28 research outputs found
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
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 (R<sup>2</sup> 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 (R<sup>2</sup> 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
Baseline characteristics of participants, by all-cause mortality.
<p>Baseline characteristics of participants, by all-cause mortality.</p
Organization of the roquefortine/meleagrin biosynthetic gene cluster and transcriptomic analysis.
<p>(A) Roquefortine/meleagrin biosynthetic gene cluster and their orthologs in phylogenetically relative species. Homologous proteins are indicated with the same color. (B) Microarray analysis of the roquefortine biosynthetic genes in <i>P. chrysogenum</i> DS54555 using shake flask culture conditions in the absence (−) or presence (+) phenylacetic acid (PAA). (C) Correlation between the expression level of <i>roqA</i> and the concentration of the product HTD (<b>1)</b> present in the growth media. The concentration of <b>1</b> was determined by HPLC-UV-MS.</p
Adjusted hazard ratios (HR) and 95% confidence interval (CI) for appropriate shock and all-cause mortality associated with each oxylipin-to-precursor ratio.
<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
Southern blot analysis for deletion of the genes in the roquefortine/meleagrin pathway.
<p>Southern blot hybridization was performed with total DNA extracted from <i>P. chrysogenum</i> DS54555 strains with a deletion of the following genes: <i>roqA</i> (A), <i>roqR</i> (B), <i>roqD</i> (C), <i>roqM</i> (D), <i>roqO</i> (E), <i>roqN</i> (F) and <i>roqT</i> (G). The DNA was digested with the restriction enzymes as indicated in the schemes.</p
Födoval av torsk (Gadus morrhua L.) i Skagerrak och Kattegatt under februari 1981 /
<div><p>Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus <i>Penicillium chrysogenum</i> and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this pathway were identified as histidyltryptophanyldiketopiperazine (HTD), dehydrohistidyltryptophanyldi-ketopiperazine (DHTD), roquefortine D, roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step. The nonribosomal peptide synthetase RoqA accepts L-histidine and L-tryptophan as substrates leading to the production of the diketopiperazine HTD. DHTD, previously suggested to be a degradation product of roquefortine C, was found to be derived from HTD involving the cytochrome P450 oxidoreductase RoqR. The dimethylallyltryptophan synthetase RoqD prenylates both HTD and DHTD yielding directly the products roquefortine D and roquefortine C without the synthesis of a previously suggested intermediate and the involvement of RoqM. This leads to a branch in the otherwise linear pathway. Roquefortine C is subsequently converted into glandicoline B with glandicoline A as intermediates, involving two monooxygenases (RoqM and RoqO) which were mixed up in an earlier attempt to elucidate the biosynthetic pathway. Eventually, meleagrin is produced from glandicoline B involving a methyltransferase (RoqN). It is concluded that roquefortine C and meleagrin are derived from a branched biosynthetic pathway.</p></div