Three-Phase Electroextraction: A New (Online) Sample Purification and Enrichment Method for Bioanalysis

The migration and at the same time enrichment of analytes from a liquid aqueous sample donor phase through an immiscible organic solvent layer acting as a filter phase into a liquid aqueous acceptor phase is enabled by the application of an electric field between the donor and acceptor phase. The organic filter phase acts as a purification filter, which prevents, for example, proteins from migrating into the acceptor phase. Moreover, the composition of the organic filter phase influences the selectivity of the extraction. We show that analytes can be rapidly enriched from a 50 μL donor phase at the bottom of a sample vial, via an immiscible organic filter phase, into a 2 μL acceptor phase which consists of a droplet that is hanging from a (conductive) pipet tip in the organic filter phase. Acylcarnitines spiked to human plasma as a donor phase were extracted reproducibly with good linearity and a 10-fold improved limit of detection and, importantly, resulted in a stable, protein-free nanoelectrospray signal. Finally, a proof of principle toward the online integration in an automated nanoelectrospray-direct infusion-mass spectrometry platform has been realized. This makes 3-phase electroextraction (3-phase EE) a novel sample purification and enrichment method, with straightforward online integration possibility. We envision that 3-phase EE will enable new possibilities using electrokinetic sample pretreatment for fully automated, high-throughput bioanalysis purposes

Three-Phase Electroextraction: A New (Online) Sample Purification and Enrichment Method for Bioanalysis

The migration and at the same time enrichment of analytes from a liquid aqueous sample donor phase through an immiscible organic solvent layer acting as a filter phase into a liquid aqueous acceptor phase is enabled by the application of an electric field between the donor and acceptor phase. The organic filter phase acts as a purification filter, which prevents, for example, proteins from migrating into the acceptor phase. Moreover, the composition of the organic filter phase influences the selectivity of the extraction. We show that analytes can be rapidly enriched from a 50 μL donor phase at the bottom of a sample vial, via an immiscible organic filter phase, into a 2 μL acceptor phase which consists of a droplet that is hanging from a (conductive) pipet tip in the organic filter phase. Acylcarnitines spiked to human plasma as a donor phase were extracted reproducibly with good linearity and a 10-fold improved limit of detection and, importantly, resulted in a stable, protein-free nanoelectrospray signal. Finally, a proof of principle toward the online integration in an automated nanoelectrospray-direct infusion-mass spectrometry platform has been realized. This makes 3-phase electroextraction (3-phase EE) a novel sample purification and enrichment method, with straightforward online integration possibility. We envision that 3-phase EE will enable new possibilities using electrokinetic sample pretreatment for fully automated, high-throughput bioanalysis purposes

Three-Phase Electroextraction: A New (Online) Sample Purification and Enrichment Method for Bioanalysis

The migration and at the same time enrichment of analytes from a liquid aqueous sample donor phase through an immiscible organic solvent layer acting as a filter phase into a liquid aqueous acceptor phase is enabled by the application of an electric field between the donor and acceptor phase. The organic filter phase acts as a purification filter, which prevents, for example, proteins from migrating into the acceptor phase. Moreover, the composition of the organic filter phase influences the selectivity of the extraction. We show that analytes can be rapidly enriched from a 50 μL donor phase at the bottom of a sample vial, via an immiscible organic filter phase, into a 2 μL acceptor phase which consists of a droplet that is hanging from a (conductive) pipet tip in the organic filter phase. Acylcarnitines spiked to human plasma as a donor phase were extracted reproducibly with good linearity and a 10-fold improved limit of detection and, importantly, resulted in a stable, protein-free nanoelectrospray signal. Finally, a proof of principle toward the online integration in an automated nanoelectrospray-direct infusion-mass spectrometry platform has been realized. This makes 3-phase electroextraction (3-phase EE) a novel sample purification and enrichment method, with straightforward online integration possibility. We envision that 3-phase EE will enable new possibilities using electrokinetic sample pretreatment for fully automated, high-throughput bioanalysis purposes

Tunable Ionic Mobility Filter for Depletion Zone Isotachophoresis

We present a novel concept of filtering based on depletion zone isotachophoresis (dzITP). In the micro/nanofluidic filter, compounds are separated according to isotachophoretic principles and simultaneously released selectively along a nanochannel-induced depletion zone. Thus, a tunable low-pass ionic mobility filter is realized. We demonstrate quantitative control of the release of fluorescent compounds through the filter using current and voltage actuation. Two modes of operation are presented. In continuous mode, supply, focusing, and separation are synchronized with continuous compound release, resulting in trapping of specific compounds. In pulsed mode, voltage pulses result in release of discrete zones. The dzITP filter was used to enhance detection of 6-carboxyfluorescein 4-fold over fluorescein, even though it had 250× lower starting concentration. Moreover, specific high-mobility analytes were extracted and enriched from diluted raw urine, using fluorescein as an ionic mobility cutoff marker and as a tracer for indirect detection. Tunable ionic filtering is a simple but essential addition to the capabilities of dzITP as a versatile toolkit for biochemical assays

