Nuclear Magnetic Resonance (NMR) spectroscopy is a non-invasive analytical technique which allows for the study of intact samples. Comprehensive Multiphase NMR (CMP-NMR) combines techniques and hardware from solution state and solid state NMR to allow for the holistic analysis of all phases (i.e. solutions, gels and solids) in unaltered samples. This study is the first to apply CMP-NMR to deceased, intact organisms and uses 13 C enriched Daphnia magna (water fleas) as an example. D. magna are commonly used model organisms for environmental toxicology studies. As primary consumers, they are responsible for the transfer of nutrients across trophic levels, and a decline in their population can potentially impact the entire freshwater aquatic ecosystem. Though in vivo research is the ultimate tool to understand an organism’s most biologically relevant state, studies are limited by conditions (i.e. oxygen requirements, limited experiment time and reduced spinning speed) required to keep the organisms alive, which can negatively impact the quality of the data collected. In comparison, ex vivo CMP-NMR is beneficial in that; organisms do not need oxygen (eliminating air holes in rotor caps and subsequent evaporation); samples can be spun faster, leading to improved spectral resolution; more biomass per sample can be analyzed; and experiments can be run for longer. In turn, higher quality ex vivo NMR, can provide more comprehensive NMR assignments, which in many cases could be transferred to better understand less resolved in vivo signals. This manuscript is divided into three sections: 1) multiphase spectral editing techniques, 2) detailed metabolic assignments of 2D NMR of 13 C enriched D. magna and 3) multiphase biological changes over different life stages, ages and generations of D. magna . In summary, ex vivo CMP-NMR proves to be a very powerful approach to study whole organisms in a comprehensive manner and should provide very complementary information to in vivo based research.

The superior mass sensitivity of micro-coil technology in Nuclear Magnetic Resonance (NMR) Spectroscopy provides potential for the analysis of extremely small mass-limited samples such as eggs, cells, and tiny organisms. For optimal performance and efficiency, the size of the micro-coil should be tailored to the size of the mass-limited sample of interest, which can be costly as mass-limited samples come in many shapes and sizes. Therefore, rapid and economic micro-coil production methods are needed. One method with great potential is 5-axis Computer Numerical Control (CNC) micro-milling, commonly used in the jewelry industry. Most CNC milling machines are designed to process larger objects and commonly have a precision >25 µm (making the machining of common spiral micro-coils, for example, impossible). Here, a 5-axis MiRA6 CNC milling machine, specifically designed for the jewelry industry, with a 0.3 µm precision was used to produce working planar micro-coils, microstrips, and novel micro-sensor designs, with some tested on the NMR in less than 24 hours after the start of the design process. Sample wells could be built into the micro-sensor and could be machined at the same time as the sensors themselves, in some cases leaving a sheet of Teflon as thin as 10 µm between the sample and sensor. This provides the freedom to produce a wide array of designs and demonstrates 5-axis CNC micro-milling as a versatile tool for the rapid prototyping of NMR micro-sensors. This approach allowed the experimental optimization of a prototype microstrip for the analysis of two intact adult Daphnia magna organisms. In addition, a 3D volume slotted tube resonator was produced that allowed for 2D 1 H-13 C NMR of D. magna neonates and exhibited 1 H sensitivity (nLOD ꙍ 600 = 1.49 nmol s 1/2 ) close to that of double striplines, which themselves offer the best compromise between concentration and mass sensitivity published to date.

Fragment-based screening has evolved as a remarkable approach within the drug discovery process both in the industry and academia. Fragment screening has become a more structure-based approach to inhibitor development, but also towards development of pathway-specific clinical probes. However, it is often witnessed that the availability, immediate and longterm, of a high quality fragment-screening library is still beyond the reach of most academic laboratories. Within iNEXT (Infrastructure for NMR, EM and X-rays for Translational research), a EU-funded Horizon 2020 program, a collection of 782 fragments were assembled utilizing the concept of "poised fragments" with the aim to facilitate downstream synthesis of ligands with high affinity by fragment ligation. Herein, we describe the analytical procedure to assess the quality of this purchased and assembled fragment library by NMR spectroscopy. This quality assessment requires buffer solubility screening, comparison with LC/MS quality control and is supported by state-of-the-art software for high throughput data acquisition and on-the-fly data analysis. Results from the analysis of the library are presented as a prototype of fragment progression through the quality control process.

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Microcoil nuclear magnetic resonance (NMR) has been interfaced with digital microfluidics (DMF) and is applied to monitor organic reactions in organic solvents as a proof of concept. DMF permits droplets to be moved and mixed inside the NMR spectrometer to initiate reactions while using sub‐microliter volumes of reagent, opening up the potential to follow the reactions of scarce or expensive reagents. By setting up the spectrometer shims on a reagent droplet, data acquisition can be started immediately upon droplet mixing and is only limited by the rate at which NMR data can be collected, allowing the monitoring of fast reactions. Here we report a cyclohexene carbonate hydrolysis in dimethylformamide and a Knoevenagel condensation in methanol/water. This is to our knowledge the first time rapid organic reactions in organic solvents have been monitored by high field DMF‐NMR. The study represents a key first step towards larger DMF‐NMR arrays that could in future serve as discovery platforms, where computer controlled DMF automates mixing/titration of chemical libraries and NMR is used to study the structures formed and kinetics in real time.