Direct Analysis In Real Time Mass Spectrometry Research Articles

Evaluation of the Deterioration State of Archaeological Wooden Artifacts: A Nondestructive Protocol based on Direct Analysis in Real Time - Mass Spectrometry (DART-MS) Coupled to Chemometrics.

Evaluating the deterioration state of archeological wood is obligatory before the preservation of archeological wooden artifacts. Herein, a nondestructive, accurate, and rapid methodology is first developed via direct analysis in real time-mass spectrometry (DART-MS) with chemometrics to classify archeological wood and recent wood into 3 groups according to their deterioration states. As water in wooden artifacts probably affected the ion fragmentation process during DART-MS, ions responsible for evaluating the deterioration state were separately screened toward waterlogged archeological wood and dried archeological wood by partial least-squares discriminant analysis (PLS-DA). The well-defined separation of severely decayed archeological wood, moderately decayed archeological wood and recent wood was revealed in PLS-DA models. Twenty and 27 wood fragment ions were further screened as key variables to evaluate the deterioration state of waterlogged archeological wood and dried archeological wood, respectively. They were tentatively identified as ions of lignin monomeric compositions, lignin dimers, lignin trimers, and oligosaccharides. Results strongly suggested that differences in the structure and relative abundances of wood cell wall components accounts for the evaluation of deterioration state by DART-MS coupled to chemometrics. PLS-DA models provided R2Y = 0.836, Q2 = 0.817, and R2Y = 0.754, Q2 = 0.682 were then established separately using mass spectral fingerprints of respective potential predictive wood fragment ions. Furthermore, archeological woods, consisting of Castanopsis, Quercus, Idesia, Populus, and Cunninghamia species and with an average MWC range of 103-465%, were used as an external validation set and evaluated with the methodology developed herein and the MWC criteria. Results showed that DART-MS coupled to chemometrics could accurately predict the inhomogeneous deterioration states of archeological wooden artifacts and avoid the interference of inorganic deposits, in comparison with the MWC criteria.

Rapid determination of chemical composition in the particulate matter of cigarette mainstream smoke

Particulate matter from mainstream smoke (MSS) is significantly hazardous when inhaled into the human body. An ambient ionization mass spectrometric method, direct analysis in real time mass spectrometry (DART-MS), was applied to rapidly and simultaneously measure multiple particulate components in MSS. A variety of compounds were obtained in seconds, where different types of cigarettes and different solvent extracts generated distinct chemical constituents as validated by principle component analysis. Chemical formula assignment and compound identification were based on accurate m/z values with mass errors <10 ppm. Quantitation of nicotine was achieved using an isotope internal standard with DART-MS. Method validation with chromatographic-MS analysis further proved the advantages of DART-MS with respect to analysis speed and operational simplicity for the direct evaluation of complex samples. DART-MS is feasible for the rapid acquisition of cigarette fingerprints for quality control as well as for quantitative assessment of carcinogens for harm reduction.

Fragmentation pathways of α-pyrrolidinophenone synthetic cathinones and their application to the identification of emerging synthetic cathinone derivatives

The expanding use of emerging synthetic drugs is creating a growing problem for both seized drug analysts and toxicologists because the clandestine suppliers continually tweak the chemical structures to keep one step ahead of the law. Synthetic cathinones, commonly referred to as bath salts, are a specific class of emerging synthetic drugs. These substances are derivatives of cathinone, which is the psychoactive component of the Catha edulis plant, commonly referred to as khat. Of the synthetic cathinone class of compounds, the α-pyrrolidinophenone synthetic cathinone derivatives stand out as one of the most abused designer drugs.The fragmentation behavior of a series of α-pyrrolidinophenone synthetic cathinones was studied with three different ionization and fragmentation techniques to enhance the current understanding of α-pyrrolidinophenone synthetic cathinones in mass spectrometers. Gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) fragmentation is commonly used by seized drug analysts, whereas liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is more commonly used in toxicological analyses. Direct analysis in real time mass spectrometry (DART-MS) is becoming more popular as a screening technique, especially in national laboratories. Each ionization and activation method encourage particular pathways of fragmentation, and whereas some pathways are conserved across all platforms, other pathways are unique to a particular instrument. This study combines isotope-labeling, multi-stage mass spectrometry (MSn) and accurate mass measurements with high-resolution mass spectrometry (HRMS) to enhance the current understanding about α-pyrrolidinophenone synthetic cathinones. This manuscript provides characteristic protonated tandem mass spectrometry fragmentation pathways and the mechanistic origins of the EI-MS fragmentation observed for this class of synthetic cathinones and provides examples of how this knowledge can be applied to the identification of novel synthetic cathinones.

