor and carcass characteristics. Specifically, the lean and fat tissue collected at 45 min postmortem can be used to predict carcass characteristics and postrigor meat flavor. Models for diet, flavor intensity acceptance, off-flavor presence, overall acceptance, age, and flavor acceptance achieved prediction accuracy higher than 80%. In addition, models generated using data from the Meat Probe had similar or better prediction accuracies for carcass background (age, diet, and gender) and consumer preference (intensity acceptance, flavor acceptance, off-flavor presence, and overall acceptance) compared to models based on the I-Knife data. Overall, these results demonstrated the potential for REIMS to predict and classify cooked sheep meat flavor accurately and validated the use of the Meat Probe for REIMS analysis.

Robust high-throughput screening (HTS) approaches for discovering new chemical entities are desirable for research and translation. Applications for which high-throughput (HT) methods are particularly required also include the screening of potential therapeutics for drug discovery and development, profiling of biofluids for disease biomarker discovery, and clinical diagnostics. Complementing the demand for HTS from specific application areas are substantial technological advancements in the fields of automation, microfluidics, and ambient ionization that facilitate highly automated and sophisticated analytical workflows. The time period spanning 2000-2025 has witnessed a significant expansion in the mass spectrometry (MS) capabilities and technology. This has included novel ionization approaches that can achieve rapid analysis with minimal solvent and sample consumption, while retaining high sensitivity and specificity in the absence of chromatography. Despite the demand for HTS methods and the well-documented analytical capabilities of MS, optical methods dominate as the HTS detection methods of choice. This perspective provides an overview of the evolution of HTS-MS over the last 25 years, focusing on emerging approaches that also provide efficient and sustainable workflows that compete with optical detection. Additionally, this perspective will highlight challenges in the field that may hinder widespread adoption and consider lessons from the COVID-19 pandemic, as well as the impact of sustainability on the future of HTS-MS and analytical chemistry.

ConspectusMass spectrometry imaging (MSI) has transformed our ability to explore molecular distributions in biological tissues with high chemical specificity and sensitivity. Despite significant advances in this field, the absence of separation prior to analysis leads to isomeric and isobaric overlaps, posing a major analytical challenge. To enhance chemical specificity and enable isomer differentiation, tandem mass spectrometry, ion mobility spectrometry, chemical complexation, and derivatization strategies are increasingly integrated into MSI workflows.Ambient ionization MSI techniques provide both chemical and spatial information under native or near-native conditions, enabling rapid, label-free molecular imaging of complex biological samples with minimal sample pretreatment. Among the most promising ambient MSI techniques is nanospray desorption electrospray ionization (nano-DESI), a method that relies on localized liquid extraction directly from biological tissue sections. We have successfully implemented custom-designed nano-DESI platforms on multiple commercial mass spectrometers to enable molecular identification at each pixel of the image and facilitate isomer-selective mass spectrometry imaging (iMSI).This Account highlights recent advances in iMSI using nano-DESI. Key developments include the integration of nano-DESI with multiple reaction monitoring on a triple quadrupole mass spectrometer to differentiate isomeric lipids in biological tissues. We also describe the integration of photoinitiated derivatization and metal ion complexation strategies to enable isomer-selective imaging using structure-specific fragments generated by collision induced dissociation. Furthermore, high-resolution separation of lipid isomers was achieved by coupling nano-DESI with trapped ion mobility spectrometry, demonstrating the value of gas-phase separation for iMSI. These innovations have significantly expanded the analytical capabilities of MSI critical to probing the spatial organization of isomeric lipids and metabolites in biological systems. We also discuss future directions, including new complexation strategies and the integration of nano-DESI with data-independent acquisition and parallel accumulation serial fragmentation technologies. Collectively, these advances establish nano-DESI iMSI as a powerful and versatile tool in the evolving field of spatial metabolomics and lipidomics.

