Charge inversion under plasma-nanodroplet reaction conditions excludes Fischer esterification for unsaturated fatty acids: a chemical approach for type II isobaric overlap.

Direct infusion ionization methods provide the highest throughput strategy for mass spectrometry (MS) analysis of low-volume samples. But the trade-off includes matrix effects, which can significantly reduce analytical performance. Herein, we present a novel chemical approach to tackle a special type of matrix effect, namely type II isobaric overlap. We focus on detailed investigation of a nanodroplet-based esterification chemistry for differentiating isotopologue [M + 2] signal due to unsaturated fatty acid (FA) from the monoisotopic signal from a saturated FA. The method developed involves the online fusion of nonthermal plasma with charged nanodroplets, enabling selective esterification of saturated FAs. We discovered that unsaturated FAs undergo spontaneous intramolecular reaction via a novel mechanism based on a carbocation intermediate to afford a protonated lactone moiety (resonance stabilized cyclic carbonium ion), whose mass is the same as the original protonated unsaturated FA. Therefore, the monoisotopic signal from any saturated FA can be selectively shifted away from the mass-to-charge position where the isobaric interference occurs to enable effective characterization by MS. The mechanism governing the spontaneous intramolecular reactions for unsaturated FAs was validated with DFT calculations, experimentation with standards, and isotope labeling. This novel insight achieved via the ultrafast plasma-nanodroplet reaction environment provides a potentially useful synthetic pathway to achieve catalyst-free lactone preparation. Analytically, we believe the performance of direct infusion MS can be greatly enhanced by combining our approach with prior sample enrichment steps for applications in biomedicine and food safety. Also, combination with portable mass spectrometers can improve the efficiency of field studies since front-end separation is not possible under such conditions.

High-Throughput Nanoliter Sampling and Direct Analysis of Biological Fluids Using Droplet Imbibition Mass Spectrometry.

A high-throughput droplet imbibition mass spectrometry (MS) experiment is reported for the first time that allows direct analysis of ultra-small volumes of complex mixtures. In this experiment, an array of optimized tips of glass capillaries containing the analyte solution is sampled by rapidly moving charged microdroplets, which picks up (i.e., imbibes) the analyte and transfers it to a proximal mass spectrometer. The advantages associated with this droplet imbibition experiment include (1) ultra-small sample consumption (1.3 nL/min), which reduces the matrix effect in complex mixture analysis, and (2) high surface activity, which eliminates ion suppression effects caused by competition for the space charge on the droplet surface. Collectively, the enhanced surface effect and small flow rates dramatically increase the sensitivity of the droplet imbibition MS approach. This was experimentally shown by constructing calibration curves for cocaine analysis in human raw urine and whole blood, achieving 2 and 7 pg/mL limits of detection, respectively. The high-throughput feature was demonstrated by analyzing five structurally different compounds in 20 s intervals. With the measured flow rate of 1.3 nL/min on a 5 µm glass tip size, the results described in the current study showcase droplet imbibition MS to be a powerful and high-throughput alternative for conventional nano-electrospray ionization (flow rate <100 nL/min), which is the most efficient method for transferring small sample volumes to mass spectrometers.

Mass Spectrometry Approach for Differentiation of Positional Isomers of Saccharides: Toward Direct Analysis of Rare Sugars.

Rare sugars have gained popularity in recent years due to their use in antiaging treatments, their ability to sweeten with few calories, and their ability to heal infections. Rare sugars are found in small quantities in nature, and they exist typically as isomeric forms of traditional sugars, rendering some challenges in their isolation, synthesis, and characterization. In this work, we present the first direct mass spectrometric approach for differentiating structural isomers of sucrose that differ only by their glycosidic linkages. The method employed a noncontact nanoelectrospray (nESI) platform capable of analyzing minuscule volumes (5 µL) of saccharides via the formation of halide adducts ([M+X]-; X = Cl and Br). Tandem mass spectrometry analysis of the five structural isomers of sucrose afforded diagnostic fragment ions that can be used to distinguish each isomer. Detailed mechanisms showcasing the distinct fragmentation pattern for each isomer are discussed. The method was applied to characterize and confirm the presence of all five selected rare sugars in raw honey complex samples. Aside from the five natural α isomers of sucrose, the method was also suitable for differentiating some ß isomers of the same glycosidic linkages, provided the monomeric sugar units are different. The halide adduct formation via the noncontact nESI source was also proven to be effective for oligosaccharides such as raffinose, ß-cyclodextrin, and maltoheptaose. The results from this study encourage the future development of methods that function with simple operation to enable straightforward characterization of small quantities of rare sugars.