Polymeric Sorbent Sheets Coupled to Direct Analysis in Real Time Mass Spectrometry for Trace-Level Volatile Analysis-A Multi-Vineyard Evaluation Study.

Etched polymeric sorbent sheets (solid-phase mesh-enhanced sorption from headspace (SPMESH) sheets) were recently described as an alternative to solid-phase microextraction (SPME) for rapid, parallel, multi-sample extraction and pre-concentration of headspace volatiles. In this report, a workflow was evaluated based on SPMESH sheet extraction followed by direct analysis in real time-mass spectrometry (DART-MS) using grape samples harvested from multiple commercial vineyards at different maturities. SPMESH sheet-DART-MS(-MS) was performed on two grape-derived odorants related to wine quality: 3-isobutyl-2-methoxypyrazine (IBMP) in Cabernet Sauvignon and Merlot grape homogenate (n = 86 samples) and linalool in Muscat-type grape juice samples (n = 18 samples). As part of the optimization process, an MS-MS method was developed for IBMP and an equilibration procedure prior to extraction was established for homogenate samples. Following optimization, we achieved good correlation between SPMESH sheet-DART-MS and SPME-GC-MS for both IBMP (range by GC-MS = < 2 ng/L to 28 ng/L, R2 = 0.70) and linalool (range by GC-MS = 135 to 415 μg/L, R2 = 0.66). The results indicate SPMESH sheet-DART-MS is suitable for rapid measurements of trace level volatiles in grapes.

Rapid and sensitive analysis of progesterone by solid-phase extraction with amino-functionalized metal-organic frameworks coupled to direct analysis in real-time mass spectrometry.

Progesterone is the representative progestogens in five major classes of steroid hormones and plays important roles in mammalian pregnancy and animal growth and development. Conventional available analytical methods for progesterone involve immunoassay, gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography-mass spectrometry (HPLC-MS), which lack specificity or usually require sophisticated operations and relatively long time. Herein, we developed a novel strategy for rapid analysis of progesterone via direct analysis in real time mass spectrometry (DART-MS) combined with solid-phase extraction (SPE) using an amino functionalized metal-organic frameworks (MOFs). Under optimized conditions, a wide linear range of 0.5-500ngmL-1 was achieved, with a satisfactory correlation coefficient (R2 = 0.9992). The relative standard deviations (RSDs) were in the range from 2.4 to 8.4%, demonstrating good precision. The applicability was then confirmed by analyzing spiked lake water and synthetic urine samples, and recoveries are between 92.0 and 117.8% in all three spiked levels (5, 25, and 100ngmL-1). The sensitivity was notably improved compared with solely DART-MS and obtained detection limit decreased by about 50 times. This research provided a rapid, simple, highly sensitive, and efficient approach for analysis of hormones through combination of advantages of ambient mass spectrometry and porous nanomaterials. Graphical abstract.

Rapid identification of polyamides using direct analysis in real time mass spectrometry.

Polyamide (PA) is the generic name of polymers synthesized by linking monomers via amide bonds, and various types of PAs with different monomer compositions are known. Distinguishing PA polymers is useful in directing monomer residual testing, product testing, and reverse engineering, but is analytically challenging and cumbersome. To simplify this, we explored the applicability of direct analysis in real time mass spectrometry (DART-MS) for screening PA polymers. A DART ion source coupled to a quadrupole Orbitrap (high-resolution (HR) mass spectrometer) was employed for this study. Ten types of PA polymers and four retail samples were evaluated. The DART-HRMS data for these samples, as well as the DART-MS/MS (MS2 ) data for PA6 and PA66, were obtained, and their repeatability was assessed across days/calibrations, operators, and equipments. Ions corresponding to the cyclic or linear monomers and oligomers of each PA polymer were detected in each DART-HR mass spectrum. Although similar DART-HR mass spectra were obtained for PA6, PA66, and PA6/PA66 (polymer blends of PA6 and PA66), their DART tandem mass spectra were completely different. The analysis was repeatable, and nearly identical DART tandem mass spectra were obtained on different days, by different operators, and with different equipment. This technique was successfully applied to commercially available samples. Ten types of PA polymers were distinguished using DART-HRMS and DART-MS2 , and their identification using these techniques was straightforward as the characteristic ions for each PA polymer were identified and detected. Furthermore, the spectra were obtained rapidly. Therefore, DART-HRMS can be considered an efficient screening technique for the rapid identification and differentiation of PA polymers.