The rapid, sensitive, selective, and real-time measurement of n-alkanes in mixtures is significant for safety, health, and the environment. However, detecting n-alkanes is challenging due to their optoelectronic and chemical inertness, particularly in ambient mass spectrometry (MS) measurements, which often necessitate hardware modifications or environmentally harmful derivatization reagents. Here, an ambient MS approach was proposed for direct n-alkane detection in complex mixtures, leveraging water dimer radical cations ((H2O)2+•) generated by a low-energy ambient corona discharge to activate C-H bonds and form detectable dihydroxyalkane adducts (R-C+(OH)2). Using hexane as a model, the mechanism involves the following: (a) (H2O)2+• reacts with hexane to produce a stable complex, followed by (b) stepwise hydroxylation reaction forming R-C+(OH)2. The method was successfully extended to other n-alkanes (e.g., pentane, heptane, octane, nonane, and decane) with their corresponding dihydroxyalkane adducts R-C+(OH)2 detected as anticipated. The presented MS approach enables simple and real-time analysis of n-alkanes in complex matrices, with a detection limit down to 0.22 parts per trillion. By leveraging (H2O)2+•-induced C-H activation, this work provides an environmentally friendly strategy for direct n-alkane determination in mixtures under ambient conditions without sample pretreatment, offering a novel pathway for direct n-alkane determination across various fields including environmental monitoring, petroleum analysis, etc.

Most ambient ionization methods for mass spectrometry require externally applied high-voltage fields to generate charged droplets, limiting their portability and energy efficiency. We report a fully three-dimensional (3D)-printed ionization source, in which a standalone atomizer functions as the droplet-generating ionizer by enabling spontaneous charging through gas-solid triboelectric interactions. The device features a coaxial flow configuration, in which high-velocity sheath gas interacts with a BaTiO3-doped poly(lactic acid) (PLA) nozzle surface, generating interfacial charges that are transferred to emerging droplets. Computational fluid dynamics simulations confirm sustained wall shear stress near the outlet, supporting the proposed charge-generation mechanism. By tuning the nozzle's dielectric composition and outlet geometry, droplet charge density and size can be precisely modulated up to 0.3 nC/μL under optimized conditions. Faraday cup measurements reveal a clear correlation between the structural parameters and total droplet charge. Using methyl viologen (MV2+) as a single-electron probe, mass spectra revealed efficient electron transfer and secondary product formation, confirming enhanced interfacial electron availability. These results demonstrate that triboelectric enhancement via a 3D-printed design enables voltage-free ion generation and controllable electron transfer, offering a structurally simple, low-cost, and power-free approach to ambient chemical analysis.

Ambient Mass Spectrometry (AMS) has revolutionized forensic analysis by enabling rapid and direct detection of chemical compounds on complex surfaces with minimal or no sample preparation. Among AMS techniques, desorption electrospray ionization (DESI) and direct analysis in real time (DART) mass spectrometry have emerged as powerful analytical tools due to their speed, sensitivity, and versatility. This review examines the major applications of DESI and DART in forensic science, including the detection of explosives, gunshot residue, illicit drugs, inks, biological fluids, and latent fingerprints. The principles of each technique are briefly discussed, followed by a comparative analysis of their advantages, limitations, and performances in various forensic scenarios. Recent developments, practical considerations for implementation, and future perspectives are also addressed, highlighting the growing impact of DESI and DART in real-time forensic investigations and evidence processing.

Development of boronic-acid-functionalized magnetic metal organic framework coupled with ambient ionization mass spectrometry for the analysis of neonicotinoid insecticides in fruits and vegetables.

Improved detection of acephate residues in tomatoes using PSI-MS/MS with functionalized polyacrylamide modified substrate.

ABSTRACTMass calibration techniques are vital in achieving high mass measurement accuracy (MMA) of large biomolecules. Variable ion populations that shift the axial frequencies due to space charge effects have been a significant challenge in achieving sub‐parts‐per‐million (sub‐ppm) MMA of glycans on a high‐resolution accurate mass (HRAM) orbitrap instrument without the activation of automatic gain control. As the role of glycans is critical to our understanding of diverse biological processes, accurate identification of glycans using sub‐ppm MMA is critical for biological interpretations. Hence, this study aims to achieve sub‐ppm MMA of glycans by exploring the impact of different ion accumulation times, data collection modes, in addition to custom calibration strategies and external mass correction to optimize accurate mass measurements. Using infrared matrix‐assisted laser desorption electrospray ionization (IR‐MALDESI), direct analysis was performed on N‐linked glycans cleaved from bovine fetuin in negative polarity, where 17 N‐linked glycans were detected and annotated. Our results indicate the significance of implementing a custom calibration external lock mass and other techniques, including the effect of external mass correction in achieving sub‐ppm MMA of large biomolecules. Implementing such approaches in mass spectrometry imaging (MSI) of biological tissue will enhance the confidence of glycan annotation and enable more accurate biological conclusions.

