Polystyrene micro- and nanoplastics cause placental dysfunction in mice†.

Maternal exposure to microplastics and nanoplastics has been shown to result in fetal growth restriction in mice. In this study, we investigated the placental and fetal hemodynamic responses to plastics exposure in mice using high-frequency ultrasound. Healthy, pregnant CD-1 dams were given either 106 ng/L of 5 µm polystyrene microplastics or 106 ng/L of 50 nm polystyrene nanoplastics in drinking water throughout gestation and were compared with controls. Maternal exposure to both microplastics and nanoplastics resulted in evidence of placental dysfunction that was highly dependent on the particle size. The umbilical artery blood flow increased by 48% in the microplastic-exposed group and decreased by 25% in the nanoplastic-exposed group compared to controls (p < 0.05). The microplastic- and nanoplastic-exposed fetuses showed a significant decrease in the middle cerebral artery pulsatility index of 10% and 13%, respectively, compared to controls (p < 0.05), indicating vasodilation of the cerebral circulation, a fetal adaptation that is part of the brain sparing response to preserve oxygen delivery. Hemodynamic markers of placental dysfunction and fetal hypoxia were more pronounced in the group exposed to polystyrene nanoplastics, suggesting nanoplastic exposure during human pregnancy has the potential to disrupt fetal brain development, which in turn may cause suboptimal neurodevelopmental outcomes.

Metabolomics revealed disruptions in amino acid and antioxidant biochemistry in Daphnia magna exposed to industrial effluents associated with plastic and polymer production.

Industrial wastewater effluents are a major source of chemicals in aquatic environments, and many of these chemicals may negatively impact aquatic life. In this study, the crustacean Daphnia magna, a common model organism in ecotoxicity studies, was exposed for 48 h to nine different industrial effluent samples from manufacturing facilities associated with the production of plastics, polymers, and coating products at a range of dilutions: 10, 25, 50, 100% (undiluted). A targeted metabolomic-based approach using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify polar metabolites from individual daphnids that survived the 48 h exposure. Multivariate analyses and metabolite changes revealed metabolic perturbations across all effluent samples studied, with non-monotonic responses and both up and downregulation relative to the unexposed control. Pathway analyses indicated the disruption of similar and distinct pathways, mostly connected to protein synthesis, amino acid metabolism, and antioxidant processes. Overall, we observed disruptions in Daphnia biochemistry that were similar across the effluent samples, but with unique features for each effluent sample. Additionally, non-monotonic heightened responses suggested additive and/or synergistic interactions between the chemicals within the industrial effluents. These findings demonstrate that targeted metabolomic approaches are a powerful tool for the biomonitoring of aquatic ecosystems in the context of complex mixtures, such as industrial wastewater effluents.

Brewing alcohol 101: An undergraduate experiment utilizing benchtop NMR for quantification and process monitoring.

In recent years there has been a renewed interest in benchtop NMR. Given their lower cost of ownership, smaller footprint, and ease of use, they are especially suited as an educational tool. Here, a new experiment targeted at upper-year undergraduates and first-year graduate students follows the conversion of D-glucose into ethanol at low-field. First, high and low-field data on D-glucose are compared and students learn both the Hz and ppm scales and how J-coupling is field-independent. The students then acquire their own quantitative NMR datasets and perform the quantification using an Electronic Reference To Access In Vivo Concentration (ERETIC) technique. To our knowledge ERETIC is not currently taught at the undergraduate level, but has an advantage in that internal standards are not required; ideal for following processes or with future use in flow-based benchtop monitoring. Using this quantitative data, students can relate a simple chemical process (fermentation) back to more complex topics such as reaction kinetics, bridging the gaps between analytical and physical chemistry. When asked to reflect on the experiment, students had an overwhelmingly positive experience, citing agreement with learning objectives, ease of understanding the protocol, and enjoyment. Each of the respondents recommended this experiment as a learning tool for others. This experiment has been outlined for other instructors to utilize in their own courses across institutions, with the hope that a continued expansion of low-field NMR will increase accessibility and learning opportunities at the undergraduate level.

Low-field, not low quality: 1D simplification, selective detection, and heteronuclear 2D experiments for improving low-field NMR spectroscopy of environmental and biological samples.

