10 research outputs found
Transgenic apple plants overexpressing the chalcone 3-hydroxylase gene of Cosmos sulphureus show increased levels of 3-hydroxyphloridzin and reduced susceptibility to apple scab and fire blight
Main conclusionOverexpression of chalcone-3-hydroxylase provokes increased accumulation of 3-hydroxyphloridzin inMalus. Decreased flavonoid concentrations but unchanged flavonoid class composition were observed. The increased 3-hydroxyphlorizin contents correlate well with reduced susceptibility to fire blight and scab.The involvement of dihydrochalcones in the apple defence mechanism against pathogens is discussed but unknown biosynthetic steps in their formation hamper studies on their physiological relevance. The formation of 3-hydroxyphloretin is one of the gaps in the pathway. Polyphenol oxidases and cytochrome P450 dependent enzymes could be involved. Hydroxylation of phloretin in position 3 has high similarity to the B-ring hydroxylation of flavonoids catalysed by the well-known flavonoid 3′-hydroxylase (F3′H). Using recombinant F3′H and chalcone 3-hydroxylase (CH3H) from Cosmos sulphureus we show that F3′H and CH3H accept phloretin to some extent but higher conversion rates are obtained with CH3H. To test whether CH3H catalyzes the hydroxylation of dihydrochalcones in planta and if this could be of physiological relevance, we created transgenic apple trees harbouring CH3H from C. sulphureus. The three transgenic lines obtained showed lower polyphenol concentrations but no shift between the main polyphenol classes dihydrochalcones, flavonols, hydroxycinnamic acids and flavan 3-ols. Increase of 3-hydroxyphloridzin within the dihydrochalcones and of epicatechin/catechin within soluble flavan 3-ols were observed. Decreased activity of dihydroflavonol 4-reductase and chalcone synthase/chalcone isomerase could partially explain the lower polyphenol concentrations. In comparison to the parent line, the transgenic CH3H-lines showed a lower disease susceptibility to fire blight and apple scab that correlated with the increased 3-hydroxyphlorizin contents.Austrian Sci-ence Fund (FWF
Liquid Chromatography, In Combination with a Quadrupole Time-of-Flight Instrument, with Sequential Window Acquisition of All Theoretical Fragment-Ion Spectra Acquisition: Validated Quantification of 39 Antidepressants in Whole Blood As Part of a Simultaneous Screening and Quantification Procedure
Sequential
window acquisition of all theoretical fragment ion spectra
(SWATH) is a data independent acquisition (DIA) method for very fast
scanning quadrupole time-of-flight (QTOF) instruments. SWATH repeatedly
cycles through 28 consecutive 20 Da precursor isolation windows detecting
all precursor ions and fragments MS ALL like and yet fast enough to
generate more than 10 data points over the chromatographic peak. It
was already shown in previous publications that SWATH, despite its
wide Q1 windows, allows the identification of different substances
and that SWATH has a higher identification rate than data dependent
acquisition approaches. The aim of this study was a proof of concept
study whether these same data sets can also enable validated quantification
according to international guidelines, exemplified for 39 antidepressants.
The validation included recovery, matrix effects, process efficiency,
ion suppression, and enhancement of coeluting ions, selectivity, accuracy,
precision, and stability. The method using SWATH acquisition proved
to be selective, sensitive, accurate, and precise enough for 33 out
of the 39 antidepressants. The applicability of SWATH for screening
and validated quantification in the same run was successfully tested
with authentic whole blood samples containing different antidepressants
and other drugs thus proving the QUAL/QUAN abilities of SWATH. In
an additional systematic investigation, it could be shown that calibration
curves injected a few days after or before the actual sample can be
used for quantification with acceptable accuracy
LC QTOF with SWATH acquisition: Systematic studies on its use for screenings in clinical and forensic toxicology and comparison with IDA and targeted MRM approaches
Forensic and clinical toxicological screening procedures are more and more employing LC-MS/MS techniques with information dependent acquisition (IDA) approaches. It is known that complexity of sample and settings of IDA might prevent important compounds from being triggered. Therefore, data independent acquisition methods (DIA) should be more suitable for systematic toxicological analysis (STA). The DIA method sequential window acquisition of all theoretical fragment-ion spectra (SWATH) which uses Q1 windows of 20-35 Da for data independent fragmentation, was systematically investigated for its suitability for STA. Quality of SWATH generated mass spectra were evaluated with regard to mass error, relative abundance of the fragments and library hits. With the Q1 window set to 20-25 Da, several precursors pass Q1 at the same time and are fragmented thus impairing the library search algorithms to a different extent: forward fit was less affected than reverse fit and purity fit. Mass error was not affected. Relative abundance of the fragments was concentration dependent for some analytes and was influenced by co-fragmentation, especially of deuterated analogues. Also the detection rate of IDA compared to SWATH was investigated in a forced coelution experiment (up to 20 analytes coeluting). Even using several different IDA settings it was observed that IDA failed to trigger relevant compounds. Screening results of 382 authentic forensic cases revealed that SWATH’s detection rate was superior to IDA, which failed to trigger about 10% of the analytes
Purple discoloration of the colon found during autopsy: Identification of betanin, its aglycone and metabolites by liquid chromatography–high-resolution mass spectrometry
