Enhanced metabolite identification with MS(E) and a semi-automated software for structural elucidation

The identification of metabolites is almost exclusively done with liquid chromatography/tandem mass spectrometry (LC/MSMS) and despite the enormous progress in the development of these techniques and software for handling of data this is a time-consuming task. In this study the use of quadrupole time-of-flight (QTOF)-generated MS(E) and MS/MS data were compared with respect to rationalization of metabolites. In addition Mass-MetaSite, a semi-automated software for metabolite identification, was evaluated. The program combines the information from MS raw data, in the form of collision-induced dissociation spectra, with a prediction of the site of metabolism in order to assign the structure of a metabolite. The aim of the software is to mimic the rationalization of fragment ions performed by a biotransformation scientist in the process of structural elucidation. For this evaluation, metabolite identification in human liver microsomes was accomplished for 19 commercially available compounds and 15 in-house compounds. The results were very encouraging and for 96% of the metabolites the same structures were assigned using MS(E) compared with MSMS acquired data. The possibility of using MS(E) could considerably reduce the analysis time. Moreover, Mass-MetaSite performed well and the correct assigned structure, compared to manual inspection of the data, was picked in the first rank in ～80% of the cases. In conclusion MS(E) could be successfully used for metabolite identification in order to reduce time of analysis and Mass-MetaSite could alleviate the work of a biotransformation scientist and decrease the workload by assigning the structure for a majority of the metabolites.     

A novel analytical tool for quantification of estrogenicity in river water based on fluorescence labelled estrogen receptor α

A novel combined procedure for estrogen-affinity purification and labelling of estrogen receptor α ligand-binding domain with Cy™ 5.5 cystein reactive dye was established. By using this procedure, mainly functional proteins are recovered. It can be easily adapted to a large variety of other proteins for which ligand-coated affinity materials are available. The labelled receptor was used in a total internal reflection fluorescence-based binding inhibition assay for determination of the impact of pollutants in river water on the receptor. The great advantage compared to conventional methods is that the total effect on the receptor is measured instead of concentrations of single compounds and that even currently unknown ligands are found as well. Therefore, the obtained signal is related to the response of the organism, which is exposed to the water. The limit of detection was found to be 0.139 nM of estradiol equivalents. The assay also provides a highly sensitive tool for pharmaceutical research and can be adapted to diagnostic applications.      

Progress in capillary electrophoresis coupled to matrix-assisted laser desorption/ionization - time of flight mass spectrometry

In this review the most important techniques developed to hyphenate CE to MALDI-TOF-MS are summarized. The principles of the different interfaces and ways to solve the hyphenation problem are explained and discussed in detail. The most important applications especially in the proteomic field are reviewed, and the advantages of CE-MALDI-TOF-MS for the analysis of these compounds compared to other techniques such as ESI-MS are exhaustingly discussed from a critical point of view. CE coupled to MALDI-TOF-MS has started to overpass traditionally used CE-coupling techniques, especially CE-ESI-MS, offering the possibility to analyze samples of interest even weeks after CE analysis and using multiplexing systems for high-sample throughput.     

Capillary electrochromatography of biologically relevant flavonoids

Flavonoids were separated utilizing CEC technique. Baseline separation of biologically relevant flavonoids was obtained using a 100 microm ID fused-silica capillary filled with 3 microm Silica-C18 material and an optimized mobile phase comprising of 20 mM Tris-HCl (pH 6.5), ACN and water at a ratio of 10/40/50 v/v/v. Separations were carried out at 25 kV and a column temperature of 25 degrees C. The influence of relevant parameters for the CEC separation, such as buffer concentration, pH, separation voltage, and ACN concentration, was investigated and optimized. Dependencies of the electroendoosmotic flow (EOF) on these parameters and effects on the resolution of the analytes were studied. During analyses the solvents used for dissolving the samples turned out to have significant effects on the separation of flavonoids. The optimized system was then successfully used for the separation of the flavonoids epicatechin, myricetin, quercetin, naringenin, and hesperetin. CEC turned out to be a useful complementary tool for the economic analysis of flavonoids in addition to common HPLC, muHPLC, and CE methodologies. This method can be used for real applications in phytomics.     

Influence of concentration and temperature on the dynamics of water in the hydrophobic hydration shell of tetramethylurea

We study the influence of the amphipilic compound tetramethylurea (TMU) on the dynamical properties of water, using dielectric relaxation spectroscopy in the regime between 0.2 GHz and 2 THz. This technique is capable of resolving different water species, their relative fractions, and their corresponding reorientation dynamics. We find that the reorientation dynamics of water molecules in the hydration shell of the hydrophobic groups of TMU is between 3 (at low concentrations) and 10 (at higher concentrations) times slower than the dynamics of bulk water. The data indicate that the effect of hydrophobic groups on water is strong but relatively short-ranged. With increasing temperature, the fraction of water contained in the hydrophobic hydration shell decreases, which implies that the overall effect of hydrophobic groups on water becomes smaller.     

Quantitative multiplex CARS spectroscopy in congested spectral regions

A novel procedure is developed to describe and reproduce experimental coherent anti-Stokes Raman scattering (CARS) data, with particular emphasis on highly congested spectral regions. The approach, exemplified here with high-quality multiplex CARS data, makes use of spontaneous Raman scattering results. It is shown that the underlying vibrational Raman response can be retrieved from the multiplex CARS spectra, so that the Raman spectrum can be reconstituted, provided an adequate signal-to-noise ratio (SNR) is present in the experimental data and sufficient a priori knowledge of the vibrational resonances involved exists. The conversion of CARS to Raman data permits a quantitative interpretation of CARS spectra. This novel approach is demonstrated for highly congested multiplex CARS spectra of adenosine mono-, di-, and triphosphate (AMP, ADP, and ATP), nicotinamide adenine dinucleotide (NAD+), and small unilamellar vesicles (SUVs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Quantitative determination of nucleotide concentrations and composition analysis in mixtures is demonstrated.     

Wetting of Alkanes on Water from a Cahn-Type Theory: Effects of Long-Range Forces

We apply the phenomenological wetting theory of Cahn to fluids with van der Waals forces, and in particular to the wetting of pentane on water. Taking into account explicitly the long-range substrate–adsorbate interaction allows us to reproduce the experimentally observed critical wetting transition, which arises from the vanishing of the Hamaker constant at T53°C. This transition is preceded by a first-order transition between a thin and a thick film at a (much) lower temperature. If long-range forces are neglected, this thin–thick transition is the only wetting transition and critical wetting is missed. Our study focuses on the development of useful theoretical tools, such as phase portraits and interface potentials adapted to systems with van der Waals forces.