Capturing Fleeting Intermediates in a Claisen Rearrangement Using Nonequilibrium Droplet Imbibition Reaction Conditions.

The Claisen rearrangement of aromatic allyl phenyl ether to 2-allyl phenol is known to be induced by heat, acid, and air-water interfacial (on-water) effects. In this work, we show that the combination of acid and interfacial effects in an "on-droplet" experiment accelerates this reaction even further (by a factor >10×). The reaction acceleration was achieved through a droplet imbibition mass spectrometry (MS) experiment that allows reactants to be deposited on rapidly moving (100 m/s), charged microdroplets while avoiding turbulent mixing. In this case, reactants are concentrated mainly at the surface of the short-lived microdroplets (microseconds), enabling enhanced interfacial effects. By doping n-butylamine in the spray solvent and subsequently exposing the resultant electrosprayed microdroplets to formic acid vapor, the ketone intermediate, 6-allylcyclohexa-2,4-dien-1-one, involved in this Claisen rearrangement was captured and characterized by tandem MS, successfully differentiating it from the corresponding isobaric reactant (allyl phenyl ether) and product (2-allyl phenol). Similar results showing rate acceleration and subsequent capture of the ketone intermediate via an instantaneous reaction with n-butylamine were demonstrated for p-methyl and p-nitro substituted allyl phenyl ether. Density functional theory calculations confirmed that the on-droplet reaction condition, with a high abundance of proton sources, is different from the neutral rearrangement. With a calculated free energy of activation of 5.2 kcal mol-1 for the protonated reactant, the on-droplet experimental condition provides a unique mechanism for catalyzing the Claisen rearrangement on the microsecond lifetime of the droplets. This experiment marks the first direct capture and detection of a short-lived ketone intermediate in the Claisen rearrangement, a task that is challenged by a thermodynamically favorable tautomerization step to give a more stabilized product (by 20 kcal/mol).

Malaria Diagnosis Using Paper-Based Immunoassay for Clinical Blood Sampling and Analysis by a Miniature Mass Spectrometer.

In this work, we have developed a paper-based microfluidic device capable of remote biofluid collection followed by an analysis of the dried clinical samples using a miniature mass spectrometer. We have evaluated a portable mass spectrometer as a possible surveillance platform by analyzing the clinical malaria samples (whole blood) collected from Ghana. We synthesized pH-sensitive ionic probes and coupled them with monoclonal antibodies specific to the Plasmodium falciparum histidine-rich protein 2 (PfHRP2) malaria antigen. We then used the antibody-ionic probe conjugates in a paper-based immunoassay to capture PfHRP2 antigen from untreated whole blood. After the immunoassay, the bound ionic probes were cleaved, and the released mass tags were analyzed through an on-chip paper spray mass spectrometry strategy. During process optimization, we determined the detection limit for PfHRP2 in untreated human serum to be 0.216 nmol/L when using the miniature mass spectrometer. This sensitivity is comparable to the World Health Organization's suggested threshold of 0.227 nmol/L for PfHRP2, proving that our method will be applicable to diagnose symptomatic malaria infection (≥200 parasites per µL blood). The paper device can be stored at room temperature for at least 25 days without affecting the clinical outcome, with each stored paper chip offering good repeatability and reproducibility (RSD = 4-12%). The stability and sensitivity of the developed paper-based immunoassay platform will allow miniature mass spectrometers to be used for point-of-care malaria detection as well as in large-scale surveillance screening to aid eradication programs.

