Koen De Cremer

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.

Hair mercury and urinary cadmium levels in Belgian children and their mothers within the framework of the COPHES/DEMOCOPHES projects

A harmonized human biomonitoring pilot study was set up within the frame of the European projects DEMOCOPHES and COPHES. In 17 European countries, biomarkers of some environmental pollutants, including urinary cadmium and hair mercury, were measured in children and their mothers in order to obtain European-wide comparison values on these chemicals. The Belgian participant population consisted in 129 school children (6-11 years) and their mothers (≤ 45 years) living in urban or rural areas of Belgium. The geometric mean levels for mercury in hair were 0.383 μg/g and 0.204 μg/g for respectively mothers and children. Cadmium in mothers and childrens urine was detected at a geometric mean concentration of respectively 0.21 and 0.04 μg/l. For both biomarkers, levels measured in the mothers and their child were correlated. While the urinary cadmium levels increased with age, no trend was found for hair mercury content, except the fact that mothers hold higher levels than children. The hair mercury content increased significantly with the number of dental amalgam fillings, explaining partially the higher levels in the mothers by their higher presence rate of these amalgams compared to children. Fish or seafood consumption was the other main parameter determining the mercury levels in hair. No relationship was found between smoking status and cadmium or mercury levels, but the studied population included very few smokers. Urinary cadmium levels were higher in both mothers and children living in urban areas, while for mercury this difference was only significant for children. Our small population showed urinary cadmium and hair mercury levels lower than the health based guidelines suggested by the WHO or the JECFA (Joint FAO/WHO Expert Committee on Food Additives). Only 1% had cadmium level slightly higher than the German HBM-I value (1 μg/l for adults), and 9% exceeded the 1 μg mercury/g hair suggested by the US EPA.

Optimisation of ICP-dynamic reaction cell-MS as specific detector for the speciation analysis of vanadium at therapeutic levels in serum

Dynamic reaction cell (DRC) technology was applied in the context of the speciation analysis of vanadium at therapeutic levels in serum. This technology was necessary in order to detect vanadium on-line after size-exclusion chromatography with a buffer at physiological salinity (0.15 M NaCl). This salinity was compulsory to assure the stability of the vanadium compounds during chromatography, in other words to prevent inter-species conversion. In fact, the DRC allowed the detection of vanadium without adapting the conditions of the chromatographic separation to ICP-MS. First, the merits of various reaction gases were compared: methane, carbon monoxide, ammonia, oxygen and the combination of argon (collision gas) and hydrogen (reaction gas). In each instance, the reaction cell parameters were optimised in order to obtain the lowest detection limit for 51V (as 51V+or as 51V16O+ with O2 as the reaction gas) in chlorine-rich solution, Cl being the parent element of the 35Cl16O+ interference. Ammonia was found to offer the best detection limit (3s criterion, 10 ng L−1 with pneumatic nebulisation as the sample introduction system). The detection limit with size-exclusion chromatography-ICP-DRC-MS for vanadate, expected to be the worst among all vanadium chemical species, was found to be 40 ng L−1 serum (or 4 pg V, 100 µL serum injected) and the repeatability 7%. This on-line separation method was used in order to speciate vanadium in serum after incubation with vanadate, at a concentration level that is representative of the pharmacological concentration range of vanadium when used as an insulin-like agent.

Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Molds of the Fusarium Genus

ABSTRACT The rates of infection with Fusarium molds are increasing, and a diverse number of Fusarium spp. belonging to different species complexes can cause infection. Conventional species identification in the clinical laboratory is time-consuming and prone to errors. We therefore evaluated whether matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a useful alternative. The 289 Fusarium strains from the Belgian Coordinated Collections of Microorganisms (BCCM)/Institute of Hygiene and Epidemiology Mycology (IHEM) culture collection with validated sequence-based identities and comprising 40 species were used in this study. An identification strategy was developed, applying a standardized MALDI-TOF MS assay and an in-house reference spectrum database. In vitro antifungal testing was performed to assess important differences in susceptibility between clinically relevant species/species complexes. We observed that no incorrect species complex identifications were made by MALDI-TOF MS, and 82.8% of the identifications were correct to the species level. This success rate was increased to 91% by lowering the cutoff for identification. Although the identification of the correct species complex member was not always guaranteed, antifungal susceptibility testing showed that discriminating between Fusarium species complexes can be important for treatment but is not necessarily required between members of a species complex. With this perspective, some Fusarium species complexes with closely related members can be considered as a whole, increasing the success rate of correct identifications to 97%. The application of our user-friendly MALDI-TOF MS identification approach resulted in a dramatic improvement in both time and accuracy compared to identification with the conventional method. A proof of principle of our MALDI-TOF MS approach in the clinical setting using recently isolated Fusarium strains demonstrated its validity.