Understanding environmental change is challenging and requires molecular-level tools to explain the physicochemical phenomena behind complex processes. Nuclear magnetic resonance (NMR) spectroscopy is a key tool that provides information on both molecular structures and interactions but is underutilized in environmental research because standard "high-field" NMR is financially and physically inaccessible for many and can be overwhelming to those outside of disciplines that routinely use NMR. "Low-field" NMR is an accessible alternative but has reduced sensitivity and increased spectral overlap, which is especially problematic for natural, heterogeneous samples. Therefore, the goal of this study is to investigate and apply innovative experiments that could minimize these challenges and improve low-field NMR analysis of environmental and biological samples. Spectral simplification (JRES, PSYCHE, singlet-only, multiple quantum filters), selective detection (GEMSTONE, DREAMTIME), and heteronuclear (reverse and CH3/CH2/CH-only HSQCs) NMR experiments are tested on samples of increasing complexity (amino acids, spruce resin, and intact water fleas) at-high field (500 MHz) and at low-field (80 MHz). A novel experiment called Doubly Selective HSQC is also introduced, wherein 1H signals are selectively detected based on the 1H and 13C chemical shifts of 1H-13C J-coupled pairs. The most promising approaches identified are the selective techniques (namely for monitoring), and the reverse and CH3-only HSQCs. Findings ultimately demonstrate that low-field NMR holds great potential for biological and environmental research. The multitude of NMR experiments available makes NMR tailorable to nearly any research need, and low-field NMR is therefore anticipated to become a valuable and widely used analytical tool moving forward.

Slice through the water-Exploring the fundamental challenge of water suppression for benchtop NMR systems.

Benchtop NMR provides improved accessibility in terms of cost, space, and technical expertise. In turn, this encourages new users into the field of NMR spectroscopy. Unfortunately, many interesting samples in education and research, from beer to whole blood, contain significant amounts of water that require suppression in 1H NMR in order to recover sample information. However, due to the significant reduction in chemical shift dispersion in benchtop NMR systems, the sample signals are much closer to the water resonance compared to those in a corresponding high-field NMR spectrum. Therefore, simply translating solvent suppression experiments intended for high-field NMR instruments to benchtop NMR systems without careful consideration can be problematic. In this study, the effectiveness of several popular water suppression schemes was evaluated for benchtop NMR applications. Emphasis is placed on pulse sequences with no, or few, adjustable parameters making them easy to implement. These fall into two main categories: (1) those based on Pre-SAT including Pre-SAT, PURGE, NOESY-PR, and g-NOESY-PR and (2) those based on binomial inversion including JRS and W5-WATERGATE. Among these schemes, solvent suppression sequences based on Pre-SAT offer a general approach for easy solvent suppression for samples with higher analyte concentrations (sucrose standard and Redbull™). However, for human urine, binomial-like sequences were required. In summary, it is demonstrated that highly efficient water suppression approaches can be implemented on benchtop NMR systems in a simple manner, despite the limited spectral dispersion, further illustrating the potential for widespread implementation of these approaches in education and research.

HR-MAS DREAMTIME NMR for Slow Spinning ex Vivo and in Vivo Samples.

HR-MAS NMR is a powerful tool, capable of monitoring molecular changes in intact heterogeneous samples. However, one of the biggest limitations of 1H NMR is its narrow spectral width which leads to considerable overlap in complex natural samples. DREAMTIME NMR is a highly selective technique that allows users to isolate suites of metabolites from congested spectra. This permits targeted metabolomics by NMR and is ideal for monitoring specific processes. To date, DREAMTIME has only been employed in solution-state NMR, here it is adapted for HR-MAS applications. At high spinning speeds (>5 kHz), DREAMTIME works with minimal modifications. However, spinning over 3-4 kHz leads to cell lysis, and if maintaining sample integrity is necessary, slower spinning (<2.5 kHz) is required. Very slow spinning (≤500 Hz) is advantageous for in vivo analysis to increase organism survival; however, sidebands from water pose a problem. To address this, a version of DREAMTIME, termed DREAMTIME-SLOWMAS, is introduced. Both techniques are compared at 2500, 500, and 50 Hz, using ex vivo worm tissue. Following this, DREAMTIME-SLOWMAS is applied to monitor key metabolites of anoxic stress in living shrimp at 500 Hz. Thus, standard DREAMTIME works well under MAS conditions and is recommended for samples reswollen in D2O or spun >2500 Hz. For slow spinning in vivo or intact tissue samples, DREAMTIME-SLOWMAS provides an excellent way to target process-specific metabolites while maintaining sample integrity. Overall, DREAMTIME should find widespread application wherever targeted molecular information is required from complex samples with a high degree of spectral overlap.