Rapid Screening and Prioritization of Culture Conditions for Natural Product Discovery using the Liquid Microjunction Surface Sampling Probe.

Background/Objectives: 1,8-Cineole, an epoxy monoterpene, is a key volatile component of Sugmel-3, a traditional Mongolian medicine used for treating insomnia. Although previous studies suggest that 1,8-Cineole can cross the blood–brain barrier (BBB), its precise spatiotemporal distribution in the brain and its in situ association with alterations in neurotransmitter (NT) levels remain unclear. This study utilized ambient mass spectrometry imaging (AFADESI-MSI) to investigate the dynamic brain distribution of 1,8-Cineole and its major metabolite, as well as their correlation with NT levels. Methods: Sprague Dawley rats (n = 3 per time point) received oral administration of 1,8-Cineole (65 mg/kg). Brain tissues were harvested 5 min, 30 min, 3 h, and 6 h post dose and analyzed using AFADESI-MSI. The spatial and temporal distributions of 1,8-Cineole, its metabolite 2-hydroxy-1,8-Cineole, key neurotransmitters (e.g., 5-HT, GABA, glutamine, melatonin), and related endogenous metabolites were mapped across 13 functionally distinct brain microregions. Results: AFADESI-MSI demonstrated rapid brain entry of 1,8-Cineole and its metabolite, with distinct spatiotemporal pharmacokinetics. The metabolite exhibited higher brain exposure, with 1,8-Cineole predominant in the cortex (CTX) and hippocampus (HP), while its metabolite showed pronounced accumulation in the pineal gland (PG), alongside CTX/HP. Region-dependent alterations in neurotransmitter levels (notably in PG, HP) correlated with drug concentrations, with observed increases in key molecules of the serotonergic and GABAergic pathways. Conclusions: Using AFADESI-MSI, this study provides the first spatiotemporal map of 1,8-Cineole and its metabolite in the brain. The correlation between their region-specific distribution and local neurotransmitter alterations suggests a direct mechanistic link to Sugmel-3′s sedative–hypnotic efficacy, guiding future target identification.

The palladium-catalyzed Suzuki–Miyaura cross-coupling (SMCC) reaction is an essential technique for C–C bond formation. It is considered to occur through two distinct pathways involving homogeneous and heterogeneous mechanisms. However, there is still debate about these mechanisms due to the lack of direct structural evidence in both spatial and temporal terms, especially regarding the conversion of active Pd species. In this study, the Pd single-atom catalyst (Pd SAC) SMCC reaction was monitored in real-time using ambient mass spectrometry (AMS) to study the conversions of Pd species both on the catalyst surface and in the liquid phase. This revealed a leaching–oxidizing–landing path as a contributors to the heterogeneous process which involves heterogeneous oxidative addition, Pd leaching along with transmetallation (rather than oxidative addition), and subsequent oxidation–landing back onto the catalyst surface. The leaching–oxidizing–landing mechanism of Pd active sites during the Pd SAC-catalyzed SMCC reaction provides an explanation for the Pd migration on the catalyst surface. A crucial role of molecular oxygen during the SMCC reaction was revealed and attributed to the re-deposition of active Pd species through coordination with the catalyst support. Overall, the leaching–oxidizing–landing mechanism of the SMCC reaction has been revealed and it not only provides insights into mechanistic studies and catalyst designs but also expands AMS applications.

