PAMAM-Functionalized Paper as a New Substrate for the Paper Spray Mass Spectrometry Measurement of Proteins.

Paper surface functionalization with polyamidoamine (PAMAM) dendrimers has been developed for increased sensitivity analysis of proteins by paper spray mass spectrometry (PS-MS). PAMAM is a branched polymeric compound with an ethylenediamine core linked to repeating PAMAM units that generates an outer surface rich in primary amines. These positively charged amine groups can interact electrostatically with negatively charged residues (e.g., aspartate, glutamate) on the protein surface. PAMAM inner amide moieties can also promote hydrogen bonding with protein surface oxygens, making PAMAM a useful material for protein extraction. PAMAM-functionalized PS-MS paper strips were used to extract proteins from biofluids, dipped in acetonitrile to remove unbound constituents, dried, and then measured with PS-MS. The use of this strategy was optimized and compared with unmodified paper strips. PAMAM-functionalized paper substrates provided sixfold greater sensitivity for albumin, 11-fold for hemoglobin, sevenfold for insulin, and twofold for lysozyme. The analytical performance of the functionalized paper substrate was evaluated through the analysis of albumin in urine, achieving linearity with R2 > 0.99, LOD of 1.1 µg mL-1, LOQ of 3.8 µg mL-1, precision better than 10%, and relative recovery 70-83%. The method was applied to quantify urinary albumin from nine anonymous patient samples (concentrations ranged from 6.5 to 77.4 µg mL-1), illustrating its potential for the diagnosis of microalbuminuria. These data demonstrate the utility of paper modification with the PAMAM dendrimer for sensitive PS-MS analysis of proteins, opening a path for further applications in clinical diagnosis through the analysis of disease-related proteins.

Direct Analysis of Polyaromatic Hydrocarbons in Soil and Aqueous Samples Using Condensed Phase Membrane Introduction Tandem Mass Spectrometry with Low-Energy Liquid Electron Ionization.

Polyaromatic hydrocarbons (PAHs) are listed as priority pollutants by the United States Environmental Protection Agency (U.S. EPA). PAH-contaminated samples often require extensive sample cleanup before analysis, with the method used dependent upon the sample matrix. We present condensed phase membrane introduction mass spectrometry with liquid electron ionization (CP-MIMS-LEI) as a sensitive and universal technique that can directly analyze both aqueous and soil samples for PAHs without the need for sample clean up or instrumental modifications for different matrixes. The method uses a semipermeable hollow fiber membrane immersion probe to transfer analytes from complex samples into a solvent acceptor phase that is directly entrained at nanoliter/min flows to an LEI-interfaced mass spectrometer. The resulting aerosol is desolvated under vacuum leading to analyte vaporization and subsequent electron ionization. Electron energy and LEI vaporization capillary position were examined and optimized for PAHs. The CP-MIMS probe was directly immersed in complex aqueous matrixes, demonstrating low nanogram per liter PAH detection limits and response times of ≤1.6 min. For soil sample analysis, 2-propanol was found to be the optimal PAH sampling solvent. Soil samples were briefly sonicated in 2-propanol, followed by direct CP-MIMS measurement. Soil sample throughput was ca. 15 samples/h, with PAH quantitation successful at microgram per kilogram levels. The workflow is remarkably simple, fast, green, and leads to reproducible results that enable high-throughput screening of heterogeneous soil samples.

Automated, High-Throughput Analysis of Tire-Derived p-Phenylenediamine Quinones (PPDQs) in Water by Online Membrane Sampling Coupled to MS/MS.

The tire-derived contaminant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ) was recently identified as a potent toxin to coho salmon (Oncorhynchus kisutch). Studies investigating 6-PPDQ have employed solid-phase extraction (SPE) or liquid-liquid extraction (LLE) with liquid chromatography-mass spectrometry (LC-MS), providing excellent sensitivity and selectivity. However, cleanup and pre-enrichment steps (SPE/LLE) followed by chromatographic separation can be time- and cost-intensive, limiting sample throughput. The ubiquitous distribution of 6-PPDQ necessitates numerous measurements to identify hotspots for targeted mitigation. We recently developed condensed phase membrane introduction mass spectrometry (CP-MIMS) for rapid 6-PPDQ analysis (2.5 min/sample), with a simple workflow and low limit of detection (8 ng/L). Here, we describe improved quantitation using isotopically labeled internal standards and inclusion of a suite of PPDQ analogues. A low-cost autosampler and data processing software were developed from a three-dimensional (3D) printer and Matlab to fully realize the high-throughput capabilities of CP-MIMS. Cross-validation with a commercial LC-MS method for 10 surface waters provides excellent agreement (slope: 1.01; R2 = 0.992). We employ this analytical approach to probe fundamental questions regarding sample stability and sorption of 6-PPDQ under lab-controlled conditions. Further, the results for 192 surface water samples provide the first spatiotemporal characterization of PPDQs on Vancouver Island and the lower mainland of British Columbia.

