Quantification of glucuronidated and sulfated steroids in human urine by ultra-high pressure liquid chromatography quadrupole time-of-flight mass spectrometry

The urinary steroid profile is constituted by anabolic androgenic steroids, including testosterone and its relatives, that are extensively metabolized into phase II sulfated or glucuronidated steroids. The use of liquid chromatography coupled to mass spectrometry (LC-MS) is an issue for the direct analysis of conjugated steroids, which can be used as urinary markers of exogenous steroid administration in doping analysis, without hydrolysis of the conjugated moiety. In this study, a sensitive and selective ultra high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS) method was developed to quantify major urinary metabolites simultaneously after testosterone intake. The sample preparation of the urine (1 mL) was performed by solid-phase extraction on Oasis HLB sorbent using a 96-well plate format. The conjugated steroids were analyzed by UHPLC-QTOF-MS^E with a single-gradient elution of 36 min (including re-equilibration time) in the negative electrospray ionization mode. MS^E analysis involved parallel alternating acquisitions of both low- and high-collision energy functions. The method was validated and applied to samples collected from a clinical study performed with a group of healthy human volunteers who had taken testosterone, which were compared with samples from a placebo group. Quantitative results were also compared to GC-MS and LC-MS/MS measurements, and the correlations between data were found appropriate. The acquisition of full mass spectra over the entire mass range with QTOF mass analyzers gives promise of the opportunity to extend the steroid profile to a higher number of conjugated steroids.     

On directional multiple-output quantile regression

This paper sheds some new light on projection quantiles. Contrary to the sophisticated set analysis used in Kong and Mizera (2008) [13], we adopt a more parametric approach and study the subgradient conditions associated with these quantiles. In this setup, we introduce Lagrange multipliers which can be interpreted in various interesting ways, in particular in a portfolio optimization context. The corresponding projection quantile regions were already shown to coincide with the halfspace depth ones in Kong and Mizera (2008) [13], but we provide here an alternative proof (completely based on projection quantiles) that has the advantage of leading to an exact computation of halfspace depth regions from projection quantiles. Above all, we systematically consider the regression case, which was barely touched in Kong and Mizera (2008) [13]. We show in particular that the regression quantile regions introduced in Hallin, Paindaveine, and Šiman (2010) [6] and [7] can also be obtained from projection (regression) quantiles, which may lead to a faster computation of those regions in some particular cases.     

Intermolecular interaction studies using small volumes

We present the use of 1-mm room-temperature probe technology to perform intermolecular interaction studies using chemical shift perturbation methods and saturation transfer difference (STD) spectroscopy using small sample volumes. The use of a small sample volume (5-10 μl) allows for an alternative titration protocol where individual samples are prepared for each titration point, rather than the usual protocol used for a 5-mm probe setup where the ligand is added consecutively to the solution containing the protein or host of interest. This allows for considerable economy in the consumption and cost of the protein and ligand amounts required for interaction studies. For titration experiments, the use of the 1-mm setup consumes less than 10% of the ligand amount required using a 5-mm setup. This is especially significant when complex ligands that are only available in limited quantities, typically because they are obtained from natural sources or through elaborate synthesis efforts, need to be investigated. While the use of smaller volumes does increase the measuring time, we demonstrate that the use of commercial small volume probes allows the study of interactions that would otherwise be impossible to address by NMR.     