Lenz Lenses in a Cryoprobe: Boosting NMR Sensitivity Toward Environmental Monitoring of Mass-Limited Samples.

Nuclear magnetic resonance (NMR) spectroscopy is commonly employed in a wide range of metabolomic research. Unfortunately, due to its relatively low sensitivity, smaller samples become challenging to study by NMR. Cryoprobes can be used to increase sensitivity by cooling the coil and preamplifier, offering sensitivity improvements of ∼3 to 4x. Alternatively, microcoils can be used to increase mass sensitivity by improving sample filling and proximity, along with decreased electrical resistance. Unfortunately, combining the two approaches is not just technically challenging, but as the coil decreases, so does its thermal fingerprint, reducing the advantage of cryogenic cooling. Here, an alternative solution is proposed in the form of a Lenz lens inside a cryoprobe. Rather than replacing the detection coil, Lenz lenses allow the B1 field from a larger coil to be refocused onto a much smaller sample area. In turn, the stronger B1 field at the sample provides strong coupling to the cryocoil, improving the signal. By combining a 530 I.D. Lenz lens with a cryoprobe, sensitivity was further improved by 2.8x and 3.5x for 1H and 13C, respectively, over the cryoprobe alone for small samples. Additionally, the broadband nature of the Lenz lenses allowed multiple nuclei to be studied and heteronuclear two-dimensional (2D) NMR approaches to be employed. The sensitivity improvements and 2D capabilities are demonstrated on 430 nL of hemolymph and eight eggs (∼350 µm O.D.) from the model organismDaphnia magna. In summary, combining Lenz lenses with cryoprobes offers a relatively simple approach to boost sensitivity for tiny samples while retaining cryoprobe advantages.

Cutting without a Knife: A Slice-Selective 2D 1H-13C HSQC NMR Sequence for the Analysis of Inhomogeneous Samples.

Nuclear magnetic resonance (NMR) is a powerful technique with applications ranging from small molecule structure elucidation to metabolomics studies of living organisms. Typically, solution-state NMR requires a homogeneous liquid, and the whole sample is analyzed as a single entity. While adequate for homogeneous samples, such an approach is limited if the composition varies as would be the case in samples that are naturally heterogeneous or layered. In complex samples such as living organisms, magnetic susceptibility distortions lead to broad 1H line shapes, and thus, the additional spectral dispersion afforded by 2D heteronuclear experiments is often required for metabolite discrimination. Here, a novel, slice-selective 2D, 1H-13C heteronuclear single quantum coherence (HSQC) sequence was developed that exclusively employs shaped pulses such that only spins in the desired volume are perturbed. In turn, this permits multiple volumes in the tube to be studied during a single relaxation delay, increasing sensitivity and throughput. The approach is first demonstrated on standards and then used to isolate specific sample/sensor elements from a microcoil array and finally study slices within a living earthworm, allowing metabolite changes to be discerned with feeding. Overall, slice-selective NMR is demonstrated to have significant potential for the study of layered and other inhomogeneous samples of varying complexity. In particular, its ability to select subelements is an important step toward developing microcoil receive-only arrays to study environmental toxicity in tiny eggs, cells, and neonates, whereas localization in larger living species could help better correlate toxin-induced biochemical responses to the physical localities or organs involved.

Application of 15N-Edited 1H-13C Correlation NMR Spectroscopy─Toward Fragment-Based Metabolite Identification and Screening via HCN Constructs.

