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.

Welcome to the next generation of Malaria Rapid Diagnostic Tests: Comparative Analysis of NxTek Eliminate Malaria P.f, Biocredit Malaria Ag Pf, and SD Bioline Malaria Ag Pf for Plasmodium falciparum Diagnosis in Ghana.

Background: Accurate diagnosis and timely treatment are crucial in combating malaria. Methods: We evaluated the diagnostic performance of three Rapid Diagnostic Tests (RDTs) in diagnosing febrile patients, namely: Abbott NxTek Eliminate Malaria Ag Pf (detecting HRP2), Rapigen Biocredit Malaria Ag Pf (detecting HRP2 and LDH on separate bands), and SD Bioline Malaria Ag Pf (detecting HRP2). Results were compared to qPCR. Results: Among 449 clinical patients, 45.7% (205/449) tested positive by qPCR for P. falciparum with a mean parasite density of 12.5parasites/µL. The sensitivity of the Biocredit RDT was 52.2% (107/205), NxTek RDT was 49.3% (101/205), and Bioline RDT was 40.5% (83/205). When samples with parasite densities lower than 20 parasites/uL were excluded (n=116), a sensitivity of 88.8% (79/89, NxTek), 89.9% (80/89, Biocredit), and 78.7% (70/89, Bioline) was obtained. All three RDTs demonstrated specificity above 95%. The limits of detection was 84 parasites/µL (NxTek), 56 parasites/µL (Biocredit, considering either HRP2 or LDH), and 331 parasites/µL (Bioline). None of the three qPCR-confirmed P. falciparum positive samples, identified solely through the LDH target, carried hrp2/3 deletions. Conclusion: The Biocredit and NxTek RDTs demonstrated comparable diagnostic efficacies and both RDTs performed better than Bioline RDT.

Dual Tunability for Uncatalyzed N-Alkylation of Primary Amines Enabled by Plasma-Microdroplet Fusion.

The fusion of non-thermal plasma with charged microdroplets facilitates catalyst-free N-alkylation for a variety of primary amines, without halide salt biproduct generation. Significant reaction enhancement (up to >200×) is observed over microdroplet reactions generated from electrospray. This enhancement for the plasma-microdroplet system is attributed to the combined effects of energetic collisions and the presence of reactive oxygen species (ROS). The ROS (e.g., O2 ⋅- ) act as a proton sink to increase abundance of free neutral amines in the charged microdroplet environment. The effect of ROS on N-alkylation is confirmed through three unique experiments: (i) utilization of radical scavenging reagent, (ii) characterization of internal energy distribution, and (iii) controls performed without plasma, which lacked reaction acceleration. Establishing plasma discharge in the wake of charged microdroplets as a green synthetic methodology overcomes two major challenges within conventional gas-phase plasma chemistry, including the lack of selectivity and product scale-up. Both limitations are overcome here, where dual tunability is achieved by controlling reagent concentration and residence time in the microdroplet environment, affording single or double N-alkylated products. Products are readily collected yielding milligram quantities in eight hours. These results showcase a novel synthetic strategy that represents a straightforward and sustainable C-N bond-forming process.

Two-dimensional isomer differentiation using liquid chromatography-tandem mass spectrometry with in-source, droplet-based derivatization.

Saccharides are increasingly used as biomarkers and for therapeutic purposes. Their characterization is challenging due to their low ionization efficiencies and inherent structural heterogeneity. Here, we illustrate how the coupling of online droplet-based reaction, in a form of contained electrospray (ES) ion source, with liquid chromatography (LC) tandem mass spectrometry (MS/MS) allows the comprehensive characterization of sucrose isomers. We used the reaction between phenylboronic acid and cis-diols for on-the-fly derivatization of saccharides eluting from the LC column followed by in situ MS/MS analysis, which afforded diagnostic fragment ions that enabled differentiation of species indistinguishable by chromatography or mass spectrometry alone. For example, chromatograms differing only by 2% in retention times were flagged to be different based on incompatible MS/MS fragmentation patterns. This orthogonal LC-contained-ES-MS/MS method was applied to confirm the presence of turanose, palatinose, maltulose, and maltose, which are structural isomers of sucrose, in three different honey samples. The reported workflow does not require modification to existing mass spectrometers, and the contained-ES platform itself acts both as the ion source and the reactor, all promising widespread application.

