FTC Forensic Toxicological Laboratory Ltd

Vienna, Austria

FTC Forensic Toxicological Laboratory Ltd

Vienna, Austria
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Stubiger G.,Medical University of Vienna | Belgacem O.,Shimadzu Biotech | Rehulka P.,University of Hradec Kralove | Bicker W.,University of Vienna | And 3 more authors.
Analytical Chemistry | Year: 2010

6-Aza-2-thiothymine (ATT) is introduced as novel matrix system for the analysis of oxidized phospholipids (OxPLs) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). A systematic evaluation comparing different established and novel matrix substances, especially 2,4,6-THAP matrix (Stübiger, G.; Belgacem O. Anal. Chem. 2007, 79, 3206-3213) as reference compound for phospholipid analysis, and specific matrix additives was performed. Thereby, ATT turned out to be the reagent of choice for MALDI analysis of major biologically relevant OxPL classes (e.g., OxPC, OxPE, and OxPS) in positive and negative ionization mode. ATT used together with specific chaotropic reagents at low concentration (0.5-2 mM) acting as OxPL ionization enhancers revealed an excellent comatrix system for application with MALDI instrument types employing UV- and Nd:YAG laser systems (337 and 355 nm). Moreover, disposable MALDI targets surfaces with specific physicochemical properties (e.g., metallized glass or polymeric substrates) were revealed as superior over stainless steel in terms of reduced chemical background noise (∼10-fold better S/N ratios), increased mass spectral reproducibility, and enhanced sensitivity (LOD ∼250-500 fg on target). The combination of these parameters offers a significant advantage for highly sensitive OxPL profiling by MALDI-MS of biological samples (e.g., human plasma) at trace levels. © 2010 American Chemical Society.


Stubiger G.,Medical University of Vienna | Wuczkowski M.,Technoclone GmbH | Bicker W.,FTC Forensic Toxicological Laboratory Ltd. | Belgacem O.,Shimadzu
Analytical Chemistry | Year: 2014

In this paper we present a pioneering approach exploiting nanoparticles (NPs) for the "on-probe" (i.e., directly from the NP-surface) monitoring of OxPLs by MALDI-MS (i.e., the Nano-MALDI approach). The "electrophilic interaction" with either metal oxide (e.g., ZrO 2) or surface-functionalized Fe3O4 core-shell superparamagnetic NPs (100 nm diameter) was exploited for the direct enrichment of short-chain carboxylic (CARBO)-OxPLs, whereas detection of aldehydic (ALDO)-OxPLs was enabled by prior derivatization with bifunctional carbonyl-reactive reagents containing a negatively charged moiety (e.g., 4-AA) followed by NP-binding. Polyetheramine (PEA)-NPs were found best suited in terms of solvent stability, binding efficiency and compatibility with MALDI-MS analysis. For quantitative analysis of the OxPLs a recently introduced MALDI-QIT-TOF-MS/MS platform (Stübiger et al. Atherosclerosis 2012, 224, 177-186) was employed and cross-validated by LC-ESI-SRM-MS/MS. The sensitivity was found in the sub-nanomolar range (LOD ∼200 pM), which is 1-4 orders of magnitude higher than necessary for detection of individual OxPLs under normal and diseased conditions in vivo (e.g., in mouse plasma or human lipoproteins). Consequently, the Nano-MALDI approach shows the potential to serve as novel platform for the screening of OxPLs in biological samples and the development of clinical diagnostic tests in the future. © 2014 American Chemical Society.


Chen Y.,Nanchang University | Bicker W.,University of Vienna | Bicker W.,FTC Forensic Toxicological Laboratory Ltd. | Wu J.,University of Vienna | And 2 more authors.
Journal of Agricultural and Food Chemistry | Year: 2012

In order to develop a simple, efficient, and sensitive method for comprehensive analysis of the nucleosides and nucleobases in natural products, a zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) method for the simultaneous determination of 16 nucleosides and nucleobases has been studied. A mechanistic study confirmed that ZIC-HILIC separation showed a mixed-mode effect of both hydrophilic and electrostatic interactions. This method was validated to be precise, accurate, and sensitive with overall precision (intra- and interday) less than 1.8% (RSD), and LOD and LOQ was in the range of 0.005-0.029 μg/mL and 0.018-0.096 μg/mL, respectively. With this method, the nucleosides and nucleobases in Ganoderma of different species (G. atrum, G. lucidum, and G. sinense) and origins were quantified. The results showed that the contents varied with the species and origins. With the aid of hierarchical cluster analysis (HCA), cultivated Ganoderma from different origins and species were successfully discriminated. It is for the first time that the content of nucleosides and nucleobases in G. atrum is reported and compared. Our data showed that HILIC had advantages as a useful and potential tool for the study of the bioactive components in Ganoderma as well as their quality control, and could therefore be used for the determination of the analytes in other natural products. © 2012 American Chemical Society.


