Emmie Dumont

Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry

An LC-MS based method for the profiling and characterization of ceramide species in the upper layer of human skin is described. Ceramide samples, collected by tape stripping of human skin, were analyzed by reversed-phase liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry operated in both positive and negative electrospray ionization mode. All known classes of ceramides could be measured in a repeatable manner. Furthermore, the data set showed several undiscovered ceramides, including a class with four hydroxyl functionalities in its sphingoid base. High-resolution MS/MS fragmentation spectra revealed that each identified ceramide species is composed of several skeletal isomers due to variation in carbon length of the respective sphingoid bases and fatty acyl building blocks. The resulting variety in skeletal isomers has not been previously demonstrated. It is estimated that over 1000 unique ceramide structures could be elucidated in human stratum corneum. Ceramide species with an even and odd number of carbon atoms in both chains were detected in all ceramide classes. Acid hydrolysis of the ceramides, followed by LC-MS analysis of the end-products, confirmed the observed distribution of both sphingoid bases and fatty acyl groups in skin ceramides. The study resulted in an accurate mass retention time library for targeted profiling of skin ceramides. It is furthermore demonstrated that targeted data processing results in an improved repeatability versus untargeted data processing (72.92% versus 62.12% of species display an RSD < 15%).

Biomarkers of human exposure to personal care products: Results from the Flemish Environment and Health Study (FLEHS 2007–2011)

Personal care products (PCPs), such as soaps, perfumes, cosmetics, lotions, etc., contain a variety of chemicals that have been described as potentially hormone disrupting chemicals. Therefore, it is important to assess the internal exposure of these chemicals in humans. Within the 2nd Flemish Environment and Health Study (FLEHS II, 2007-2011), the human exposure to three classes of pollutants that are present in a wide variety of PCPs--i.e. polycyclic musks (galaxolide, HHCB and tonalide, AHTN in blood), parabens (urinary para-hydroxybenzoic acid, HBA) and triclosan (urinary TCS)--was assessed in 210 Flemish adolescents (14-15 years) and in 204 adults (20-40 years) randomly selected from the general population according to a stratified two stage clustered study design. The aim of this study was to define average levels of exposure in the general Flemish population and to identify determinants of exposure. Average levels (GM (95% CI)) in the Flemish adolescents were 0.717 (0.682-0.753) μg/L for blood HHCB; 0.118 (0.108-0.128) μg/L for blood AHTN; 1022 (723-1436) μg/L for urinary HBA and 2.19 (1.64-2.92) μg/L for urinary TCS. In the adults, levels of HBA were on average 634 (471-970) μg/L. Inter-individual variability was small for HHCB and AHTN, intermediate for HBA, and large for TCS. All biomarkers were positively associated with the use of PCPs. Additionally, levels of HHCB and AHTN increased with higher educational level of the adolescents. Both in adults and adolescents, urinary HBA levels were negatively correlated with BMI. We define here Flemish exposure values for biomarkers of PCPs, which can serve as baseline exposure levels to identify exposure trends in future biomonitoring campaigns.

Separation and detection of Se-compounds by ion pairing liquid chromatography-microwave assisted hydride generation-atomic fluorescence spectrometry

Liquid chromatography coupled to a hydride generation atomic fluorescence spectrometer has been applied for the speciation of Se in extracts of Saccharomyces cerevisiae. In order to develop a method which allows the separation of the compounds and detection of the element, seven Se standards were used: Se-methionine (Se-Met), Se-cystine (Se-(Cys)2), Se-cystamine (Se-Cya), Se-methylselenocysteine (Se-MeSeCys), Se-ethionine (Se-Et), selenate (SeVI), selenite (SeIV). Optimal chromatographic results were obtained with reversed-phase chromatography on an XTerra C18 column using a positively charged ion-pairing agent. It was observed that for these standards precise control of the pH was of utmost importance. Attention was devoted to the compatibility of the mobile phase with hydride generation. Efficient formation of the hydrides was obtained by optimisation of different parameters. The redox mixture which allowed optimum conversion of all different species was HBr–KBrO3. To assist in the conversion of the compounds, on-line microwave digestion was applied. The detection limits obtained for the standards were: 0.8 µg Se l−1 for selenite(IV); 1.3 µg Se l−1 for selenate(VI); 1.2 µg Se l−1 for Se-methionine; 1.2 µg Se l−1 for Se-cystine; 1.3 µg Se l−1 for Se-cystamine; and 1.1 µg Se l−1 for Se-methylselenocysteine, respectively. Se-compounds in Saccharomyces cerevisiae were extracted by hot water (50 °C) or proteolytic digestion with protease XIV (37 °C). The method developed for separation and elemental detection was applied to these extracts in order to distinguish between the different species extracted from the yeast matrix. Total Se concentration in the extracts was measured with pneumatic nebulization-inductively coupled plasma-mass spectrometry (PN-ICP-MS). Species transformation was investigated by analysing extracts preserved at 2 different temperatures (−20 °C and 4 °C). Only those extracts kept at −20 °C proved to be unchanged.