Many key building blocks of life contain nitrogen moieties. Despite the prevalence of nitrogen-containing metabolites in nature, 15N nuclei are seldom used in NMR-based metabolite assignment due to their low natural abundance and lack of comprehensive chemical shift databases. However, with advancements in isotope labeling strategies, 13C and 15N enriched metabolites are becoming more common in metabolomic studies. Simple multidimensional nuclear magnetic resonance (NMR) experiments that correlate 1H and 15N via single bond 1JNH or multiple bond 2-3JNH couplings using heteronuclear single quantum coherence (HSQC) or heteronuclear multiple bond coherence are well established and routinely applied for structure elucidation. However, a 1H-15N correlation spectrum of a metabolite mixture can be difficult to deconvolute, due to the lack of a 15N specific database. In order to bridge this gap, we present here a broadband 15N-edited 1H-13C HSQC NMR experiment that targets metabolites containing 15N moieties. Through this approach, nitrogen-containing metabolites, such as amino acids, nucleotide bases, and nucleosides, are identified based on their 13C, 1H, and 15N chemical shift information. This approach was tested and validated using a [15N, 13C] enriched Daphnia magna (water flea) metabolite extract, where the number of clearly resolved 15N-containing peaks increased from only 11 in a standard HSQC to 51 in the 15N-edited HSQC, and the number of obscured peaks decreased from 59 to just 7. The approach complements the current repertoire of NMR techniques for mixture deconvolution and holds considerable potential for targeted metabolite NMR in 15N, 13C enriched systems.

Targeted Compound Selection with Increased Sensitivity in 13C-Enriched Biological and Environmental Samples Using 13C-DREAMTIME in Both High-Field and Low-Field NMR.

Chemical characterization of complex mixtures by Nuclear Magnetic Resonance (NMR) spectroscopy is challenging due to a high degree of spectral overlap and inherently low sensitivity. Therefore, NMR experiments that reduce overlap and increase signal intensity hold immense potential for the analysis of mixtures such as biological and environmental media. Here, we introduce a 13C version of DREAMTIME (Designed Refocused Excitation And Mixing for Targets In Vivo and Mixture Elucidation) NMR, which, when analyzing 13C-enriched materials, allows the user to selectively detect only the compound(s) of interest and remove all other peaks in a 13C spectrum. Selected peaks can additionally be "focused" into sharp "spikes" to increase sensitivity. 13C-DREAMTIME is first demonstrated at high field strength (500 MHz) with simultaneous selection of eight amino acids in a 13C-enriched cell free amino acid mixture and of six metabolites in an extract of 13C-enriched green algae and demonstrated at low field strength (80 MHz) with a standard solution of 13C-d-glucose and 13C-l-phenylalanine. 13C-DREAMTIME is then applied at high-field to analyze metabolic changes in 13C-enrichedDaphnia magna after exposure to polystyrene "microplastics," as well as at low-field to track fermentation of 13C-d-glucose using wine yeast. Ultimately, 13C-DREAMTIME reduces spectral overlap as only selected compounds are recorded, resulting in the detection of analyte peaks that may otherwise not have been discernable. In combination with focusing, up to a 6-fold increase in signal intensity can be obtained for a given peak. 13C-DREAMTIME is a promising experiment type for future reaction monitoring and for tracking metabolic processes with 13C-enriched compounds.

Integrated Digital Microfluidics NMR Spectroscopy: A Key Step toward Automated In Vivo Metabolomics.

Toxicity testing is currently undergoing a paradigm shift from examining apical end points such as death, to monitoring sub-lethal toxicity in vivo. In vivo nuclear magnetic resonance (NMR) spectroscopy is a key platform in this endeavor. A proof-of-principle study is presented which directly interfaces NMR with digital microfluidics (DMF). DMF is a "lab on a chip" method allowing for the movement, mixing, splitting, and dispensing of µL-sized droplets. The goal is for DMF to supply oxygenated water to keep the organisms alive while NMR detects metabolomic changes. Here, both vertical and horizontal NMR coil configurations are compared. While a horizontal configuration is ideal for DMF, NMR performance was found to be sub-par and instead, a vertical-optimized single-sided stripline showed most promise. In this configuration, three organisms were monitored in vivo using 1H-13C 2D NMR. Without support from DMF droplet exchange, the organisms quickly showed signs of anoxic stress; however, with droplet exchange, this was completely suppressed. The results demonstrate that DMF can be used to maintain living organisms and holds potential for automated exposures in future. However, due to numerous limitations of vertically orientated DMF, along with space limitations in standard bore NMR spectrometers, we recommend future development be performed using a horizontal (MRI style) magnet which would eliminate practically all the drawbacks identified here.