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.

Plasma-Droplet Reaction Systems: A Direct Mass Spectrometry Approach for Enhanced Characterization of Lipids at Multiple Isomer Levels.

Neutral triacylglyceride (TG) lipids are critical in cellular function, signaling, and energy storage. Multiple molecular pathways control TG structure via nonselective routes making them structurally complex and analytically challenging to characterize. The presence of C=C bond positional isomers exacerbates this challenge as complete structural elucidation is not possible by conventional tandem mass spectrometric methods such as collision-induced dissociation (CID), alone. Herein, we report a custom-made coaxial contained-electrospray ionization (ESI) emitter that allows the fusion of plasma discharge with charged microdroplets during electrospray (ES). Etched capillaries were incorporated into this contained-ES emitter, facilitating the generation of reactive oxygen species (ROS) at low (3 kV) ESI voltages and allowing stable ESI ion signal to be achieved at an unprecedented high (7 kV) spray voltage. The analytical utility of inducing plasma discharge during electrospray was investigated using online ionization of neutral TGs, in situ epoxidation of unsaturation sites, and C=C bond localization via conventional CID mass spectrometry. Collisional activation of the lipid epoxide generated during the online plasma-droplet fusion experiment resulted in a novel fragmentation pattern that showed a quadruplet of diagnostic ions for confident assignment of C=C bond positions and subsequent isomer differentiation. This phenomenon enabled the identification of a novel TG lipid, composed of conjugated linoleic acid, that is isomeric with two other TG lipids naturally found in extra virgin olive oil. To validate our findings, we analyzed various standards of TG lipids, including triolein, trilinolein, and trilinolenin, and isomeric mixtures in the positive-ion mode, each of which produced the expected quadruplet diagnostic fragment ions. Further validation was obtained by analyzing standards of free fatty acids expected from the hydrolysis of the TG lipids in the negative-ion mode, together with isomeric mixtures. The chemistry governing the gas-phase fragmentation of the lipid epoxides was carefully elucidated for each TG lipid analyzed. This comprehensive shotgun lipidomic approach has the potential to impact biomedical research since it can be accomplished on readily available mass spectrometers without the need for instrument modification.

Online Cross-Linking of Peptides and Proteins during Contained-Electrospray Ionization Mass Spectrometry.

Recent advancements in mass spectrometry (MS) now enable all levels of protein structures to be characterized, including primary protein sequence, post-translational modifications, and three-dimensional protein conformations. However, protein conformational studies by MS require the use of many separate techniques that are performed independently of each other. Herein, we described a contained-electrospray (ES) experiment that has potential to integrate peptide/protein cross-linking with the general MS workflow. In our experiment, cross-linking of protein/peptide occurs simultaneously with ionization after analytes, and cross-linkers are sprayed from two separate ES emitters. The online cross-linking process occurring in the charged microdroplet environment was optimized using trilysine peptide and bis(sulfosuccinimidyl)suberate cross-linker. We detected the electrostatic complex between analyte and cross-linker, the mono-linked intermediate, and the fully cross-linked product, allowing us to correctly predict the sequence of reaction events in the cross-linking process. Importantly, we observed that the terminal fully cross-linked product is composed of two distinct conformations. In one form, the product involved cross-linking between two ε-NH2 amines in lysine residues, while the other conformer was formed by a reaction between one ε-NH2 amine and the N-terminus. The experimental conditions for selecting one cross-linked species over others during the online ES ionization-MS analysis have been detailed. Appropriate parameters enabled the reaction between α-lactalbumin proteins and cross-linkers using a non-denaturing spray condition. These results establish a framework for a future development in high-throughput structural MS method, where all levels of protein information can be gathered in a single experiment.

Mass spectrometry for metabolomics analysis: Applications in neonatal and cancer screening.