Reactive Thread Spray Mass Spectrometry for Localization of C═C Bonds in Free Fatty Acids: Applications for Obesity Diagnosis.

Cellulose thread substrates offer a platform for microsampling and reactive ionization of free fatty acid (FFA) isomers for direct differentiation by mass spectrometry. Ambient corona discharge forms when direct current high voltage is applied to the tiny subfibers on the thread substrate in the presence of a polar spray solvent (MeOH/H2O, 2:1, v/v), facilitating chemical reactions across a C═C bond of unsaturated fatty acids. The process was applied for diagnosis of obesity, which we observed to show better discriminatory power when compared to determinations based on body mass index. Overall, the integrated reactive thread-based platform is capable of (i) microsampling and dry-state, room-temperature storage (>30 days) of the biofluids, (ii) in-capillary liquid/liquid extraction, and (iii) in situ epoxidation reactions to locate the C═C bond position in unsaturated fatty acids via reactions with reactive oxygen species present in ambient corona discharge. The study showcased the ability to correctly characterize FFAs, including degree of unsaturation, and the determination of their relative concentrations in clinical biofluid samples.

Microsampling with a Solid-Phase Extraction Cartridge: Storage and Online Mass Spectrometry Analysis.

This study aims to introduce the concept of utilizing a solid-phase extraction (SPE) cartridge for remote biofluid collection, followed by direct sample analysis at a later time. For this, a dried matrix spot was prepared in a syringe, in the form of SPE cartridge for the first time to enable small biofluid collection (microsampling), storage, shipment, and online electrospray ionization (ESI) mass spectrometry (MS) analysis of the stored dried samples. The SPE sorbents were packed into an ESI syringe and the resultant cartridge was used for sampling small volumes (<20 µL) of different complex biological fluids including blood, plasma, serum, and urine. The collected sample was stored in the dry state within the confinement of the SPE sorbent at room temperature, and analyte stability (e.g., diazepam) was maintained for more than a year. Direct coupling of the SPE cartridge to MS provides excellent accuracy, precision, and sensitivity for analyzing illicit drugs present in the biofluid. The corresponding mechanism of wrong-way positive ion generation from highly basic elution solvents was explored. Without chromatography, our direct SPE-ESI-MS analysis technique afforded detection limits as low as 26 and 140 pg/mL for raw urine and untreated plasma, respectively. These promising results proved that the new syringe-based SPE cartridge can serve as a good alternative to conventional microsampling techniques in terms of analyte stability, ease of operation and versatility, and analytical sensitivity and speed.

Uncatalyzed N-Alkylation of Amines in Ionic Wind from Ambient Corona Discharge.

Ionic wind comprising of the drag of bulk air in the presence of electrical discharge enabled N-alkylation reactions under ambient conditions. By introducing reactant vapor as part of the discharge gas during the stages of electron acceleration, both neutral and charged species of the selected organic reactant gain energy through ion-neutral collisions, which is identified to facilitate chemical reactions. By performing this experiment in front of a mass spectrometer, chemical reactions occurring in the ionic wind were examined in real time. Reaction energetics were characterized via the use of benzylamine, which freely dissociates at a critical energy of 3.6 eV to give the resonance-stabilized benzyl cation as reaction intermediate. Benzylamine and many other primary amines were observed to undergo N-alkylation reactions by engaging in self-cross-coupling ion-molecule reactions. Because of the high energies of species involved and the fact that the ionic wind is generated at atmospheric pressure, it was straightforward to collect the ensuing reaction products without the use of complicated instrumentation. Water served as an effective collecting solvent allowing >0.1 mg of intact N-alkylated products to be collected under ambient conditions using a single plasma emitter. A novel N-alkylation reaction pathway involving the synthesis of N-benzyl-1-(methyleneamino)-1-phenylmethanaime was discovered through this offline product collection experiment, providing new insight into benzylamine dissociation in the ionic wind.