Thermal desorption and pyrolysis direct analysis in real time mass spectrometry for qualitative characterization of polymers and polymer additives.

Direct analysis in real time mass spectrometry (DART-MS) provides qualitative information about additives and polymer composition. However, the observed mass spectra are dependent on sampling conditions, in particular the DART gas temperature. This report describes the combination of a heated sample stage with DART-MS for polymer characterization. Industrial polymers with different compositions were examined by thermal desorption and pyrolysis (TDPy) DART. Samples were heated on disposable copper stages from ambient temperature to 600°C, and the evolved gases were introduced directly into a DART ion source through a glass tee. Time- and temperature-dependent mass spectra were acquired using a high-resolution time-of-flight mass spectrometer. Kendrick mass analysis was applied to the interpretation of complex mass spectra observed for fluorinated polymers. Positive-ion DART mass spectra of common polymers exhibited peak series differing by monomer masses, often accompanied by a peak corresponding to the protonated monomer. Even polymers that did not exhibit a clear series of peaks produced characteristic mass spectra. Positive-ion and negative-ion mass spectra were recorded for fluorinated polymers, with polytetrafluoroethylene (PTFE) producing only negative ions. Thermal desorption provided characteristic temperature profiles for volatile species such as polymer additives and polymer pyrolysis products. In comparison with direct analysis by positioning sample directly in the heated DART gas stream, TDPy DART provides a more versatile sampling method and provides thermal separation and profiling of polymer additives, intact short polymer chains, and pyrolysis fragments.

Determination of ethanol concentration in alcoholic beverages by direct analysis in real time mass spectrometry (DART-MS)

Determination of the ethanol concentration in beverages remains an occasional request to forensic laboratories. While traditionally determined using headspace gas chromatography (HS-GC), this process involves lengthy run times and the need for specialized headspace sampling equipment for the GC system. The work presented here highlights a potential alternative to HS-GC analysis, using direct analysis in real time mass spectrometry (DART-MS). By incorporating a simple T-junction to the orifice of the mass spectrometer, analysis of the headspace of a beverage can be completed, resulting in measurement of the ethanol concentration in a matter of seconds with greater than 99% accuracy. This work presents the development of a method for ethanol quantitation as well as results from the analysis of both ethanol reference material standards and in-house created ethanol-containing solutions to evaluate the precision and accuracy of the technique. Analysis of ethanolic beverages via DART-MS is shown to be a rapid and reliable alternative to traditional analysis by HS-GC.

Emerging techniques for the detection of pyrotechnic residues from seized postal packages containing fireworks

High volume screening of parcels with the aim to trace the illegal distribution and selling of fireworks using postal services is challenging. Inspection services have limited manpower and means to perform extensive visual inspection. In this study, the presence of solid pyrotechnic residues collected from cardboard shipping parcels containing fireworks was investigated for direct in-field chemical detection. Two emerging trace detection techniques, i.e., capillary electrophoresis (CE)-based inorganic oxidizer detector and infrared thermal desorption (IRTD) coupled with direct analysis in real time mass spectrometry (DART-MS), were investigated for their potential as screening tools. Detection of non-visible pyrotechnic trace residues from real-case seized parcels was demonstrated using both screening techniques. However, the high nitrate background in the commercial CE system complicated its screening for black powder traces. IRTD-DART-MS allowed differentiation between flash and black powder by identification of the molecular inorganic ions. Compared to the portable CE instrument, rapid screening using IRTD-DART-MS is currently limited to laboratory settings. The capabilities of these emerging techniques established solid particle and trace residue chemical detection as interesting options for parcel screening in a logistic setting.

Optimization of confined direct analysis in real time mass spectrometry (DART-MS).

Direct analysis in real time mass spectrometry (DART-MS) is seeing increased use in many fields, including forensic science, environmental monitoring, food safety, and healthcare. With increased use, novel configurations of the system have been created to either aid in detection of traditionally difficult compounds or surfaces, provide a more reproducible analysis, and/or chemically image surfaces. This work focuses on increasing the fundamental understanding of one configuration, where the DART ionization gas is confined in a junction, such as with thermal desorption (TD) DART-MS. Using five representative compounds and a suite of visualization tools, the role of the DART ionization gas, Vapur flow rate, gas back pressure, and exit grid voltage were examined to better understand both the chemical and physical processes occurring inside the confined configuration. The use of nitrogen as a DART ionization gas was found to be more beneficial than helium because of enhanced mixing with the analyte vapors, providing a more reproducible response. Lower Vapur flow rates were also found to be advantageous as they increased the analyte residence time in the junction, thus increasing the probability of its ionization. Operation at even lower Vapur flow rates was achieved by modifying the junction to restrict the DART gas flow. The DART exit grid voltage and gas back pressure had little observed impact on analyte response. These results provide the foundation to better understand and identify best practices for using a confined DART-MS configuration.