Chemical analysis by analytical instrumentation has played a major role in disease diagnosis, which is a necessary step for disease treatment. While the treatment process often targets specific organs or compounds, the diagnostic step can occur through various means, including physical or chemical examination. Chemically, the genome may be evaluated to give information about potential genetic outcomes, the transcriptome to provide information about expression actively occurring, the proteome to offer insight on functions causing metabolite expression, or the metabolome to provide a picture of both past and ongoing physiological function in the body. Mass spectrometry (MS) has been elevated among other analytical instrumentation because it can be used to evaluate all four biological machineries of the body. In addition, MS provides enhanced sensitivity, selectivity, versatility, and speed for rapid turnaround time, qualities that are important for instance in clinical procedures involving the diagnosis of a pediatric patient in intensive care or a cancer patient undergoing surgery. In this review, we provide a summary of the use of MS to evaluate biomarkers for newborn screening and cancer diagnosis. As many reviews have recently appeared focusing on MS methods and instrumentation for metabolite analysis, we sought to describe the biological basis for many metabolomic and additional omics biomarkers used in newborn screening and how tandem MS methods have recently been applied, in comparison to traditional methods. Similar comparison is done for cancer screening, with emphasis on emerging MS approaches that allow biological fluids, tissues, and breath to be analyzed for the presence of diagnostic metabolites yielding insight for treatment options based on the understanding of prior and current physiological functions of the body.

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.

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).

Liquid Chromatography-Tandem Mass Spectrometry with Online, In-Source Droplet-Based Phenylboronic Acid Derivatization for Sensitive Analysis of Saccharides.

The ability to identify abnormalities in the body's saccharide profile is a promising means for early disease detection but requires analytical tools capable of detecting saccharides at low concentrations and/or for volume-limited samples. The preferred analysis approach for these compounds, liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), often lacks sensitivity due to poor ionization efficiency. In this work, we employ a modified electrospray interface-termed contained-electrospray (contained-ESI) to couple accelerated droplet chemistry to conventional LC-MS for the online and automated separation, derivatization, and detection of saccharides. The chromatographic component enables complex sample and mixtures analysis with low sample volume requirements, while the enhanced reaction kinetics afforded by electrosprayed microdroplets facilitates rapid, on-the-fly derivatization to boost sensitivity. Derivatization occurs during ion formation as analytes elute from the column, eliminating the need for superfluous post-column derivatization hardware or complicated benchtop protocols. A grounded coupler was incorporated to shield the LC from the high-voltage ion source, and method conditions were optimized to accommodate the low flow rates preferred for microdroplet reactions. The new LC-contained-ESI-MS/MS platform was demonstrated for the analysis of several mono-, di-, and oligosaccharides using in-source droplet-based phenylboronic acid derivatization. Femtomole limits of detection were achieved for a 1 µL injection, representing sensitivity enhancement of 1-2 orders of magnitude over conventional LC-ESI-MS/MS without derivatization. In addition, isobaric saccharides that are difficult to differentiate by MS alone were easily distinguished. Method precision, accuracy, and linearity were established, and the ability to detect oligosaccharides at trace levels in human urine and plasma was demonstrated.

Assessment of creatinine concentration in whole blood spheroids using paper spray ionization-tandem mass spectrometry.

Accurate quantification of blood creatinine is important to estimate the glomerular filtration rate. Existing techniques using liquid chromatography tandem mass spectrometry (LC-MS/MS) have a high accuracy and eliminate most interferences encountered in routine enzymatic and Jaffé methods. However, they require laborious and time-consuming sample treatment and data acquisition. The aim of this study is to develop a fast and simple method to enable a direct analysis of whole blood creatinine with performance measures that are comparable to conventional LC-MS/MS. 5µL whole blood is formed as a three-dimensional spheroid on hydrophobic silanized paper substrates which then undergoes paper-spray ionization-tandem mass spectrometry (PSI-MS/MS). The method is validated using real human samples and compared with LC-MS/MS. PSI-MS/MS whole blood analysis exhibited a lower limit of quantification of 2.5 µg/mL, precision ≤ 6.3%, recovery in the range of 88-94% and excellent linearity (R2 > 0.99; 2.5-20 µg/mL) covering the normal range for creatinine levels. Creatinine levels were comparable to those measured by LC-MS/MS with small deviations of less than 0.3 µg/mL. This simple, fast and accurate microsampling technique for direct analysis of creatinine from whole blood shows promise for routine clinical screening and monitoring. This approach can be readily extended for other analytes of interest and, due to inherent advantages relating to cost, storability, speed, and simplicity, it can be especially advantageous for use in resource-limited settings.