High-Throughput Mass Spectrometry Screening Platform for Discovering New Chemical Reactions under Uncatalyzed, Solvent-Free Experimental Conditions.

A gas-phase high-throughput reaction screening platform was developed for the first time to study chemical structures of closely related functional groups and for the discovery of novel organic reaction pathways. Experiments were performed using the contained atmospheric pressure chemical ionization (APCI) source that enabled nonthermal, nonequilibrium plasma chemistry to be monitored by mass spectrometry (MS) in real time. This contained-APCI MS platform allowed an array of reagents to be tested, resulting in the studies of multiple gas-phase reactions in parallel. By exposing headspace vapor of the selected reagents to corona discharge, solvent-free Borsche-Drecsel cyclization reaction, Katritzky chemistry, and Paal-Knorr pyrrole synthesis were examined in the gas phase, outside the high vacuum environment of the mass spectrometer. A new radical-mediated hydrazine coupling reaction was also discovered, which provided a selective pathway to synthesize secondary amines without using a catalyst. The mechanisms of these atmospheric pressure gas-phase reactions were explored through the direct capture of intermediates and via comparison with the corresponding bulk solution and droplet-phase reactions.

Direct Analysis of Doping Agents in Raw Urine Using Hydrophobic Paper Spray Mass Spectrometry.

In this study, the direct analysis of doping agents in urine samples with no sample preparation by a modified paper spray mass spectrometry (PS-MS) methodology has been demonstrated for the first time. We have described a paper surface treatment with trichloromethylsilane using a gas-phase reaction to increase the ionization of target compounds. This approach was applied for the analysis of two classes of banned substances in urine samples: anabolic agents (trenbolone and clenbuterol) and diuretics (furosemide and hydrochlorothiazide). Under optimized conditions, the developed methods presented satisfactory repeatability, and an analysis of variance showed linearity without lack-of-fit. Highly sensitive detections as low as sub-nanogram per milliliter levels, which is below the minimum required performance levels proposed by the World Anti-Doping Agency, have been reached using the hydrophobic PS-MS analysis without any preconcentration and cleanup step.

Droplet Imbibition Enables Nonequilibrium Interfacial Reactions in Charged Microdroplets.

A droplet imbibition experiment is proposed to study interfacial effects, which appears to be the main factor influencing reaction acceleration in charged microdroplets produced by electrospray ionization (ESI). One reagent is deposited onto the surface of rapidly moving microdroplets containing the second reagent to be reacted. In this manner, reactions are hindered from reaching equilibrium and monitored in real time by mass spectrometry. We demonstrated this phenomenon using Katritzky chemistry, which is known to proceed either by the solvent-stabilized 2H-pyran intermediate or via the surface-active pseudobase intermediate. Comparisons with reactions performed using ESI show obvious surface effects in favor of the droplet imbibition experiment. By keeping reactant mole ratio constant, it was demonstrated that similar interfacial effects observed in the droplet imbibition experiment can be reached by allowing ESI microdroplets containing premixed reagents to traverse a distance >16 mm. At such spray distance, molecular diffusion and droplet lifetime become comparable allowing reactants to be enriched at droplet surface. Reactions were also conducted in rapid mixing, theta capillary-based droplets, which showed markedly reduced yields compared with the interfacial droplet imbibition experiment.

Direct Mass Spectrometry Analysis of Complex Mixtures by Nanoelectrospray with Simultaneous Atmospheric Pressure Chemical Ionization and Electrophoretic Separation Capabilities.