Direct and quantitative monitoring of catalytic organic reactions under heterogeneous conditions using direct analysis in real time mass spectrometry.

This new method overcomes problems of conventional analytical methodologies such as light scattering and sampling reproducibility issues. We used this method for mechanistic studies of catalytic reactions under heterogeneous conditions. Direct-type hydroxymethylation reactions and Mukaiyama-type hydroxymethylation reactions both catalyzed by a scandium–bipyridine ligand complex under micellar conditions were employed as examples of heterogeneous reactions. For direct-type hydroxymethylation reactions, initial reaction rate assays revealed first-order dependency on both substrate and catalyst. On the other hand, Mukaiyama-type hydroxymethylation reactions showed first-order rate dependency on substrate, zero-order on catalyst and saturation kinetics on formaldehyde.

Salting Out: A Simple and Reliable Method to Distinguish Between Common Fluid Preservatives and Estimate Alcohol Concentration

Abstract This paper details the salting-out method, which uses the salts potassium carbonate and sodium chloride to distinguish between the three most commonly used fluid preservatives: ethanol, isopropanol, and formalin. A summary of other methods to identify fluid preservative type and a review of the salting-out method published by Mayfield (2013, Distinguishing between ethanol and isopropanol in natural history collection fluid storage, Society for the Preservation of Natural History Collections, https://spnhc.org/wp-content/uploads/2018/11/Mayfieldfinalwithtablechanges.pdf) are provided. A new salting-out method is presented, which requires a small fluid sample (2–4 ml). It is simple, quick, and relatively inexpensive to implement, making it a viable method to distinguish between common fluid preservatives. The materials and equipment for the salting-out test cost just over $100 US, and tests take approximately 3 minutes per container. Results of testing on known concentrations and combinations of ethanol, isopropanol, and formalin (a solution of formaldehyde in water) and on samples of fluid preservatives from specimen containers in the Smithsonian National Museum of Natural History and Bernice Pauahi Bishop Museum collections are presented. The results of salting-out tests have been verified by direct analysis in real time mass spectrometry (DART-MS) (Cody et al., 2005, Versatile new ion source for the analysis of materials in open air under ambient conditions, Analytical Chemistry 77(8):2297–302), which confirmed the results of salting-out tests but also highlighted some limitations, particularly when combinations of fluid preservative are encountered.

Direct Quantification of Natural Moisturizing Factors in Stratum Corneum using Direct Analysis in Real Time Mass Spectrometry with Inkjet-Printing Technique

Proper hydration of the stratum corneum, the skin’s outermost layer, is essential for healthy skin. Water-soluble substances called natural moisturizing factors (NMF) are responsible for maintaining adequate moisture in the skin and are closely associated with a variety of the skin’s functions. Therefore, quantitative analysis methods for NMF are indispensable when attempting to clarify one of the mechanisms of hydration and its effect on the skin. This study sought to develop a quick and simple analytical technique, which can quantify NMF from the skin without the need for extraction or separation, using direct analysis in real time-mass spectrometry (DART-MS). The goal was to deliver a high quantitative capability, so a unique inkjet printing technique was employed to evenly coat a stable isotope-labeled internal standard (SIL-IS) on tape-stripped skin. This technique allowed for the quantification of 26 NMF with established calibration curves and comparatively high linear correlations. The speed of measurement was found to be advantageous as 100 strips of tape can be measured in roughly 2 hours. The effectiveness of the inkjet coating was also verified by comparing its precision with that of conventional pipetting. This new technique can be an alternative method to quantify NMF rapidly and perhaps allow for a clearer elucidation of their function in skin.

Dispersive Magnetic Solid-Phase Extraction Coupled to Direct Analysis in Real Time Mass Spectrometry for High-Throughput Analysis of Trace Environmental Contaminants.