Reischl R.J.,University of Vienna | Bicker W.,University of Vienna | Bicker W.,FTC Forensic Toxicological Laboratory Ltd. | Keller T.,University of Salzburg | And 2 more authors.
Analytical and Bioanalytical Chemistry | Year: 2012

Acetaldehyde is a strongly electrophilic compound that is endogenously produced as a first intermediate in oxidative ethanol metabolism. Its high reactivity towards biogenic nucleophiles has toxicity as a consequence. Acetaldehyde readily undergoes a non-enzymatic condensation reaction and consecutive ring formation with cysteine to form 2-methylthiazolidine-4- carboxylic acid (MTCA). For analytical purposes, N-acetylation of MTCAwas required for stabilization and to enable its quantification by reversed-phase chromatography combined with electrospray ionization-tandem mass spectrometry. Qualitative screening of post mortem blood samples with negative blood alcohol concentration (BAC) mostly showed low basal levels of MTCA. In BACpositive post mortem samples, but not in corresponding urine specimens, strongly increased levels were present. To estimate the association between ethanol consumption and the occurrence ofMTCA in human blood, the time curves of BAC and MTCA concentration were determined after a single oral dose of 0.5 g ethanol per kilogram of body weight. The blood elimination kinetics ofMTCAwas slower than that of ethanol. The peak concentration of MTCA (12.6 mgL-1) was observed 4 h after ethanol intake (BAC 0.07%) and MTCA was still detectable after 13 h. Although intermediary acetaldehyde scavenging by formation of MTCA is interesting from a toxicological point of view, lack of hydrolytic stability under physiological conditions may hamper the use of MTCA as a quantitative marker of acetaldehyde exposure, such as resulting from alcohol consumption. © Springer-Verlag 2012.


Bicker W.,University of Vienna | Bicker W.,FTC Forensic Toxicological Laboratory Ltd. | Wu J.,University of Vienna | Yeman H.,University of Tübingen | And 2 more authors.
Journal of Chromatography A | Year: 2011

The separation properties of five silica packings bonded with 1-[3-(trimethoxysilyl)propyl]urea in the range of 0-3.67μmolm-2 were investigated in the hydrophilic interaction chromatography (HILIC) elution mode. An increase of the ligand surface density promoted retention of non-charged polar compounds and even more so for acids. An opposite trend was observed for bases, while the amphoteric compound tyrosine exhibited a U-shaped response profile. An overall partitioning retention mechanism was incompatible with these observations; rather, the substantial involvement of adsorptive interactions was implicated. Support for the latter was provided by column-specific changes in analyte retention and concomitant selectivity effects due to variations of salt concentration, type of salt, pH value, organic modifier content, and column temperature. Silica was more selective for separating compounds differing in charge state (e.g. tyramine vs. 4-hydroxybenzoic acid), while in cases where structural differences of solutes resided in non-charged polar groups (e.g. tyramine vs. 5-hydroxydopamine, nucleoside vs. nucleobase) more selective separations were obtained on bonded phases. Hierarchical cluster analysis of the home-made urea-type and three commercial amide-type bonded packings evinced considerable differences in separation properties. The present data emphasise that the role of the packing material under HILIC elution conditions is hardly just the polar support for a dynamic coating with a water-enriched layer. Three major retention mechanisms are claimed to be relevant on bare silica and the urea-type bonded packings: (i) HILIC-type partitioning, (ii) HILIC-type weak adsorption such as hydrogen bonding between solutes and ligands or solutes and silanols (potentially influenced by individual degrees of solvation, salt bridging, etc.), (iii) strong electrostatic (ionic) solute-silanol interactions (attractive/repulsive). Even when non-charged polar bonded phases are used, solute-silanol interactions should not be discounted, which makes them a prime parameter to be characterised by HILIC column tests. Multi/mixed-mode type separations seem to be common under HILIC elution conditions, associated with a great deal of selectivity increments. They are accessible and controllable by a careful choice of the type of packing, the mobile phase composition, and the temperature. © 2010 Elsevier B.V.