Accurate and rapid analysis of complex microsamples are challenging tasks in translational research. Nanoelectrospray ionization (nESI) is the method of choice for analyzing small sample volumes by mass spectrometry (MS), but this technique works well only for polar analytes. Herein, we describe a versatile dual noncontact nESI/nAPCI (nanoatmospheric pressure chemical ionization) source that allows simultaneous detection of both polar and nonpolar analytes in microliter quantities of samples under ambient conditions and without pretreatment. The same device can be activated to enable electrophoretic separation. The noncontact nESI/nAPCI MS platform was applied to analyze different samples, including high sensitive direct analysis of biofluids and the efficient detection of proteins in buffers with high concentration of nonvolatile salts. Excellent linearity, accuracy and limits of detection were achieved for compounds with different chemical properties in different matrices. The high sensitivity, universality, simplicity, and ease of operation make this MS technique promising for use in clinical and forensic applications.

Reactive Olfaction Ambient Mass Spectrometry.

Chemical ionization of organic compounds with negligible vapor pressures (VP) is achieved at atmospheric pressure when the proximal sample is exposed to corona discharge. The vapor-phase analyte is produced through a reactive olfaction process, which is determined to include electrostatic charge induction in the proximal condensed-phase sample, resulting in the liberation of free particles. With no requirement for physical contact, a new contained nano-atmospheric pressure chemical ionization (nAPCI) source was developed that allowed direct mass spectrometry analysis of complex mixtures at a sample consumption rate less than nmol/min. The contained nAPCI source was applied to analyze a wide range of samples including the detection of 1 ng/mL cocaine in serum and 200 pg/mL caffeine in raw urine, as well as the differentiation of chemical composition of perfumes and beverages. Polar (e.g., carminic acid; estimated VP 5.1 × 10-25 kPa) and nonpolar (e.g., vitamin D2; VP 8.5 × 10-11 kPa) compounds were successfully ionized by the contained nAPCI ion source under ambient conditions, with the corresponding ion types of 78 other organic compounds characterized.

Re-configurable, multi-mode contained-electrospray ionization for protein folding and unfolding on the millisecond time scale.

A new reconfigurable contained-electrospray (ES) ion source is described that can be operated in three unique modes (Types I, II and III) and was applied to control the charge state of proteins for subsequent online characterization by mass spectrometry. Using this device, proteins prepared in 100% water were highly charged after exposure to hydrochloric acid vapor. For myoglobin, the shift to a higher charge state occurred faster than the heme cofactor could escape the unfolding protein. This effect reflected in the detection of highly charged holo-myoglobin intermediates (+26), which suggests the modification process occurred on a millisecond time scale (Type I mode). By introducing a cavity in the contained-ES emitter (Type II mode), increased protein denaturation was observed where only apo-myoglobin ions were detected. A similar increase in the charge effect was observed for less pH sensitive proteins such as ubiquitin and cytochrome c. In a third type of experiment, acidified aqueous-based droplets containing unfolded proteins were briefly exposed to aqueous ammonium acetate/triethylammonium acetate solution mixtures to increase the droplet pH and to induce protein folding during electrospray ionization. Charge-state distributions showed protein folding occurred, including the appearance of charged reduced species that were not observed when sprayed in pure water. The three operational modes of contained-electrospray allow online modification of an analyte during charge droplet formation using both vapor and non-volatile liquid reagents, without a direct addition of the modifying reagent to the analyte solution.

Reactive Charged Droplets for Reduction of Matrix Effects in Electrospray Ionization Mass Spectrometry.

A new quantitative contained-electrospray (ES) process is described here that employs a movable ES emitter to control the reactivity of charged microdroplets by varying their exposure time with acid vapor. The method allows elimination of ion suppression effects caused by the presence of various surface active compounds that coelute with the analyte. For mixtures, contained-ESI mass spectrometric analysis produces relative ion intensities that reflect the true concentrations of analytes in solution. The mechanism for this effect has been elucidated and ascribed to the generation of fine initial droplets in the presence of a high abundance of protons; together, these two factors eliminate competition for charge and space during ion formation. Examples of analytes tested include steroids, phospholipids, phosphopeptides, and sialylated glycans. At least 1 order of magnitude improvement in detection limits, sensitivity, and accuracy of detection was observed when compared to conventional electrospray.