Single hair analysis of small molecules using MALDI-triple quadru-pole MS imaging and LC-MS/MS – investigations on opportunities and pitfalls
Single hair analysis normally requires extensive sample preparation micro scale protocols including time consuming steps like segmentation and extraction. Matrix assisted laser desorption and ionization mass spectrometric imaging (MALDI-MSI) was shown to be an alternative tool in single hair analysis but still questions remain. Therefore an investigation of MALDI-MSI in single hair analysis concerning extraction process, usage of internal standard (IS) and influences on ionization processes were systematically investigated to enable the reliable application to hair analysis. Furthermore, single dose detection, quantitative correlation to single hair and hair strand LC-MS/MS results was performed and performance compared to LC-MS/MS single hair monitoring. MALDI process showed to be independent from natural hair color and not being influenced by the presence of melanin. Ionization was shown to be reproducible along and in between different hair samples. MALDI image intensities in single hair and hair snippets showed good semi-quantitative correlation to zolpidem hair concentrations obtained from validated routine LC-MS/MS methods. MALDI-MSI is superior to LC-MS/MS analysis when a fast, easy and cheap sample preparation is necessary, whereas LC-MS/MS showed higher sensitivity with the ability of single dose detection for zolpidem. MALDI-MSI and LC-MS/MS segmental single hair analysis showed good correlation and are both suitable for consumption monitoring of drugs of abuse with a high time resolution
Liquid Chromatography, in Combination with a Quadrupole Time-of-Flight Instrument (LC QTOF), with Sequential Window Acquisition of All Theoretical Fragment-Ion Spectra (SWATH) Acquisition: Systematic Studies on Its Use for Screenings in Clinical and Forensic Toxicology and Comparison with Information-Dependent Acquisition (IDA)
Forensic and clinical toxicological
screening procedures are employing
liquid chromatography–tandem mass spectrometry (LC-MS/MS) techniques
with information-dependent acquisition (IDA) approaches more and more
often. It is known that the complexity of a sample and the IDA settings
might prevent important compounds from being triggered. Therefore,
data-independent acquisition (DIA) methods should be more suitable
for systematic toxicological analysis (STA). The DIA method sequential
window acquisition of all theoretical fragment-ion spectra (SWATH),
which uses Q1 windows of 20–35 Da for data-independent fragmentation,
was systematically investigated for its suitability for STA. Quality
of SWATH-generated mass spectra were evaluated with regard to mass
error, relative abundance of the fragments, and library hits. With
the Q1 window set to 20–25 Da, several precursors pass Q1 at
the same time and are fragmented, thus impairing the library search
algorithms to a different extent: forward fit was less affected than
reverse fit and purity fit. Mass error was not affected. The relative
abundance of the fragments was concentration dependent for some analytes
and was influenced by cofragmentation, especially of deuterated analogues.
Also, the detection rate of IDA compared to SWATH was investigated
in a forced coelution experiment (up to 20 analytes coeluting). Even
using several different IDA settings, it was observed that IDA failed
to trigger relevant compounds. Screening results of 382 authentic
forensic cases revealed that SWATH’s detection rate was superior
to IDA, which failed to trigger ∼10% of the analytes
Comparison of conventional liquid chromatography–tandem mass spectrometry versus microflow liquid chromatography–tandem mass spectrometry within the framework of full method validation for simultaneous quantification of 40 antidepressants and neuroleptics in whole blood
Liquid Chromatography, In Combination with a Quadrupole Time-of-Flight Instrument, with Sequential Window Acquisition of All Theoretical Fragment-Ion Spectra Acquisition: Validated Quantification of 39 Antidepressants in Whole Blood As Part of a Simultaneous Screening and Quantification Procedure
Single Hair Analysis of Small Molecules Using MALDI-Triple Quadrupole MS Imaging and LC-MS/MS: Investigations on Opportunities and Pitfalls
Single hair analysis normally requires
extensive sample preparation
microscale protocols including time-consuming steps like segmentation
and extraction. Matrix assisted laser desorption and ionization mass
spectrometric imaging (MALDI-MSI) was shown to be an alternative tool
in single hair analysis, but still, questions remain. Therefore, an
investigation of MALDI-MSI in single hair analysis concerning the
extraction process, usage of internal standard (IS), and influences
on the ionization processes were systematically investigated to enable
the reliable application to hair analysis. Furthermore, single dose
detection, quantitative correlation to a single hair, and hair strand
LC-MS/MS results were performed, and the performance was compared
to LC-MS/MS single hair monitoring. The MALDI process was shown to
be independent from natural hair color and not influenced by the presence
of melanin. Ionization was shown to be reproducible along and in between
different hair samples. MALDI image intensities in single hair and
hair snippets showed good semiquantitative correlation to zolpidem
hair concentrations obtained from validated routine LC-MS/MS methods.
MALDI-MSI is superior to LC-MS/MS analysis when a fast, easy, and
cheap sample preparation is necessary, whereas LC-MS/MS showed higher
sensitivity with the ability of single dose detection for zolpidem.
MALDI-MSI and LC-MS/MS segmental single hair analysis showed good
correlation, and both are suitable for consumption monitoring of drugs
of abuse with a high time resolution