Calcite-Assisted Localization and Kinetics (CLocK) Microscopy.

Localization-based super-resolution imaging techniques have improved the spatial resolution of optical microscopy well below the diffraction limit, yet encoding additional information into super-resolved images, such as anisotropy and orientation, remains a challenge. Here we introduce calcite-assisted localization and kinetics (CLocK) microscopy, a multiparameter super-resolution imaging technique easily integrated into any existing optical microscope setup at low cost and with straightforward analysis. By placing a rotating calcite crystal in the infinity space of an optical microscope, CLocK microscopy provides immediate polarization and orientation information while maintaining the ability to localize an emitter/scatterer with <10 nm resolution. Further, kinetic information an order of magnitude shorter than the integration time of the camera is encoded in the unique point spread function of a CLocK image, allowing for new mechanistic insight into dynamic processes such as single-nanoparticle dissolution and single-molecule surface-enhanced Raman scattering.

Aqueous naphthenic acids and polycyclic aromatic hydrocarbons in a meso-scale spill tank affected by diluted bitumen analyzed directly by membrane introduction mass spectrometry.

With the increasing use of unconventional, heavy crude oils there is growing interest in potential impacts of a diluted bitumen (DB) spill in marine and freshwater environments. DB has the potential to release several toxic, trace organic contaminants to the water column. Here, the aqueous concentrations and compositions of two classes of organic contaminants, naphthenic acids (NAs) and polycyclic aromatic hydrocarbons (PAHs), are followed over 8 weeks after a simulated spill of DB (10 L) into a freshwater mesocosm (1200 L) with river sediment (2.4 kg). These complex samples contain biogenic dissolved organic matter, inorganic ions, petroleum contaminants, suspended sediments, and oil droplets. We report the first use of condensed phase membrane introduction mass spectrometry (CP-MIMS) as a direct sampling platform in a complex multi-phase mesocosm spill tank study to measure trace aqueous phase contaminants with little to no sample preparation (dilution and/or pH adjustment). CP-MIMS provides complementary strengths to conventional analytical approaches (e.g., gas- or liquid chromatography mass spectrometry) by allowing the entire sample series to be screened quickly. Trace NAs are measured as carboxylates ([M-H]-) using electrospray ionization and PAHs are detected as radical cations (M+•) using liquid electron ionization coupled to a triple quadrupole mass spectrometer. The DB-affected mesocosm exhibits NA concentrations from 0.3 to 1.2 mg/L, which rise quickly over the first 2 - 5 days , then decrease slowly over the remainder of the study period. The NA profile (measured as the full scan in negative-electrospray ionization at nominal mass resolution) shifts to lower m/z with weathering, a process followed by principal component analysis of the normalized mass spectra. We couple CP-MIMS with high-resolution mass spectrometry to follow changes in molecular speciation over time, which reveals a concomitant shift from classical 'O2' naphthenic acids to more oxidized analogues. Concentrations of PAHs and alkylated analogues (C1 - C4) in the DB-affected water range from 0 to 5 µg/L. Changes in PAH concentrations depend on ring number and degree of alkylation, with small and/or lightly alkylated (C0 - C2) PAH concentrations rising to a maximum in the first 4 - 8 days (100 - 200 h) before slowly decaying over the remainder of the study period. Larger and heavily alkylated (C3 - C4) PAH concentrations generally rise slower, with some species remaining below the detection limit throughout the study period (e.g., C20H12 class including benzo[a]pyrene). In contrast, a control mesocosm (without oil) exhibited NA concentrations below 0.05 mg/L and PAHs were below detection limit. Capitalizing on the rapid analytical workflow of CP-MIMS, we also investigate the impacts of sample filtration at the time of sampling (on NA and PAH data) and sample storage time (on NA data only).

Direct mass spectrometric analysis of naphthenic acids and polycyclic aromatic hydrocarbons in waters impacted by diluted bitumen and conventional crude oil.

Crude oil spills have well-documented, deleterious impacts on the hydrosphere. In addition to macroscopic effects on wildlife and waterscapes, several classes of petroleum derived compounds, such as naphthenic acids (NAs) and polycyclic aromatic hydrocarbons (PAHs), may be released into the water and present aquatic contamination hazards. The concentrations of these contaminants may be affected by both oil type and water chemistry. We characterize the concentrations of NAs and PAHs in natural and constructed waters, spanning a range of pH and salinity, and directly compare the influence of diluted bitumen (DB) and conventional crude (CC) oil, using condensed-phase membrane introduction mass spectrometry (CP-MIMS) as a direct sampling, on-line technique. The concentration and isomer class profiles of classical NAs in the aqueous phase were assessed using electrospray ionization in negative-ion mode as [M-H]- whereas PAH concentrations were monitored using liquid electron ionization (LEI) in positive-ion mode as [M+•]. NA concentrations (0.03-25 ppm) were highly pH-dependent, and an order of magnitude greater in water samples contaminated with DB than CC. Conversely, concentrations of naphthalene (10-130 ppb) and alkyl-naphthalenes (10-90 ppb) were three to four-fold higher in water samples exposed to CC. We demonstrate that naturally occurring dissolved organic matter does not bias results from the membrane sampling approach employed, and that DB and CC contaminated waters can be differentiated using principal component analysis of the NA isomer class distribution in both constructed and natural waters. Finally, we describe the first demonstration of the concurrent analysis of trace NAs and PAHs in the same water sample by controlling perm-selectivity at the membrane and the ionization mode of the mass spectrometer. The techniques employed here for trace analysis of petroleum derived compounds in water can be applied to rapid screening and real-time monitoring of contamination and remediation processes.