Development of Low-Magnetic Susceptibility Microcoils via 5-Axis Machining for Analysis of Biological and Environmental Samples.

In environmental research, it is critical to understand how toxins impact invertebrate eggs and egg banks, which, due to their tiny size, are very challenging to study by conventional nuclear magnetic resonance (NMR) spectroscopy. Microcoil technology has been extensively utilized to enhance the mass-sensitivity of NMR. In a previous study, 5-axis computer numerical control (CNC) micromilling (shown to be a viable alternative to traditional microcoil production methods) was used to create a prototype copper slotted-tube resonator (STR). Despite the excellent limit of detection (LOD) of the resonator, the quality of the line shape was very poor due to the magnetic susceptibility of the copper resonator itself. This is best solved using magnetic susceptibility-matched materials. In this study, approaches are investigated that improve the susceptibility while retaining the versatility of coil milling. One method involves machining STRs from various copper/aluminum alloys, while the other involves machining ones from an aluminum 2011 alloy and electroplating them with copper. In all cases, combining copper and aluminum to produce resonators resulted in improved line shape and SNR compared to pure copper resonators due to their reduced magnetic susceptibility. However, the copper-plated aluminum resonators showed optimal performance from the devices tested. The enhanced LOD of these STRs allowed for the first 1H-13C heteronuclear multiple quantum coherence (HMQC) of a single intact 13C-labeled Daphnia magna egg (∼4 µg total biomass). This is a key step toward future screening programs that aim to elucidate the toxic processes in aquatic eggs.

Maternal exposure to polystyrene nanoplastics alters fetal brain metabolism in mice.

INTRODUCTION: Plastics used in everyday materials accumulate as waste in the environment and degrade over time. The impacts of the resulting particulate micro- and nanoplastics on human health remain largely unknown. In pregnant mice, we recently demonstrated that exposure to nanoplastics throughout gestation and during lactation resulted in changes in brain structure detected on MRI. One possible explanation for this abnormal postnatal brain development is altered fetal brain metabolism. OBJECTIVES: To determine the effect of maternal exposure to nanoplastics on fetal brain metabolism. METHODS: Healthy pregnant CD-1 mice were exposed to 50 nm polystyrene nanoplastics at a concentration of 106 ng/L through drinking water during gestation. Fetal brain samples were collected at embryonic day 17.5 (n = 18-21 per group per sex) and snap-frozen in liquid nitrogen. Magic angle spinning nuclear magnetic resonance was used to determine metabolite profiles and their relative concentrations in the fetal brain. RESULTS: The relative concentrations of gamma-aminobutyric acid (GABA), creatine and glucose were found to decrease by 40%, 21% and 30% respectively following maternal nanoplastic exposure when compared to the controls (p < 0.05). The change in relative concentration of asparagine with nanoplastic exposure was dependent on fetal sex (p < 0.005). CONCLUSION: Maternal exposure to polystyrene nanoplastics caused abnormal fetal brain metabolism in mice. The present study demonstrates the potential impacts of nanoplastic exposure during fetal development and motivates further studies to evaluate the risk to human pregnancies.

A holistic NMR framework to understand environmental impact: Examining the impacts of superparamagnetic iron oxide nanoparticles (SPIONs) in Daphnia magna via imaging, spectroscopy, and metabolomics.

Superparamagnetic iron oxide nanoparticles (SPIONs) are a contaminant of emerging interest, often used in the medical field as an imaging contrast agent, with additional uses in wastewater treatment and as food additives. Although the use of SPIONs is increasing, little research has been conducted on the toxic impacts to living organisms beyond traditional lethal concentration endpoints. Daphnia magna are model organisms for aquatic toxicity testing with a well understood metabolome and high sensitivity to SPIONs. Thus, as environmental concentrations continue to increase, it is becoming critical to understand their sub-lethal toxicity. Due to the paramagnetic nature of SPIONs, a range of potential nuclear magnetic resonance spectroscopy (NMR) experiments are possible, offering the potential to probe the physical location (via imaging), binding (via relaxation weighted spectroscopy), and the biochemical pathways impacted (via in vivo metabolomics). Results indicate binding to carbohydrates, likely chitin in the exoskeleton, along with a decrease in energy metabolites and specific biomarkers of oxidative stress. The holistic NMR framework used here helps provide a more comprehensive understanding of SPIONs impacts on D. magna and showcases NMR's versatility in providing physical, chemical, and biochemical insights.