Coupling dispersive magnetic solid-phase extraction (DMSPE) to direct analysis in real time mass spectrometry (DART-MS) with a newly developed metal iron probe enables high-throughput, sensitive detection of herbicides such as triazine in environmental waters. Magnetic graphene oxide was used as a dispersive sorbent because it increased adsorption capacity in the DMSPE process. The planar structure and excellent thermal conductivity of graphene oxide facilitated the desorption and ionization of target analytes in DART-MS analysis. The iron probe, which is designed to fit into the moving trail of the DART interface, served as the sorbent collector as well as the support for the magnetic graphene oxide after DMSPE, and was put directly into the DART system. The ratio of magnetic core to graphene oxide in the nanoparticles and other key parameters in DMSPE and DART-MS procedures were systematically investigated and optimized. In addition, the presence of water on the sorbent proves to have a significant effect on DART-MS analysis. No organic solvents are used in this method, and the reusable iron probe is of low cost. Under the optimal conditions, limits of detection were found in the range of 1.6-152.1 ng/L for the triazines. Recovery and reproducibility were found to be in the ranges of 87.5-115.0% and 1.9-10.2%, respectively, for the six herbicides studied. The analytical performance of the DMSPE-DART-MS method indicated that applications for trace analysis of other compounds in liquid samples are also possible.

Nitrogen-Activated Oxidation in Nitrogen Direct Analysis in Real Time Mass Spectrometry (DART-MS) and Rapid Detection of Explosives Using Thermal Desorption DART-MS.

Direct analysis in real time mass spectrometry (DART-MS) was used to analyze an array of explosives including nitro-based explosives, peroxide explosives, and energetic heterocyclic compounds with different DART discharge gases (helium, argon, and nitrogen). Profound analyte oxidation was observed for particular compounds (TNT (9) and 2, 4-DNT (10)), whose mass spectra were completely dominated by the oxidation products when nitrogen was substituted for helium in DART analysis. This interesting phenomenon suggested that a highly oxidative environment provided by N2 DART ion source. A possible mechanism involved in nitrogen DART was proposed which may help further understanding the different chemistry involved in the ionization process. This work also presents a thermal desorption DART (TD-DART) configuration that can enable rapid, specific analysis of explosives from swipes. The screening of swipes with three different compositions (fiberglass, Hybond N+ membrane, and filter paper) showed that fiberglass swipe has the best performance which was then used for the subsequent TD-DART analysis. A direct comparison of TD-DART with traditional DART demonstrated that TD-DART indeed gives better response than traditional DART (provided that the distance between the DART source and mass spectrometer is the same) and will have wider applications than traditional DART.

Rapid Evaluation of the Debromination Mechanism of Eosin in Oil Paint by Direct Analysis in Real Time and Direct Infusion-Electrospray Ionization Mass Spectrometry.

Eosin is a synthetic organic colorant prone to fading under the influence of light. On the basis of the growing interest in the understanding of the discoloration mechanism of eosin-based lakes, this study compares the ability of two ultrafast and ultrasensitive mass spectrometry techniques to detect eosin derivatives in complex matrices, such as oil media without the use of conventional separation columns or additional sample preparation protocols. Direct analysis in real time mass spectrometry (DART-MS) and direct infusion electrospray ionization mass spectrometry (DI-ESI-MS) were used to characterize the degradation pathway of eosin in oil media. The analysis protocols developed in this study are applied to discern the degradation mechanism of the lake pigment eosin (comprising the molecule per se complexed to an inorganic substrate) dispersed in linseed oil to create an oil paint. The analysis of oil paints by high resolution MS without an extraction methodology that modifies the system chemistry allowed us to identify the degradation forms without causing any additional fragmentation. Both techniques revealed the primary photodegradation pathway of eosin in linseed oil, and DI-ESI-MS provided additional information on the native conformation of the lake.

Mass Spectrometry-Sensitive Probes Coupled with Direct Analysis in Real Time for Simultaneous Sensing of Chemical and Biological Properties of Botanical Drugs.

The development of botanical materials as therapeutic agents involves the meticulous assessment of safety, efficacy, and quality. Compared with small-molecule drugs, quality control of botanical drugs confronts with more significant challenges due to their inherent complexity. Current quality control methods for botanical drugs, either prevailing chemical tests or emerging biological assays, are not able to meet recent demands of multiplexing, sensitivity, and speed. Here, we propose an on-demand strategy based on a direct analysis in real time-mass spectrometry (DART-MS) platform, which is capable of simultaneously analyzing multiple constituents and bioactivities of botanical drugs. Notably, the bioactivities are assessed by a multiple-enzyme assay that adopts cleavable mass spectrometry probes as enzymatic substrates: these probes labeled with a piperazine tag make possible sensitive, multiplexed, and quantitative enzyme activity measurements. The concept is successfully demonstrated via a case study of Danshen (Salvia miltiorrhiza) Injection where simultaneous detection of 34 constituents and inhibitory activities on two target enzymes can be achieved in just minutes. This proof-of-concept application also gives evidence that combining MS-sensitive probes with DART-MS can provide an environmentally friendly, highly sensitive analytical approach for botanical quality control.

