ALBERT

Description: Emission test chamber measurement is necessary to proof building materials as sources of volatile organic compounds (VOCs). The results of such measurements are used to evaluate materials and label them according to their potential to emit harmful substances, polluting indoor air. If only labelled materials were installed indoors, this would improve indoor air quality and prevent negative impacts on human health. Because of the complex testing procedure, reference materials for the quality assurance are mandatory. Currently, there is a lack of such materials because most building products show a broad variation of emissions even within one batch. A previous study indicates lacquers, mixed with volatile organic pollutants, as reproducible emission source for a wide range of substances. In the present study, the curing of the lacquer-VOC mixture inside micro-chambers was optimised. Therefore, the humidity and the chamber flow were varied. Typical indoor air pollutants with a wide range of volatilities, for example, styrene, n -hexadecane, dimethyl and dibutyl phthalate were selected. It turned out that, under optimised curing parameters inside the micro-chamber, their emission can be reproduced with variations of less than 10 %. With this, a next important step towards a reference material for emission testing was achieved.

Description: A sensitive liquid chromatography/electrospray ionization-tandem mass spectrometry method for the determination of free and conjugated estrogens in cow milk is described. Milk samples were diluted with water and then extracted and cleaned up by solid-phase extraction using graphitized carbon black as adsorbent material, without any enzymatic cleavage, derivatization, and/or protein precipitation step. Two fractions were collected (free and conjugated estrogens) and analyzed separately. Mass spectrometry analysis was performed in negative ion mode using selected reaction monitoring acquisition mode. For all compounds, the coefficients of determination ( R 2 ) ranged between 0.9892 and 0.9997. Analytical recoveries were in the range of 86–109 % for free estrogens and 85–118 % for conjugates, with relative standard deviations below 10 %, and the method detection limit ranged between 3 and 80 ng L −1 . Finally, the developed method was used to determine the presence of free and conjugated estrogens in six retail milk samples (five cow milk samples and one goat milk sample) and one goat milk sample provided by a local shepherd. Estrone was found to be the major free estrogen present in commercial milk samples, and estrone 3-sulfate showed the highest concentration among the target conjugated estrogens. Estriol was also observed in some analyzed milk samples, but the concentrations were always below the limit of quantification. Graphical abstract Free and conjugated estrogens were extracted from milk samples and cleaned-up by solid-phase extraction using graphitized carbon black as adsorbent material, without any enzymatic cleavage, derivatization and/or protein precipitation step. Two distinct fractions were collected and analyzed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry.

Description: A novel dual-function mixed-mode stationary phase based on poly(glycidyl methacrylate- co -ethylene dimethacrylate) microspheres was synthesized by thiol–ene click chemistry and characterized by infrared spectroscopy and elemental analysis. The new system displays both hydrophobic interaction chromatography (HIC) character in a high salt concentration mobile phase, and weak cation exchange (WCX) chromatography character in a low salt concentration mobile phase. It can be used to separate proteins in both ion-exchange chromatography (IEC) mode and HIC mode. The resolution and selectivity of the stationary phase were evaluated in both HIC mode and IEC mode using protein standards. In comparison with the conventional WCX and HIC columns, the results were satisfactory and acceptable. Protein mass and bioactivity recoveries of more than 96 % can be achieved in both HIC mode and IEC mode using this column. The results indicate that the novel dual-function mixed-mode column in many cases can replace the use of two individual WCX and HIC columns. In addition, the effects on protein separation of different ligand structures in the dual-function stationary phase and the pH of the mobile phase used were also investigated in detail. The results show that electrostatic interaction of the ligand with proteins must match the hydrophobicity of the ligand, which is an important factor to prepare the dual-function stationary phase. On the basis of this dual-function mixed-mode chromatography column, a new two-dimensional liquid chromatography technology with a single column system was also developed in this study, and was used to renature and purify recombinant human interferon-γ from inclusion bodies. The mass recovery, purity, and specific bioactivity obtained for the purified recombinant human interferon-γ were 87.2 %, 92.4 %, and 2.8 × 10 7  IU/mg, respectively, in IEC mode, and 83.4 %, 95.2 %, and 4.3 × 10 7  IU/mg, respectively, in HIC mode. The results indicate that the dual-function mixed-mode stationary phase prepared in this study may aid in the development of new two-dimensional liquid chromatography technology with a single column for intensive proteomic studies, and it may also find applications in recombinant protein drug production since it can save column costs and simplify the biotechnological processes.