The Effect of the Physical Morphology of Dried Biofluids on the Chemical Stability of Analytes Stored in Paper and Direct Analysis by Mass Spectrometry.

Three-dimensional (3D) dried blood spheroids formed on hydrophobic paper are a new microsampling platform that can stabilize labile molecules in whole blood stored in ambient air at room temperature. In this study, we define the ideal conditions for preparing the dried blood spheroids. The physical morphology of 3D dried blood spheroids is found to be largely impacted by the unregulated relative humidity of the surrounding environment. A solution of KOH placed in an enclosed chamber offers a facile way to control humidity. We also report a general polymer coating strategy that serves to stabilize dried biofluids when prepared under ordinary ambient conditions without regulation of humidity. Dried blood spheroids coated in xanthan gum polymer exhibited enhanced chemical and physical stability. The same xanthan gum polymer provided chemical stability for 2D dried blood spots when compared with the conventional noncoated samples. We have expanded the application of xanthan gum to less viscous biofluids such as urine to induce an artificial protective barrier that also provides enhanced stability for labile performance-enhancing drugs stored at room temperature. The impact of polymer coating on direct biofluid analysis via paper spray mass spectrometry was determined by comparing the relative ionization efficiency, percent difference of ionization efficiency, and matrix effects of performance-enhancing drugs that were spiked in undiluted raw urine. Acceptable analytical performance was recorded for all three criteria, including high ionization efficiencies that ranged from 77 to 93% in the presence of the xanthan gum polymer.

Automated Immunoassay Performed on a 3D Microfluidic Paper-Based Device for Malaria Detection by Ambient Mass Spectrometry.

Microfluidic paper-based analytical devices (µPADs) are emerging as a prominent platform for disease detection, specifically in developing countries. This paper device offer simplicity and affordability not typically seen in centralized laboratory settings. However, detection limits in µPADs are inadequate and often require test results to be read within a specific time interval to ensure accuracy. To overcome these challenges, we are developing an on-chip mass spectrometry (MS) detection strategy for immunoassays performed on paper substrates. Herein, we present our initial results from a proof-of-concept study toward the development of µPADs capable of storing immunoassay reagents within the confinements of the 3D device, automatic splitting of biofluid into four individual test zones, immuno-capture of the disease biomarker, and on-chip MS detection of the captured species. The reported study encourages the development of point-of-care and direct-to-customer testing using disposable µPADs to collect samples, followed by sensitive analysis using portable MSs. We demonstrate this capability using malaria Plasmodium falciparum histidine-rich protein 2 (PfHRP2) antigen detection.

Utility of machine learning in developing a predictive model for early-age-onset colorectal neoplasia using electronic health records.

