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.
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.
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.
Accelerated droplet chemistry aids chromatographic retention time data and tandem mass spectrometry to differentiate saccharide isomers.
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.
This study aims to understand the fate and transport of per- and polyfluoroalkyl substances (PFAS) and inorganic fluoride (IF) at an undisclosed municipal wastewater treatment plant (WWTP) operating ...a sewage sludge incinerator (SSI). A robust statistical analysis characterized concentrations and mass flows at all WWTP and SSI primary influents/effluents, including thermal-treatment derived airborne emissions. WWTP-level net mass flows (NMFs) of total PFAS were not statistically different from zero. SSI-level NMFs indicate that PFAS, and specifically perfluoroalkyl acids (PFAAs), are being broken down. The NMF of perfluoroalkyl sulfonic acids (PFSAs; −274 ± 34 mg/day) was statistically significant. The observed breakdown primarily occurred in the sewage sludge. However, the total PFAS destruction and removal efficiency of 51 % indicates the SSI may inadequately remove PFAS. The statistically significant IF source (NMF = 16 ± 4.2 kg/day) compared to the sink of PFAS as fluoride (NMF = −0.00036 kg/day) suggests that other fluorine-containing substances are breaking down in the SSI. WWTP PFAS mass discharges were primarily to the aquatic environment (>99 %), with <0.5 % emitted to the atmosphere/landfill. Emission rates for formerly phased-out PFOS and PFOA were compared to previously reported levels. Given the environmental persistence of these compounds, the observed decreases in PFOS and PFOA discharge rates from prior reports implies regional/local differences in emissions or possibly their accumulation elsewhere. PFAS were observed in stack gas emissions, but modestly contributed to NMFs and showed negligible contribution to ambient air concentrations observed downwind.
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•The fate and transport of PFAS at WWTPs operating SSIs is an important data gap.•Study measured PFAS and IF at all influents/effluents of a WWTP operating an SSI.•Statistical analyses characterized concentrations and mass flows at the WWTP/SSI.•Most PFAS mass discharged in aqueous effluent; small contributions to air/landfill.•The SSI may inadequately destroy PFAS, as the observed thermal removal was 51 %.
Diisopropylfluorophosphate (DFP) is a potent acetylcholinesterase inhibitor commonly used in toxicological studies as an organophosphorus nerve agent surrogate. However, LD50 values for DFP in the ...same species can differ widely even within the same laboratory, possibly due to the use of degraded DFP. The objectives here were to identify an efficient synthesis route for high purity DFP and assess the storage stability of both the in-house synthesized and commercial source of DFP at the manufacturer-recommended storage temperature of 4°C, as well as −10°C and −80°C. After 393 days, the commercial DFP stored at 4°C experienced significant degradation, while only minor degradation was observed at −10°C and none was observed at −80°C. DFP prepared using the newly identified synthesis route was significantly more stable, exhibiting only minor degradation at 4°C and none at −10°C or −80°C. The major degradation product was the monoacid derivative diisopropylphosphate, formed via hydrolysis of DFP. It was also found that storing DFP in glass containers may accelerate the degradation process by generating water in situ as hydrolytically generated hydrofluoric acid attacks the silica in the glass. Based on the results here, it is recommended that DFP be stored at or below −10°C, preferably in air-tight, nonglass containers.
As Analytical Chemists we are constantly demanding more of our methods: greater selectivity, lower detection limits, wider utility, reduced complexity, etc. We are frequently tasked with meeting the ...ever-changing demands of the many evolving fields that require analytical expertise including the medical, environmental, forensic, and consumer industries. In many cases, new techniques are introduced, or old methods are refined and matured to tackle these challenges. However, there are still times when the development of new and improved instrumentation lags behind demand, and traditional chemical principles must be used to bolster current methods. Chemical derivatization is a well-established approach to overcoming the limitations of available instrumentation and methods. It is commonly used to expand detectability into adjacent areas in the chemical space, to drive signal enhancement for analytes that exhibit low sensitivity, and to improve selectivity in separations. However, conventional protocols can be resource intensive, complex, and time-consuming. The overarching aim of this dissertation is to leverage the virtues of derivatization while mitigating its shortcomings by shifting the procedure from the benchtop to the analytical platform. We do this using a specialized electrospray platform that allows us to carry out chemical reactions rapidly and online during the analysis. Although this approach can be useful for a wide range of compounds, we focus on saccharides due to their broad physiological importance. Recent potential applications in disease diagnosis and monitoring have brought about a need for new analytical tools capable of detecting saccharides at low concentrations and/or for volume-limited samples. Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) is the preferred methodology for these compounds due its inherent specificity. However, saccharides generally exhibit limited sensitivity in LC-ESI-MS because they are not easily ionized. In Chapter 2, we employ a coaxially configured, contained-electrospray ionization (contained-ESI) source to carry out online, droplet-based chemical derivatization to enhance sensitivity for the direct infusion analysis of saccharides. Derivatization is carried out in real-time within the electrospray microdroplets during ion formation. This is possible due to the reaction rate acceleration afforded by desolvating electrospray microdroplets, allowing reactions to occur in high yield on the seconds to milliseconds timescale. Signal enhancements of greater than two orders of magnitude were observed for many mono- and disaccharides using in-source, droplet-based phenylboronic acid (PBA) derivatization, resulting in parts-per-trillion (picomolar) limits of detection. In addition, isobaric sugars such as lactose and sucrose were easily distinguished based on their unique product ion spectra. In Chapter 3, we couple the contained-ESI source to a traditional LC-MS platform for the online and automated separation, derivatization, and detection of various mono-, di-, and oligosaccharides. The LC-contained-ESI-MS platform with in-source, droplet-based derivatization provides a complete analytical system capable of highly specific and sensitive analysis of complex matrices. PBA derivatization is performed online, within the contained-ESI source as the sugars elute from the LC column. The new platform offers a significant enhancement in sensitivity when compared to conventional LC-ESI-MS/MS without derivatization and is found to be accurate and precise over linear ranges of at least two orders of magnitude. In Chapter 4, the utility of the LC-contained-ESI-MS platform for complex sample analysis is demonstrated in two separate applications: (1) the targeted analysis of select oligosaccharides in complex physiological samples including human urine and plasma, and (2) the differentiation of several disaccharide isomers in two orthogonal dimensions: chromatographic retention and tandem MS product ion formation.
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