Exploring Proton-Only NMR Experiments and Filters for Daphnia In Vivo: Potential and Limitations.

Environmental metabolomics provides insight into how anthropogenic activities have an impact on the health of an organism at the molecular level. Within this field, in vivo NMR stands out as a powerful tool for monitoring real-time changes in an organism's metabolome. Typically, these studies use 2D 13C-1H experiments on 13C-enriched organisms. Daphnia are the most studied species, given their widespread use in toxicity testing. However, with COVID-19 and other geopolitical factors, the cost of isotope enrichment increased ~6-7 fold over the last two years, making 13C-enriched cultures difficult to maintain. Thus, it is essential to revisit proton-only in vivo NMR and ask, "Can any metabolic information be obtained from Daphnia using proton-only experiments?". Two samples are considered here: living and whole reswollen organisms. A range of filters are tested, including relaxation, lipid suppression, multiple-quantum, J-coupling suppression, 2D 1H-1H experiments, selective experiments, and those exploiting intermolecular single-quantum coherence. While most filters improve the ex vivo spectra, only the most complex filters succeed in vivo. If non-enriched organisms must be used, then, DREAMTIME is recommended for targeted monitoring, while IP-iSQC was the only experiment that allowed non-targeted metabolite identification in vivo. This paper is critically important as it documents not just the experiments that succeed in vivo but also those that fail and demonstrates first-hand the difficulties associated with proton-only in vivo NMR.

Exploring the Potential of Broadband Complementary Metal Oxide Semiconductor Micro-Coil Nuclear Magnetic Resonance for Environmental Research.

With sensitivity being the Achilles' heel of nuclear magnetic resonance (NMR), the superior mass sensitivity offered by micro-coils can be an excellent choice for tiny, mass limited samples such as eggs and small organisms. Recently, complementary metal oxide semiconductor (CMOS)-based micro-coil transceivers have been reported and demonstrate excellent mass sensitivity. However, the ability of broadband CMOS micro-coils to study heteronuclei has yet to be investigated, and here their potential is explored within the lens of environmental research. Eleven nuclei including 7Li, 19F, 31P and, 205Tl were studied and detection limits in the low to mid picomole range were found for an extended experiment. Further, two environmentally relevant samples (a sprouting broccoli seed and a D. magna egg) were successfully studied using the CMOS micro-coil system. 13C NMR was used to help resolve broad signals in the 1H spectrum of the 13C enriched broccoli seed, and steady state free precession was used to improve the signal-to-noise ratio by a factor of six. 19F NMR was used to track fluorinated contaminants in a single D. magna egg, showing potential for studying egg-pollutant interactions. Overall, CMOS micro-coil NMR demonstrates significant promise in environmental research, especially when the future potential to scale to multiple coil arrays (greatly improving throughput) is considered.

SASSY NMR: Simultaneous Solid and Solution Spectroscopy.

Synergism between different phases gives rise to chemical, biological or environmental reactivity, thus it is increasingly important to study samples intact. Here, SASSY (SimultAneous Solid and Solution spectroscopY) is introduced to simultaneously observe (and differentiate) all phases in multiphase samples using standard, solid-state NMR equipment. When monitoring processes, the traditional approach of studying solids and liquids sequentially, can lead to information in the non-observed phase being missed. SASSY solves this by observing the full range of materials, from crystalline solids, through gels, to pure liquids, at full sensitivity in every scan. Results are identical to running separate 13 C CP-MAS solid-state and 13 C solution-state experiments back-to-back but requires only a fraction of the spectrometer time. After its introduction, SASSY is applied to process monitoring and finally to detect all phases in a living freshwater shrimp. SASSY is simple to implement and thus should find application across all areas of research.

Comparing the Potential of Helmholtz and Planar NMR Microcoils for Analysis of Intact Biological Samples.