Multiphase reactivity of polycyclic aromatic hydrocarbons is driven by phase separation and diffusion limitations

Benzo[a]pyrene (BaP), a key polycyclic aromatic hydrocarbon (PAH) often associated with soot particles coated by organic compounds, is a known carcinogen and mutagen. When mixed with organics, the kinetics and mechanisms of chemical transformations of BaP by ozone in indoor and outdoor environments are still not fully elucidated. Using direct analysis in real-time mass spectrometry (DART-MS), kinetics studies of the ozonolysis of BaP in thin films exhibited fast initial loss of BaP followed by a slower decay at long exposure times. Kinetic multilayer modeling demonstrates that the slow decay of BaP over long times can be simulated if there is slow diffusion of BaP from the film interior to the surface, resolving long-standing unresolved observations of incomplete PAH decay upon prolonged ozone exposure. Phase separation drives the slow diffusion time scales in multicomponent systems. Specifically, thermodynamic modeling predicts that BaP phase separates from secondary organic aerosol material so that the BaP-rich layer at the surface shields the inner BaP from ozone. Also, BaP is miscible with organic oils such as squalane, linoleic acid, and cooking oil, but its oxidation products are virtually immiscible, resulting in the formation of a viscous surface crust that hinders diffusion of BaP from the film interior to the surface. These findings imply that phase separation and slow diffusion significantly prolong the chemical lifetime of PAHs, affecting long-range transport of PAHs in the atmosphere and their fates in indoor environments.

Solvent-free high-throughput analysis of herbicides in environmental water

A high throughput, and eco-friendly method based on solid-phase microextraction (SPME) combined with direct analysis in real time mass spectrometry (DART-MS) was established for the determination of trace pollutants in water. A laboratory-made SPME device coated with a metal organic framework (MIL-100 (M)) was fabricated for the on-site enrichment of triazine herbicides in environmental water, and the device was directly subjected to DART ionization (<20 s) for high resolution MS. The interactions between the target analytes and sorbents were investigated to improve the SPME device targeting specific pollutants, as well as to improve the desorption and ionization processes with DART-MS. Other factors were also systematically studied to obtain the optimal conditions, including pH, salinity, extraction temperature, pressure at the Vapur® interface, linear rail moving speed, gas temperature, extraction time, and sample volume. The limit of detection of target compounds were 5.0–50.0 ng L−1 and the recoveries ranged from of 92.4%–125.7%.

Areca alkaloids measured from buccal cells using DART‐MS serve as accurate biomarkers for areca nut chewing

Areca nut (AN) chewing is carcinogenic and biomarkers reflecting it are urgently needed to determine the effectiveness of emergent cessation programs. Buccal cells (BCs) may serve as an ideal matrix to measure such biomarkers; however, their utility for this purpose is unknown. Direct analysis in real time-mass spectrometry (DART-MS) is a sensitive technique that analyzes materials in the open air and requires minimal/no sample preparation. We utilized DART-MS to analyze BCs to test the usefulness of this method in measuring areca alkaloids as biomarkers for AN chewing. We applied DART-MS in positive-ion mode to quantitate over time human BCs: (a) exposed ex vivo to betel quid extracts (BQE) consisting of young AN, Piper betle L. leaf, slaked lime, and tobacco; and (b) obtained from seven chewers before and after BQ chewing. Quantification was performed by normalizing DART-MS alkaloid signal intensities to cholesterol intensities. Signals for areca alkaloids arecoline and arecaidine-guvacoline were detected in BCs exposed ex vivo to BQE up to 7 days (the last day tested) after exposure and in BCs from chewers up to 3 days (the last day tested) post chewing. The presence of alkaloid signals in BQ-exposed BCs verified BCs as a valid matrix and DART-MS as a suitable technique to measure biomarkers for AN chewing and provided reliable information on AN chewing timing. DART-MS analyses of BCs can be used to accurately determine areca alkaloids as AN chewing biomarkers up to 3days post chewing and possibly longer.