Exposure determinants of cadmium in European mothers and their children

The metal cadmium (Cd) is a widespread environmental pollutant with documented adverse effects on the kidneys and bones from long-term environmental exposure, but with insufficiently elucidated public health consequences such as risk of cardiovascular disease, hormone-related cancer in adults and developmental effects in children. This study is the first pan-European human biomonitoring project that succeeded in performing harmonized measurements of Cd in urine in a comparable way in mother-child couples from 16 European countries. The aim of the study was to evaluate the overall Cd exposure and significant determinants of Cd exposure. A study population of 1632 women (24-52 years of age), and 1689 children (5-12 years of age), from 32 rural and urban areas, was examined within a core period of 6 months in 2011-2012. Women were stratified as smokers and non-smokers. As expected, smoking mothers had higher geometric mean (gm) urinary cadmium (UCd; 0.24 µg/g crea; n=360) than non-smoking mothers (gm 0.18 µg/g crea; n=1272; p<0.0001), and children had lower UCd (gm 0.065 µg/g crea; n=1689) than their mothers at the country level. Non-smoking women exposed to environmental tobacco smoke (ETS) at home had 14% (95% CI 1-28%) higher UCd than those who were not exposed to ETS at home (p=0.04). No influence of ETS at home or other places on UCd levels was detected in children. Smoking women with primary education as the highest educational level of the household had 48% (95% CI 18-86%) higher UCd than those with tertiary education (p=0.0008). The same observation was seen in non-smoking women and in children; however they were not statistically significant. In children, living in a rural area was associated with 7% (95% CI 1-13%) higher UCd (p=0.03) compared to living in an urban area. Children, 9-12 years had 7% (95% CI 1-13%) higher UCd (p=0.04) than children 5-8 years. About 1% of the mothers, and 0.06% of the children, exceeded the tolerable weekly intake (TWI) appointed by EFSA, corresponding to 1.0 µg Cd/g crea in urine. Poland had the highest UCd in comparison between the 16 countries, while Denmark had the lowest. Whether the differences between countries are related to differences in the degree of environmental Cd contamination or to differences in lifestyle, socioeconomic status or dietary patterns is not clear.

Capability of flatbed electrophoresis (IEF and native PAGE) combined with sector field ICP-MS and autoradiography for the speciation of Cr, Ga, In, Pt and V in incubated serum samples

The capability of flatbed electrophoresis combined with sector field ICP-MS (ICP-SFMS) and autoradiography is reported. Human serum and rabbit serum were separated by IEF and native PAGE using a PhastSystem unit (Amersham Pharmacia Biotech, Uppsala, Sweden). Rabbits (Flemish Giant) received intraperitoneal injections of ‘cold’ Ga(III) and In(III) in physiological buffer. Human serum was incubated in vitro with the radiotracers 51 Cr(III), 191 Pt(II) or carrier-free 48 V(V). Measurement of the protein-bound metals followed two different strategies: (A) ICP-SFMS, in which each rabbit serum sample was run twofold and, after separation, one gel was silver stained and the other replicate was cut into segments, which were digested and measured for Ga and In by ICP-SFMS; (B) autoradiography, in which, after separation, the gels were exposed to a phosphorus screen at –20 °C, an autoradiogram was obtained by laser densitometry and subsequently the proteins were detected by silver staining. Both strategies have advantages and drawbacks, but both are powerful and highly sensitive and allow the attribution of the respective metals to serum proteins. The suitability of these methods for trace element speciation depends strongly on the stability of the trace element-protein binding.