Description: In order to monitor creatinine levels or to adjust the dosage of renally excreted or nephrotoxic drugs, the analysis of creatinine in dried blood spots (DBS) could be a useful addition to DBS analysis. We developed a LC-MS/MS method for the analysis of creatinine in the same DBS extract that was used for the analysis of tacrolimus, sirolimus, everolimus, and cyclosporine A in transplant patients with the use of Whatman FTA DMPK-C cards. The method was validated using three different strategies: a seven-point calibration curve using the intercept of the calibration to correct for the natural presence of creatinine in reference samples, a one-point calibration curve at an extremely high concentration in order to diminish the contribution of the natural presence of creatinine, and the use of creatinine-[ 2 H 3 ] with an eight-point calibration curve. The validated range for creatinine was 120 to 480 μmol/L (seven-point calibration curve), 116 to 7000 μmol/L (1-point calibration curve), and 1.00 to 400.0 μmol/L for creatinine-[ 2 H 3 ] (eight-point calibration curve). The precision and accuracy results for all three validations showed a maximum CV of 14.0 % and a maximum bias of −5.9 %. Creatinine in DBS was found stable at ambient temperature and 32 °C for 1 week and at −20 °C for 29 weeks. Good correlations were observed between patient DBS samples and routine enzymatic plasma analysis and showed the capability of the DBS method to be used as an alternative for creatinine plasma measurement. Graphical Abstract From blood spot to chromatogram

Description: We established an optimized biofilm observation method using a hydrophilic ionic liquid (IL), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF 4 ]). In the present study, a biofilm was formed by Staphylococcus epidermidis . Using field emission (FE) scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the colonization of assemblages formed by microbial cells was observed as a function of the cultivation time. FE-TEM analysis revealed that the fibril comprises three types of protein. In addition, the ultrastructure of each protein monomer was visualized. It was expected that the curly-structured protein plays an important role in extension during fibril formation. Compared to the conventional sample preparation method for electron microscopy, a fine structure was easily obtained by the present method using IL. This observation technique can provide valuable information to characterize the ultrastructure of the fibril and biofilm that has not been revealed till date. Furthermore, these findings of the molecular architecture of the fibril and the colonization behavior of microbial cells during biofilm formation are useful for the development of antibacterial drugs and microbial utilization.

Description: Fabry disease is an X-linked lysosomal storage disease due to deficient α-galactosidase A (α-Gal A) activity and the resultant lysosomal accumulation of globotriaosylceramide (Gb3) and related lipids primarily in blood vessels, kidney, heart, and other organs. The renal distribution of stored glycolipid species in the α-Gal A knockout mouse model was compared to that in mice to assess relative distribution and absolute amounts of accumulated sphingolipid isoforms. Twenty isoforms of five sphingolipid groups were visualized by mass spectrometry imaging (MSI), and their distribution was compared with immunohistochemical (IHC) staining of Gb3, the major stored glycosphingolipid in consecutive tissue sections. Quantitative bulk lipid analysis of tissue sections was assessed by electrospray ionization with tandem mass spectrometry (ESI-MS/MS). In contrast to the findings in wild-type mice, all three analytical techniques (MSI, IHC, and ESI-MS/MS) revealed increases in Gb3 isoforms and ceramide dihexosides (composed mostly of galabiosylceramides), respectively. To our knowledge, this is the first report of the distribution of individual molecular species of Gb3 and galabiosylceramides in kidney sections in Fabry disease mouse. In addition, the spatial distribution of ceramides, ceramide monohexosides, and sphingomyelin forms in renal tissue is presented and discussed in the context of their biosynthesis. Graphical Abstract Immunohistochemical images of a wild type ( left ) and Fabry mouse kidney ( right )

Description: Here, we report on a novel protocol for determining the viability of individual cells in an adherent cell culture, without adversely affecting the remaining cells in the sample. This is facilitated using a freestanding microfluidic perfusion device, the Multifunctional Pipette (MFP), which generates a virtual flow cell around selected single cells. We investigated the utility on four different cell lines, NG108-15, HEK 293, PC12, and CHO, and combined the assay with a cell poration experiment, in which we apply the pore-forming agent digitonin, followed by fluorescein diphosphate, a pre-fluorescent substrate for alkaline phosphatase, in order to monitor intracellular enzyme activity. The cell viability was instantly assessed through simultaneous perfusion with fluorescein diacetate (FDA) and propidium iodide (PI), both being dispensed through the same superfusion device used to porate and deliver the enzyme substrate. In this fluorescence assay, viable and non-viable cells were distinguished by their green and red emission, respectively, within 10 s. In addition, the enzyme activity was monitored over time as a secondary test for cellular activity. Our findings demonstrate that this microfluidic technology-assisted approach is a facile, rapid, and reliable means to determine the viability in single-cell experiments and that viability studies can be performed routinely alongside typical substrate delivery protocols. This approach would remove the need for global cell viability testing and would enable viability studies of only the cells under experimental analysis.