Determination of arylamines and aminopyridines in pharmaceutical products using in-situ derivatization and liquid chromatography-mass spectrometry

Arylamines and aminopyridines form a class of potentially genotoxic impurities (PGIs) that can be present at trace levels in active pharmaceutical ingredients (APIs). A generic method was developed that allows the analysis of a selected set of these solutes at sub-ppm level relative to the drug substance. A highly concentrated solution of the pharmaceutical compound is analyzed by LC-MS using a single quadrupole mass spectrometer in the selected ion monitoring (SIM) mode. Since a number of target compounds show little or no retention in the reversed-phase LC setup, a fast and simple derivatization procedure using hexylchloroformate was applied. The amide derivatives of the PGI result in a higher molecular weight (more specific ion for SIM) and better chromatographic behavior. The methodology, consisting of a dual run on respectively a non-derivatized and a derivatized sample, was validated and applied to a selection of pharmaceutical substances. The method was found to be sufficiently sensitive and robust and is applicable in a QA/QC environment.

Vanadium speciation in serum by means of blue native gel electrophoresis.

Slab‐gel electrophoresis has been applied to the speciation of vanadium in serum. The electrophoresis separation is an adaptation of the blue native polyacrylamide gel electrophoresis separation necessary to ensure the stability of the vanadium‐protein complex; Coomassie blue was used to shift the charges of the proteins and to stabilize the vanadium complex. The detection of the vanadium species was made possible by the use of the 48V radiotracer and the phosphor‐screen technology. The method was first developed using transferrin, incubated with 48V, as a model. After it was proved that the vanadium‐transferrin complex was stable during separation, the method was validated by separating serum incubated with 48V. The efficiency of the separation was assessed according to two parameters: resolution and conservation of the species. First, the resolution of the separation was as expected from a native separation. Second, the release of free vanadium from the transferrin complex, which was the main vanadium species expected, was negligible, which proves that the species remain intact during separation. In accordance with the literature, it was found that vanadium binds to transferrin in incubated serum at these low concentrations.

Heart-cutting two-dimensional gas chromatography in combination with isotope ratio mass spectrometry for the characterization of the wax fraction in plant material

Gas chromatography coupled to isotope ratio mass spectrometry after on-line combustion (GC-C-IRMS) and high temperature conversion (GC-HTC-IRMS) is used for compound specific isotope ratio determination. This determination can only be performed successfully if the target solutes are fully resolved from other compounds. A new instrumental set-up consisting of heart-cutting two-dimensional GC based on capillary flow technology and a low thermal mass GC oven in combination with an isotope ratio mass spectrometer is presented. Capillary flow technology was also used in all column and interface connections for robust and leak-free operation. The new configuration was applied to the characterization of wax compounds in tobacco leaf and corresponding smoke samples. It is demonstrated that high accuracy is obtained, both in the determination of δ(13)C and δ(2)H values, allowing the study of biosynthesis and delivery mechanisms of naturally occurring compounds in tobacco.

Influence of reducing agents on the integrity of selenocompounds. Exploratory work for selenoproteome analysis

Three selenium species (selenomethionine, selenocystine and selenocystamine) were studied for their behaviour in the presence of the reducing agents dithiothreitol and tributylphosphine, and of a derivatising agent, iodoacetic acid. These chemicals are commonly used in proteome analysis by means of gel electrophoresis. To study the effect of these chemicals on the selenospecies, two separation techniques were used, capillary electrophoresis and liquid chromatography, both with ICP-MS as an element-specific detector. Electrospray-mass spectrometry provided molecular information. Tributylphosphine (TBT) was shown to partly reduce selenocystine to selenocysteine, and iodoacetic acid to derivatise all the species, with conservation of the species information. Dithiothreitol (DTT), on the contrary, partly sequestered selenium from the species to form a DTT–Se molecule.

Determination of pesticides in fatty matrices using gel permeation clean-up followed by GC-MS/MS and LC-MS/MS analysis: A comparison of low- and high-pressure gel permeation columns

Two low-pressure columns (Bio-Beads SX-3) and three high-pressure GPC columns were compared for clean-up of a wide range of pesticides in fatty matrices of vegetable or animal origin. The GPC fractions were analyzed by GC-MS/MS and LC-MS/MS without additional clean-up. The performance of the GPC clean-up on the five column types was compared in terms of solvent consumption, lipid removal, pesticide recovery and repeatability. It was found that for fatty matrices, mainly consisting of high molecular weight triglycerides i.e. most vegetable oils and animal fats, good fractionation is obtained for the majority of the pesticides. On the other hand, for fats and oils containing relatively high amounts of low molecular weight triglycerides, i.e. butter fat and palm kernel oil, none of the columns provided sufficient clean-up and cause interferences and system contamination, especially in the case of GC-MS/MS analysis. For the latter case, best results in terms of lipid removal and pesticide recovery were obtained on a set (2×300mmlength) of narrow bore (7.5mm ID) columns packed with 5µm PL Gel material. Column loadability is, however, much lower on that set of columns compared the other evaluated GPC columns, impairing overall method sensitivity.