BACKGROUND AND AIMS: The incidence of colorectal cancer (CRC) is increasing in adults younger than 50, and early screening remains challenging due to cost and under-utilization. To identify individuals aged 35-50 years who may benefit from early screening, we developed a prediction model using machine learning and electronic health record (EHR)-derived factors. METHODS: We enrolled 3,116 adults aged 35-50 at average-risk for CRC and underwent colonoscopy between 2017-2020 at a single center. Prediction outcomes were (1) CRC and (2) CRC or high-risk polyps. We derived our predictors from EHRs (e.g., demographics, obesity, laboratory values, medications, and zip code-derived factors). We constructed four machine learning-based models using a training set (random sample of 70% of participants): regularized discriminant analysis, random forest, neural network, and gradient boosting decision tree. In the testing set (remaining 30% of participants), we measured predictive performance by comparing C-statistics to a reference model (logistic regression). RESULTS: The study sample was 55.1% female, 32.8% non-white, and included 16 (0.05%) CRC cases and 478 (15.3%) cases of CRC or high-risk polyps. All machine learning models predicted CRC with higher discriminative ability compared to the reference model [e.g., C-statistics (95%CI); neural network: 0.75 (0.48-1.00) vs. reference: 0.43 (0.18-0.67); P = 0.07] Furthermore, all machine learning approaches, except for gradient boosting, predicted CRC or high-risk polyps significantly better than the reference model [e.g., C-statistics (95%CI); regularized discriminant analysis: 0.64 (0.59-0.69) vs. reference: 0.55 (0.50-0.59); P<0.0015]. The most important predictive variables in the regularized discriminant analysis model for CRC or high-risk polyps were income per zip code, the colonoscopy indication, and body mass index quartiles. DISCUSSION: Machine learning can predict CRC risk in adults aged 35-50 using EHR with improved discrimination. Further development of our model is needed, followed by validation in a primary-care setting, before clinical application.

Embossed Paper Platform for Whole Blood Collection, Room Temperature Storage, and Direct Analysis by Pinhole Paper Spray Mass Spectrometry.

Dry-state microsampling techniques are convenient and advantageous for sample collection in resource-limited settings, including healthcare systems designed for the underserved population. In this work, a microsampling platform based on an embossed hydrophobic paper substrate is introduced together with three-dimensional (3D) printed cartridges that offer opportunities for rapid (<30 min) drying of the collected samples while also preserving sample integrity when the embossed paper chip is shipped at room temperature. More importantly, a new pinhole paper spray ionization method was developed that facilitates direct mass spectrometry (MS) analysis of the dried blood samples without prior sample preparation. We compared the direct pinhole paper spray MS method with a liquid chromatographic (LC) MS approach that relied upon electrospray ionization (ESI) after analytes present in the blood sample were extracted through liquid-liquid extraction. Limits of detection as low as 0.12 and 0.49 ng/mL were calculated for cocaine and its metabolite benzoylecgonine, respectively, when using the direct pinhole paper spray MS method. Analytical merits such as precision and accuracy, recovery, carryover effects, and analyte stability were all quantified for this new paper spray method and compared to the traditional LC-ESI-MS. Although LC-ESI-MS was observed to be 10× more sensitive, the linear dynamic range for both methods was determined to be the same, in the range of 1-500 ng/mL for both cocaine and benzoylecgonine analytes. When fully developed, the current microsampling strategy could offer an easy-to-use kit that can enable a more effective MS analysis of 20 µL dried blood samples delivered by mail. Both sensitivity (10×) and sample stability are found to be more superior for blood prepared in the embossed hydrophobic paper compared to samples prepared in the planar hydrophilic paper.

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.

In-Source Microdroplet Derivatization Using Coaxial Contained-Electrospray Mass Spectrometry for Enhanced Sensitivity in Saccharide Analysis.

Online, droplet-based in-source chemical derivatization is accomplished using a coaxial-flow contained-electrospray ionization (contained-ESI) source to enhance sensitivity for the mass spectrometric analysis of saccharides. Derivatization is completed in microseconds by exploiting the reaction rate acceleration afforded by electrospray microdroplets. Significant improvements in method sensitivity are realized with minimal sample preparation and few resources when compared to traditional benchtop derivatizations. For this work, the formation of easily ionizable phenylboronate ester derivatives of several mono-, di-, and oligosaccharides is achieved. Various reaction parameters including concentration and pH were evaluated, and a Design of Experiments approach was used to optimize ion source parameters. Signal enhancements of greater than two orders of magnitude were observed for many mono- and disaccharides using in-source phenylboronic acid derivatization, resulting in parts-per-trillion (picomolar) limits of detection. In addition, amino sugars such as glucosamine, which do not ionize in negative mode, were detected at low parts-per-billion concentrations, and isobaric sugars such as lactose and sucrose were easily distinguished. The new in-source derivatization approach can be employed to expand the utility of ESI-MS analysis for compounds that historically experience limited sensitivity and detectability, while avoiding resource-intensive, bulk-phase derivatization procedures.