Native mass spectrometry (MS) enables the analysis of protein interactions in complex biological mixtures. However, nonvolatile salts and buffers commonly present in such samples can cause ion adduction, peak broadening, and reduced signal intensity. Reducing the pressure surrounding the ionization emitter significantly improves native MS performance under these challenging conditions. Signal enhancements of up to 20‐fold were observed with nanoscale emitters, and up to 7‐fold with microscale emitters in high‐salt solutions. Protein ions remained detectable in solutions containing up to 300 mM NaCl, unlike ambient pressure ionization. High signal‐to‐noise was observed for the DDB1:DCAF1 complex at 50 nM using reduced pressure ionization, whereas no readily assignable signal was detected at ambient pressure, demonstrating its utility for detecting tightly bound complexes at trace levels. Coupling to native ion mobility mass spectrometry showed that arrival time distributions and collision cross sections did not depend significantly on the pressure used, indicating that structural information is preserved. These results show that reduced pressure ionization improves native MS performance under conditions that typically suppress signal at ambient pressure, such as high salt or low analyte concentration. The method is compatible with both nano‐ and microscale emitters and requires only minor modifications to existing instrumentation. Reduced pressure ionization expands the range of conditions accessible by native MS and is expected to enable automated, high‐throughput workflows in structural proteomics, biopharmaceutical characterisation, and protein‐ligand interaction studies.

Active Humidity Control Chamber for Desorption Electrospray Ionization-Mass Spectrometry Imaging Applications.

Simultaneous determination of pesticide residues and rapid discrimination of corn production origin using ambient ionization mass spectrometry combined with machine learning.

Etomidate, which is a psychoactive drug with an anesthetic effect, is used as a substitute for expensive mainstream drugs. There has been a trend toward abuse of etomidate and its emerging structural analogues now. Faced with a large number of samples, a rapid and effective detection method is needed. In this study, ambient flame ionization (AFI) coupled with LTQ-Orbitrap mass spectrometry was used to analyze etomidate and its structural analogues in urine. It can realize detection in less than 0.2min without sample preparation. Ideal analysis conditions were obtained by optimizing various parameters and analytical performance was validated. The isomers (isopropoxate and propoxate) can be distinguished by ion abundance ratios. Positive samples (n = 75) were analyzed very efficiently and successfully from plenty of authentic specimens (n = 116). Statistical analysis was conducted on drug types, age, and gender of drug users. A new structural analogue was discovered in one of the samples, which was a very crucial discovery. That meant the market may face with the emergence of new structural analogues. This study can satisfy both targeted and non-targeted screening, which provides support for timely monitoring and detection of novel drugs and offers a wider range of method choices for forensic laboratories. It can also better cope with the current situation of drug control and combat crimes related to new types of drugs.

The use of ambient ionization mass spectrometry (AI-MS)to aidin the preliminary screening of seized drug evidence has steadilyincreased over the past two decades. Unlike gas chromatography–massspectrometry (GC-MS), where electron ionization using a single quadrupoleanalyzer is commonplace, a wide range of ionization sources and massspectrometers can be used in AI-MS. Differences in instrument configurationcan lead to substantial variability in the mass spectral data obtained.An interlaboratory study, consisting of 35 participants from 17 laboratories,was conducted to begin to understand the landscape and the differencesin the data that are produced. Laboratories analyzed a series of 21solutions across multiple days using their own instrumental methods.Mass spectra were extracted and compared to understand operator, within-lab,and between-lab reproducibility for common compounds and mixturesobserved in seized drug analysis. In addition, five participants analyzedthe 21 solutions using prescribed method parameters to measure reproducibilityimprovements when using identical instrumental conditions. Mass spectralreproducibility, measured through pairwise cosine similarity, wasfound to be generally quite high, regardless of sample type, instrumenttype, method, or operator. Low-fragmentation spectra showed the lowestvariability, as they were dominated by intact protonated moleculepeaks. Several potential issues that increased variability were identified,including carryover from mass calibrants, poor sample introduction,and mass spectrometer inlets that required cleaning. The use of uniformmethod parameters was shown to increase the reproducibility of massspectra across laboratories, most notably at higher in-source collision-induceddissociation energies. This study provides initial insights into thecurrent landscape of AI-MS in seized drug analysis and lays the foundationfor future studies that can provide needed data for the developmentof documentary standards, standard methods, and possibly the establishmentof error rates.