A Direct Mass Spectrometry Method for the Rapid Analysis of Ubiquitous Tire-Derived Toxin N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine Quinone (6-PPDQ).

The oxidative transformation product of a common tire preservative, identified as N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ), has recently been found to contribute to "urban runoff mortality syndrome" in Coho salmon at nanogram per liter levels. Given the number of fish-bearing streams with multiple stormwater inputs, large-scale campaigns to identify 6-PPDQ sources and evaluate mitigation strategies will require sensitive, high-throughput analytical methods. We report the development and optimization of a direct sampling tandem mass spectrometry method for semiquantitative 6-PPDQ determinations using a thin polydimethylsiloxane membrane immersion probe. The method requires no sample cleanup steps or chromatographic separations, even in complex, heterogeneous samples. Quantitation is achieved by the method of standard additions, with a detection limit of 8 ng/L and a duty cycle of 15 min/sample. High-throughput screening provides semiquantitative concentrations with similar sensitivity and a full analytical duty cycle of 2.5 min/sample. Preliminary data and performance metrics are reported for 6-PPDQ present in representative environmental and stormwater samples. The method is readily adapted for real-time process monitoring, demonstrated by following the dissolution of 6-PPDQ from tire fragments and subsequent removal in response to added sorbents.

Direct analysis of naphthenic acids in constructed wetland samples by condensed phase membrane introduction mass spectrometry.

The application of direct mass spectrometry techniques to the analysis of complex samples has a number of advantages including reduced sample handling, higher sample throughput, in situ process monitoring, and the potential for adaptation to on-site analysis. We report the application of a semi-permeable capillary hollow fibre membrane probe (immersed directly into an aqueous sample) coupled to a triple quadrupole mass spectrometer by a continuously flowing methanol acceptor phase for the rapid analysis of naphthenic acids with unit mass resolution. The intensity of the naphthenic acid-associated peaks in the mass spectrum are normalized to an internal standard in the acceptor phase for quantitation and the relative abundance of the peaks in the mass spectrum are employed to monitor compositional changes in the naphthenic acid mixture using principle component analysis. We demonstrate the direct analysis of a synthetic oil sands process-affected water for classical naphthenic acids (CnH2n+zO2) as they are attenuated through constructed wetlands containing sedge (Carex aquatilis), cattail (Typha latifolia), or bulrush (Schoenoplectus acutus). Quantitative results for on-line membrane sampling compare favourably to those obtained by solid-phase extraction high-resolution mass spectrometry. Additionally, chemometric analysis of the mass spectra indicates a clear discrimination between naphthenic acid-influenced and natural background waters. Furthermore, the compositional changes within complex naphthenic acid mixtures track closely with the degree of attenuation. Overall, the technique is successful in following changes in both the concentration and composition of naphthenic acids from synthetic oil sands process-affected waters, with the potential for high throughput screening and environmental forensics.

Direct Measurement of Acid Dissociation Constants of Trace Organic Compounds at Nanomolar Levels in Aqueous Solution by Condensed Phase-Membrane Introduction Mass Spectrometry.

We report the use of condensed phase-membrane introduction mass spectrometry as a novel method for the determination of acid dissociation constants for hydrophobic organic acids in aqueous solution at nanomolar concentrations. The technique is based on the pH-dependent permeation of analytes through a semipermeable polydimethylsiloxane membrane probe that is immersed directly in aqueous samples. We describe the method and report the dissociation constant (pKa ) values for compounds of biological and environmental relevance, including contaminants, pharmaceuticals, and naphthenic acids. The approach can be applied to individual compounds, combined suites, and complex mixtures at parts-per-billion levels. We report pKa values for 10 carboxylic acids with precision estimates and relative errors (where reliable literature values are available) of <0.1 log units. We report acidity constants for 2-methyl-3-methoxy-4-phenyl butanoic acid (a biomarker for microcystin algal toxins) and 4-t-butylcyclohexane carboxylic acid (a model naphthenic acid) as 4.28 ± 0.03 and 5.15 ± 0.05, respectively. Furthermore, we employ this approach to measure the effect of both temperature and deuterium oxide (heavy water) on acid dissociation, reporting the enthalpy and entropy changes for the ionization of a representative carboxylic acid and substituted phenol. Environ Toxicol Chem 2019;38:1879-1889. © 2019 SETAC.