Stubiger G.,Medical University of Vienna | Aldover-Macasaet E.,Medical University of Vienna | Bicker W.,FTC Forensic Toxicological Laboratory Ltd. | Sobal G.,Medical University of Vienna | And 5 more authors.
Atherosclerosis | Year: 2012

Objectives: Phospholipids (PLs) are increasingly recognized as key molecules with potential diagnostic value in acute inflammation, CVD and atherosclerosis. We introduce a pioneer mass spectrometry (MS)-based approach aiming to investigate the relationship of specific plasma PL-subsets with atherogenic blood parameters in young patients with familial hyperlipidemia representing high-CVD-risk groups. Methods: Plasma of carefully phenotyped FH and FCH patients as well as normolipidemic subjects (age 13 ± 5 years, n = 20) was used. Clinical parameters were assessed using standard laboratory techniques and lipids were subjected to a direct targeted monitoring using LC-ESI-SRM- and MALDI-QIT-TOF-MS/MS, respectively. Statistical analysis was performed to evaluate correlations between PL data and the clinical parameters. Results: Most characteristically significant differences of SM/PC and PC/LPC ratios and positive correlations between SM vs. LDL-C (r = 0.946; p = 0.004) and LPC vs. VLDL-C (r = 0.669; p = 0.218) were observed in FH in contrast to the other study groups. OxPC levels were found in the range of ∼2-20 μmol/L with predominance of short-chain aldehydic species (e.g. SOVPC). A positive correlation of OxPCs with IMT (r = 0.952; p = 0.052) and HDL-C (r = 0.893; p = 0.016) but negative correlation with OxLDL (r = -0.910; p = 0.096) was observed. Conclusions: Our study was a first attempt to use a MALDI-QIT-TOF-MS/MS based clinical lipidomics approach to investigate atherogenic dyslipidemia in young patients with familial hyperlipidemia. This technique represents a promising platform for clinical screening of lipid biomarkers in the future. © 2012 Elsevier Ireland Ltd.


Bicker W.,FTC Forensic Toxicological Laboratory Ltd. | Monticelli F.,University of Salzburg | Bauer A.,Ludwig Maximilians University of Munich | Roider G.,Ludwig Maximilians University of Munich | Keller T.,University of Salzburg
Drug Testing and Analysis | Year: 2013

The diester-diterpene alkaloid aconitine was quantified by liquid chromatography-tandem mass spectrometry in post-mortem specimens of three cases where suicidal ingestion of Aconitum napellus L. ('monkshood') was supposed. In an attempt at rationalization, sample preparation and chromatographic conditions of plasma/serum drug analysis routine were utilized. Linearity was established from 0.5 to 20μgL-1 using newborn calf serum (NCS) as a surrogate calibration matrix for all sample types and mesaconitine as an internal standard. Validation (selectivity, sensitivity, precision, accuracy, recovery of the extraction procedure, matrix effect, processed sample stability) confirmed the applicability of the analytical method to various post-mortem matrices. Internal standard selection was based on multi-matrix process efficiency data. In human post-mortem peripheral blood a lower limit of quantification of 0.51μgL-1 and a limit of detection of 0.13μgL-1 were accomplished (0.1ml sample aliquots). Aconitine was degraded to a large extent in different sample types when being stored at +20°C for 30days, while at -20°C and for some matrices also at +4°C no appreciable degradation occurred. Aconitine concentrations in real samples were 10.3-17.9μgL-1 (peripheral blood, n=3), 14.9-87.9μgL-1 (heart blood, n=3), 317-481μgL-1 (urine, n=2), 609-4040μgL-1 (stomach content, n=3), 139-240μgL-1 (bile, n=2), 8.4μgL-1 (vitreous humor, n=1), 54.7μgL-1 (pericardial fluid, n=1), 492μgkg-1 (liver, n=1), 15.2-19.7mgL-1 (unknown liquids secured onsite, n=3). Together with concomitant circumstances the analytical data provided compelling evidence for acute Aconitum poisoning as being the cause of death. © 2013 John Wiley & Sons, Ltd.