Nuclear magnetic resonance (NMR) spectroscopy has played an integral role in medical and environmental metabolic research. However, smaller biological entities, such as eggs and small tissue samples, are becoming increasingly important to better understand toxicity, biological growth/development, and diseases. Unfortunately, their small sizes make them difficult to study using conventional 5 mm NMR probes due to limited sensitivity. The use of microcoil NMR holds great potential for the analysis of such samples, where the coil can be designed to match the sample size to significantly improve NMR mass sensitivity and the filling factor. Here, we compare the potential of planar and Helmholtz microcoil designs to execute complex experiments for the analysis of intact, mass-limited biological samples. The planar coil offers the advantage of an open access design, potentially allowing flow systems to be incorporated and varying sample sizes to be studied; however, its relatively inhomogeneous B1 field leads to reduced NMR performance. The Helmholtz microcoil overcomes this drawback with its symmetrical design, improving B1 homogeneity across the sample but with the caveat that the size and shape of the sample is limited to the spacing between the two parallel coils. The line shape, sensitivity, and RF performance are compared on both coils using standard samples and biological samples. This study found that the Helmholtz microcoil used here considerably outperforms the planar coil in multipulse experiments and has great potential to study complex biological samples in the 50-200 nL range.

Gas Chromatography-(Cyclic) Ion Mobility Mass Spectrometry: A Novel Platform for the Discovery of Unknown Per-/Polyfluoroalkyl Substances.

Per- and polyfluoroalkyl substances (PFASs) have been widely used since the 1940s in industry and everyday household products. They also persist in the environment and bioaccumulate in humans and wildlife. Despite these concerns, the identities of most PFASs in environmental and biological samples are unknown. Herein, we describe a novel cyclic ion mobility mass spectrometer (cIMS), hyphenated with gas chromatography (GC) atmospheric pressure chemical ionization, that can reveal the presence of unknown PFASs on the basis of the ratio of their mass and collision cross section (CCS). Prediction of the CCS of ca. 20,000 chemicals used in industry and commerce indicates that most compounds characterized by CCS values that are less than the sum of 100 Å2 and one-fifth of their mass are either PFASs or polybrominated flame retardants. When this filter is applied to GC-cIMS data collected from a set of 20 indoor dust samples, PFAS compounds are revealed without prior knowledge of their occurrence. Validation of this approach was performed using SRM 2585, a standard reference material of household dust, by comparing the PFASs detected with those (tentatively) identified in previous studies. Chlorofluoro phthalimides tentatively identified previously were confirmed with a synthesized standard. The method also reveals the presence of chlorofluoro n-alkanes as an emerging class of "forever chemicals" that contaminate the indoor environment.

Exploring the Applications of Carbon-Detected NMR in Living and Dead Organisms Using a 13C-Optimized Comprehensive Multiphase NMR Probe.

Comprehensive multiphase-nuclear magnetic resonance (CMP-NMR) is a non-invasive approach designed to observe all phases (solutions, gels, and solids) in intact samples using a single NMR probe. Studies of dead and living organisms are important to understand processes ranging from biological growth to environmental stress. Historically, such studies have utilized 1H-based phase editing for the detection of soluble/swollen components and 1H-detected 2D NMR for metabolite assignments/screening. However, living organisms require slow spinning rates (∼500 Hz) to increase survivability, but at such low speeds, complications from water sidebands and spectral overlap from the modest chemical shift window (∼0-10 ppm) make 1H NMR challenging. Here, a novel 13C-optimized E-Free magic angle spinning CMP probe is applied to study all phases in ex vivo and in vivo samples. This probe consists of a two-coil design, with an inner single-tuned 13C coil providing a 113% increase in 13C sensitivity relative to a traditional multichannel single-CMP coil design. For organisms with a large biomass (∼0.1 g) like the Ganges River sprat (ex vivo), 13C-detected full spectral editing and 13C-detected heteronuclear correlation (HETCOR) can be performed at natural abundance. Unfortunately, for a single living shrimp (∼2 mg), 13C enrichment was still required, but 13C-detected HETCOR shows superior data relative to heteronuclear single-quantum coherence at low spinning speeds (due to complications from water sidebands in the latter). The probe is equipped with automatic-tuning-matching and is compatible with automated gradient shimming─a key step toward conducting multiphase screening of dead and living organisms under automation in the near future.