Gas Pressure Assisted Microliquid–Liquid Extraction Coupled Online to Direct Infusion Mass Spectrometry: A New Automated Screening Platform for Bioanalysis

In the field of bioanalysis, there is an increasing demand for miniaturized, automated, robust sample pretreatment procedures that can be easily connected to direct-infusion mass spectrometry (DI-MS) in order to allow the high-throughput screening of drugs and/or their metabolites in complex body fluids like plasma. Liquid–Liquid extraction (LLE) is a common sample pretreatment technique often used for complex aqueous samples in bioanalysis. Despite significant developments that have been made in automated and miniaturized LLE procedures, fully automated LLE techniques allowing high-throughput bioanalytical studies on small-volume samples using direct infusion mass spectrometry, have not been matured yet. Here, we introduce a new fully automated micro-LLE technique based on gas-pressure assisted mixing followed by passive phase separation, coupled online to nanoelectrospray-DI-MS. Our method was characterized by varying the gas flow and its duration through the solvent mixture. For evaluation of the analytical performance, four drugs were spiked to human plasma, resulting in highly acceptable precision (RSD down to 9%) and linearity (R² ranging from 0.990 to 0.998). We demonstrate that our new method does not only allow the reliable extraction of analytes from small sample volumes of a few microliters in an automated and high-throughput manner, but also performs comparable or better than conventional offline LLE, in which the handling of small volumes remains challenging. Finally, we demonstrate the applicability of our method for drug screening on dried blood spots showing excellent linearity (R² of 0.998) and precision (RSD of 9%). In conclusion, we present the proof of principe of a new high-throughput screening platform for bioanalysis based on a new automated microLLE method, coupled online to a commercially available nano-ESI-DI-MS

Additional file 1: of Metabolic characterization of the natural progression of chronic hepatitis B

Supplementary methods. (DOCX 33 kb

Additional file 2: of Metabolic characterization of the natural progression of chronic hepatitis B

Supplementary Figures S1 and S2 and Tables S1 and S3. (DOCX 397 kb

Matrix Effect Compensation in Small-Molecule Profiling for an LC–TOF Platform Using Multicomponent Postcolumn Infusion

The possible presence of matrix effect is one of the main concerns in liquid chromatography–mass spectrometry (LC–MS)-driven bioanalysis due to its impact on the reliability of the obtained quantitative results. Here we propose an approach to correct for the matrix effect in LC–MS with electrospray ionization using postcolumn infusion of eight internal standards (PCI-IS). We applied this approach to a generic ultraperformance liquid chromatography–time-of-flight (UHPLC–TOF) platform developed for small-molecule profiling with a main focus on drugs. Different urine samples were spiked with 19 drugs with different physicochemical properties and analyzed in order to study matrix effect (in absolute and relative terms). Furthermore, calibration curves for each analyte were constructed and quality control samples at different concentration levels were analyzed to check the applicability of this approach in quantitative analysis. The matrix effect profiles of the PCI-ISs were different: this confirms that the matrix effect is compound-dependent, and therefore the most suitable PCI-IS has to be chosen for each analyte. Chromatograms were reconstructed using analyte and PCI-IS responses, which were used to develop an optimized method which compensates for variation in ionization efficiency. The approach presented here improved the results in terms of matrix effect dramatically. Furthermore, calibration curves of higher quality are obtained, dynamic range is enhanced, and accuracy and precision of QC samples is increased. The use of PCI-ISs is a very promising step toward an analytical platform free of matrix effect, which can make LC–MS analysis even more successful, adding a higher reliability in quantification to its intrinsic high sensitivity and selectivity

Adjusted hazard ratios (HR) and 95% confidence interval (CI) for appropriate shock and all-cause mortality associated with each oxylipin.

<p>Models were adjusted for age, sex, race, enrollment center, ejection fraction, NYHA class, cardiomyopathy etiology, atrial fibrillation, diabetes, hypertension, and chronic kidney disease.</p

Baseline characteristics of participants, by appropriate ICD shock.

<p>Baseline characteristics of participants, by appropriate ICD shock.</p