Uderhardt S.,Friedrich - Alexander - University, Erlangen - Nuremberg | Herrmann M.,Friedrich - Alexander - University, Erlangen - Nuremberg | Oskolkova O.V.,Medical University of Vienna | Aschermann S.,Friedrich - Alexander - University, Erlangen - Nuremberg | And 10 more authors.
Immunity | Year: 2012

Noninflammatory clearance of apoptotic cells (ACs) is crucial to maintain self-tolerance. Here, we have reported a role for the enzyme 12/15-lipoxygenase (12/15-LO) as a central factor governing the sorting of ACs into differentially activated monocyte subpopulations. During inflammation, uptake of ACs was confined to a population of 12/15-LO-expressing, alternatively activated resident macrophages (resMΦ), which blocked uptake of ACs into freshly recruited inflammatory Ly6Chi monocytes in a 12/15-LO-dependent manner. ResMΦ exposed 12/15-LO-derived oxidation products of phosphatidylethanolamine (oxPE) on their plasma membranes and thereby generated a sink for distinct soluble receptors for ACs such as milk fat globule-EGF factor 8, which were essential for the uptake of ACs into inflammatory monocytes. Loss of 12/15-LO activity, in turn, resulted in an aberrant phagocytosis of ACs by inflammatory monocytes, subsequent antigen presentation of AC-derived antigens, and a lupus-like autoimmune disease. Our data reveal an unexpected key role for enzymatic lipid oxidation during the maintenance of self-tolerance. © 2012 Elsevier Inc.


Gruber F.,Medical University of Vienna | Bicker W.,FTC Forensic Toxicological Laboratory Ltd. | Oskolkova O.V.,Medical University of Vienna | Tschachler E.,Medical University of Vienna | Bochkov V.N.,Medical University of Vienna
Journal of Lipid Research | Year: 2012

Oxidized phospholipids (OxPLs) are increasingly recognized as signaling mediators that are not only markers of oxidative stress but are also "makers" of pathology relevant to disease pathogenesis. Understanding the biological role of individual molecular species of OxPLs requires the knowledge of their concentration kinetics in cells and tissues. In this work, we describe a straightforward "fingerprinting" procedure for analysis of a broad spectrum of molecular species generated by oxidation of the four most abundant species of polyunsaturated phosphatidylcholines (OxPCs). The approach is based on liquid-liquid extraction followed by reversedphase HPLC coupled to electrospray ionization MS/MS. More than 500 peaks corresponding in retention properties to polar and oxidized PCs were detected within 8 min at 99 m/z precursor values using a single diagnostic product ion in extracts from human dermal fibroblasts. Two hundred seventeen of these peaks were fluence-dependently and statistically significantly increased upon exposure of cells to UVA irradiation, suggesting that these are genuine oxidized or oxidatively fragmented species. This method of semitargeted lipidomic analysis may serve as a simple first step for characterization of specific "signatures"of OxPCs produced by different types of oxidative stress in order to select the most informative peaks for identification of their molecular structure and biological role. Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc.


PubMed | FTC Forensic Toxicological Laboratory Ltd.
Type: Case Reports | Journal: Drug testing and analysis | Year: 2013

The diester-diterpene alkaloid aconitine was quantified by liquid chromatography-tandem mass spectrometry in post-mortem specimens of three cases where suicidal ingestion of Aconitum napellus L. (monkshood) was supposed. In an attempt at rationalization, sample preparation and chromatographic conditions of plasma/serum drug analysis routine were utilized. Linearity was established from 0.5 to 20gL using newborn calf serum (NCS) as a surrogate calibration matrix for all sample types and mesaconitine as an internal standard. Validation (selectivity, sensitivity, precision, accuracy, recovery of the extraction procedure, matrix effect, processed sample stability) confirmed the applicability of the analytical method to various post-mortem matrices. Internal standard selection was based on multi-matrix process efficiency data. In human post-mortem peripheral blood a lower limit of quantification of 0.51gL and a limit of detection of 0.13gL were accomplished (0.1ml sample aliquots). Aconitine was degraded to a large extent in different sample types when being stored at +20C for 30days, while at -20C and for some matrices also at +4C no appreciable degradation occurred. Aconitine concentrations in real samples were 10.3-17.9gL (peripheral blood, n=3), 14.9-87.9gL (heart blood, n=3), 317-481gL (urine, n=2), 609-4040gL (stomach content, n=3), 139-240gL (bile, n=2), 8.4gL (vitreous humor, n=1), 54.7gL (pericardial fluid, n=1), 492gkg (liver, n=1), 15.2-19.7mgL (unknown liquids secured onsite, n=3). Together with concomitant circumstances the analytical data provided compelling evidence for acute Aconitum poisoning as being the cause of death.

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