Metabolomics has the potential to play a pivotal role in understanding disease onset and progression, and ultimately personalized treatments. One of its major challenges is its large-scale... Show moreMetabolomics has the potential to play a pivotal role in understanding disease onset and progression, and ultimately personalized treatments. One of its major challenges is its large-scale implementation, which is necessary to deal with the high variability of the metabolome. In this work we have developed tools for automated sample handling and preparation for metabolomics analysis, and bioanalysis in general. The tools are versatile, suitable for high-throughput, and able to deal with sensitive and biomass-limited samples. Sample transfer through segmented-flow can accommodate a wide range of samples and volumes, and can work seamlessly with many downstream processing or analysis. Two sample preparation tools based on droplets; one universal preconcentration tools using controlled evaporation, and one based on simultaneous extraction and enrichment, also provide a versatile interface and can be used to bridge gaps between processing steps. The working principles of these sample handling and preparation tools are universal and can be adapted for specific applications. Show less
Wouters, B.; Miggiels, A.L.W.; Bezemer, R.P.; Cruijsen, E.A.W. van der; Leeuwen, E. van; Gauvin, J.; ... ; Hankemeier, T. 2022
High-throughput analysis in fields such as industrial biotechnology, combinatorial chemistry, and life sciences is becoming increasingly important. Nuclear magnetic resonance (NMR) spectroscopy is... Show moreHigh-throughput analysis in fields such as industrial biotechnology, combinatorial chemistry, and life sciences is becoming increasingly important. Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique providing exhaustive molecular information on complex samples. Flow NMR in particular is a cost and time-efficient method for large screenings. In this study, we have developed a novel 3.0 mm inner diameter polychlorotrifluoroethylene (PCTFE) flow cell for a segmented-flow analysis (SFA) - NMR automated platform. The platform uses FC-72 fluorinated oil and fluoropolymer components to achieve a fully fluorinated flow path. Samples were repeatably transferred from 96-deepwell plates to the flow cell by displacing a fixed volume of oil, with a transfer time of 42 s. 1H spectra were acquired fully automated with 500 and 600 MHz NMR spectrometers. The spectral performance of the novel PCTFE cell was equal to that of commercial glass cells. Peak area repeatability was excellent with a relative standard deviation of 0.1-0.5% for standard samples, and carryover was below 0.2% without intermediate washing. The sample temperature was conditioned by using a thermostated transfer line in order to reduce the equilibration time in the probe and increase the throughput. Finally, analysis of urine samples demonstrated the applicability of this platform for screening complex matrices. Show less
Sample preparation is a labor-intensive and time-consuming procedure, especially for the bioanalysis of small-volume samples with low-abundant analytes. To minimize losses and dilution, sample... Show moreSample preparation is a labor-intensive and time-consuming procedure, especially for the bioanalysis of small-volume samples with low-abundant analytes. To minimize losses and dilution, sample preparation should ideally be hyphenated to downstream on-line analysis such as liquid chromatography-mass spectrometry (LC-MS). In this study, an automated three-phase electro-extraction (EE) method coupled to machine vision was developed, integrated with a robotic autosampler hyphenated to LC-MS. Eight model compounds, i.e. amitriptyline, clemastine, clomipramine, haloperidol, loperamide, propranolol, oxeladin, and verapamil were utilized for the optimization and evaluation of the automated EE setup. The stability of automated EE was evaluated by monitoring the acceptor droplet size by machine vision and recording the current during EE. A Design of Experiment approach (Box-Behnken design) was utilized to optimize the critical parameters of the EE method, i. e., the ratio of formic acid in the sample to acceptor phase, extraction voltage, and extraction time. The developed quadratic models showed good fitness (p < 0.001, R-2 > 0.95). Automated EE could be achieved in less than 2 min with enrichment factors (EF) up to 387 and extraction recoveries (ER) up to 97% for academic samples. Finally, the optimized EE method was successfully applied to both spiked human urine and plasma samples with low-concentration (50 ng mL(-1)) analytes and a low starting sample volume of 20 mu L of plasma and urine in 10-fold diluted samples. The developed automated EE setup is easy to operate, provides a fast extraction method for analytes from volume-limited biological samples, and is hyphenated with on-line LC-MS analysis. Therefore, this method can provide fast and automated sample preparation to solve bottlenecks in high-throughput bioanalysis workflows. Show less
Sample preparation is a challenge for high-throughput analysis, especially for volume-limited samples with low-abundant analytes. Ideally, sample preparation enriches the analytes of interest while... Show moreSample preparation is a challenge for high-throughput analysis, especially for volume-limited samples with low-abundant analytes. Ideally, sample preparation enriches the analytes of interest while removing the interferents to reduce the matrix effect and improve both sensitivity and quantification. In this study, a three-phase electroextraction (EE) method hyphenated to fast online liquid chromatography -mass spectrometry (LC-MS) was developed. Four model acidic drugs of relevance for drug monitoring in plasma, i.e. naproxen, fenoprofen, flurbiprofen, and ibuprofen, were utilized for the optimization and evaluation of the method. A Design of Experiment approach (Box-Behnken design) was used to optimize the critical parameters of the method, i.e., the type of organic solvent, pH of the sample and acceptor phase, and the extraction voltage and time. Good fitness (P < 0.02, R2 > 0.95) was observed for the developed quadratic model. Extraction could be achieved in less than 2 min (115 s) with enrichment factors (EF) up to 190 and extraction recoveries (ER) up to 38% for academic samples. Additionally, the optimized three-phase EE method was successfully applied to spiked plasma samples with lowabundant (50 ng mL-1) analytes and a low sample volume of 15 mL plasma in 10-fold diluted samples. Finally, two crucial contributors to the matrix effect of three-phase EE application on plasma samples were determined. Specifically, the ion-suppression effect in the MS source was reduced by the fast LC separation, and the matrix effect during extraction was negligible for the diluted protein-precipitated plasma samples. The developed three-phase EE method is easy to operate and provides fast and online extraction of trace-level acidic analytes from volume-limited biological samples. Therefore, this method can provide a potential solution for sample-preparation bottlenecks in high-throughput bioanalysis workflows. (c) 2021 Published by Elsevier B.V. Show less