Application of LC-MS/MS MRM to Determine Staphylococcal Enterotoxins (SEB and SEA) in Milk

Staphylococcus aureus is one of the important aetiological agents of food intoxications in Europe and can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Due to their stability and ease of production and dissemination, some SEs have also been studied as potential agents for bioterrorism. Therefore, specific and accurate analytical tools are required to detect and quantify SEs. Online solid-phase extraction liquid chromatography electrospray ionization tandem mass spectrometry (online SPE-LC-ESI-MS/MS) based on multiple reaction monitoring (MRM) was used to detect and quantify two types of SE (A and B) spiked in milk and buffer solution. SE extraction and concentration was performed according to the European Screening Method developed by the European Reference Laboratory for Coagulase Positive Staphylococci. Trypsin digests were screened for the presence of SEs using selected proteotypic heavy-labeled peptides as internal standards. SEA and SEB were successfully detected in milk samples using LC-MS/MS in MRM mode. The selected SE peptides were proteotypic for each toxin, allowing the discrimination of SEA and SEB in a single run. The detection limit of SEA and SEB was approximately 8 and 4 ng/g, respectively.

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Combined Species Identification and Drug Sensitivity Testing in Mycobacteria

ABSTRACT Species identification and drug susceptibility testing (DST) of mycobacteria are important yet complex processes traditionally reserved for reference laboratories. Recent technical improvements in matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has started to facilitate routine mycobacterial identifications in clinical laboratories. In this paper, we investigate the possibility of performing phenotypic MALDI-based DST in mycobacteriology using the recently described MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA). We randomly selected 72 clinical Mycobacterium tuberculosis and nontuberculous mycobacterial (NTM) strains, subjected them to MBT-ASTRA methodology, and compared its results to current gold-standard methods. Drug susceptibility was tested for rifampin, isoniazid, linezolid, and ethambutol (M. tuberculosis, n = 39), and clarithromycin and rifabutin (NTM, n = 33). Combined species identification was performed using the Biotyper Mycobacteria Library 4.0. Mycobacterium-specific MBT-ASTRA parameters were derived (calculation window, m/z 5,000 to 13,000, area under the curve [AUC] of >0.015, relative growth [RG] of <0.5; see the text for details). Using these settings, MBT-ASTRA analyses returned 175/177 M. tuberculosis and 65/66 NTM drug resistance profiles which corresponded to standard testing results. Turnaround times were not significantly different in M. tuberculosis testing, but the MBT-ASTRA method delivered on average a week faster than routine DST in NTM. Databases searches returned 90.4% correct species-level identifications, which increased to 98.6% when score thresholds were lowered to 1.65. In conclusion, the MBT-ASTRA technology holds promise to facilitate and fasten mycobacterial DST and to combine it directly with high-confidence species-level identifications. Given the ease of interpretation, its application in NTM typing might be the first in finding its way to current diagnostic workflows. However, further validations and automation are required before routine implementation can be envisioned.

Banana infecting fungus, Fusarium musae, is also an opportunistic human pathogen: Are bananas potential carriers and source of fusariosis?

During re-identification of Fusarium strains in the BCCM™/IHEM fungal collection by multilocus sequence-analysis we observed that five strains, previously identified as Fusarium verticillioides, were Fusarium musae, a species described in 2011 from banana fruits. Four strains were isolated from blood samples or biopsies of immune-suppressed patients and one was isolated from the clinical environment, all originating from different hospitals in Belgium or France, 2001–2008. The F. musae identity of our isolates was confirmed by phylogenetic analysis using reference sequences of type material. Absence of the gene cluster necessary for fumonisin biosynthesis, characteristic to F. musae, was also the case for our isolates. In vitro antifungal susceptibility testing revealed no important differences in their susceptibility compared to clinical F. verticillioides strains and terbinafine was the most effective drug. Additional clinical F. musae strains were searched by performing BLAST queries in GenBank. Eight strains were found, of which six were keratitis cases from the U.S. multistate contact lens-associated outbreak in 2005 and 2006. The two other strains were also from the U.S., causing either a skin infection or sinusitis. This report is the first to describe F. musae as causative agent of superficial and opportunistic, disseminated infections in humans. Imported bananas might act as carriers of F. musae spores and be a potential source of infection with F. musae in humans. An alternative hypothesis is that the natural distribution of F. musae is geographically a lot broader than originally suspected and F. musae is present on different plant hosts.

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.