On the experimental validation of combustion simulations in turbulent non-premixed jets

A Reynolds averaged Navier–Stokes (RANS) based combustion model, which incorporated the conditional source-term estimation (CSE) method for the closure of the chemical source term and the trajectory generated low-dimensional manifold (TGLDM) method for the reduction of detailed chemistry, was applied to predict the OH radical distribution in a combusting non-premixed methane jet. The results of the numerical prediction were compared with the results of a complementary experimental study in which the OH radical fields of combusting non-premixed methane jets were visualized using planar laser induced fluorescence (PLIF). It is well known within the modelling community that RANS based models are unable to capture the stochastic nature of turbulent combustion and autoignition, and are therefore unable to predict individual realizations of the flame. In this study, the agreement between the predicted OH field and a well-converged ensemble average of the experimental results was also shown to be poor. The lack of agreement between the numerical results and the ensemble averaged experimental results expose the potential significance of the known weakness in the RANS method. A statistical analysis of the experimental results was also performed. The results of the analysis showed that a minimum of 100 individual realizations was required to provide a well-converged average OH field for the combusting non-premixed jet under investigation. The significance of this result with respect to the validation of large-eddy simulations (LES) of combusting jets is discussed.     

High-Throughput Screening of Drugs of Abuse in Urine by Supported Liquid–Liquid Extraction and UHPLC Coupled to Tandem MS

A qualitative method, involving supported liquid–liquid extraction (SLE) and ultra high pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS–MS), was developed for the rapid tentative identification of various drugs of abuse in urine. In this study, 28 drugs and metabolites were covered by the screening procedure. Before analysis, urine samples were extracted by SLE and good extraction recoveries were obtained for most investigated compounds. The UHPLC strategy was then selected for the rapid separation of amphetamines, cocaine, opiates and related compounds in urine. Using columns packed with sub-2 µm particles, analysis time was reduced down to 2 min, while maintaining acceptable performance. Finally, the detection was by tandem MS operating in the single reaction monitoring (SRM) mode. The most intense transition was selected for the different drugs and SRM dwell times set at 5 ms, to maintain sufficient data points across the narrow UHPLC peaks. The tentative identification of the drugs of interest, including amphetamines, opiates and cocaine, was based on both, retention times and mass spectrometry information. With the proposed method, limits of detection were estimated at about 1 ng mL^?1 and the applicability was assessed by successfully analyzing several samples of drug abusers. Finally, this study demonstrates the potential of UHPLC coupled to tandem MS for the rapid screening of drugs of abuse in urine.     

Separation of the enantiomers of fluazifop and other 2-phenoxypropionic acids using chiral metal chelate additives in reversed-phase high-performance liquid chromatography

Most of the work on the use of chiral ligand-exchange systems in reversed-phase liquid chromatography has been focussed on the resolution of the enantiomers of amino acids. This paper describes the novel use of L-prolyl-n-octylamide-Ni(II) in the mobile phase for the resolution of the enantiomers of fluazifop and other phenoxypropionic acids.     

Laser induced excitation transfer in helium glow discharge

Some experimental results on the temporal decay of the fluorescence induced by a resonant laser pulse excitation focused onto a helium gas discharge are presented. In particular, excitation transfer between singlet-singlet and singlet- triplet sub-levels has been studied when 2¹ S → 3¹ P and 2¹ P → 4¹ D transitions of He I are optically pumped.     

Automatic gravimetric apparatus for recording the adsorption isotherms of gases or vapours onto solids

The assembly described was built with the aim of obtaining high resolution and precision, together with automaticity. It was designed around a symmetrical commercial vacuum microbalance (Setaram), its main features being the following:(1) Controlled “residual” vacuum during outgassing of the sample.(2) Two possible procedures for the introduction of the adsorbate: either a conventional — but automated — point by point procedure (with discontinuous introduction of gas or vapour) or a continuous procedure, specially suited for high resolution work.(3) Measurement of pressure (0–1 atm) by a silica Bourdon gague (Texas Instruments) of 10^?5 accuracy.(4) Direct recording of the isotherms on a XY recorder.(5) Constant level (± 0.5 mm) cryogenic bath (liquid nitrogen or argon) of low consumption (4 liters per 15 hours).(6) Liquid thermostat for adsorption temperatures between ? 30 and + 100°C.(7) Temperature control (up to 50°C) of the whole assembly (manifolds, balance, gauges) allowing to handle condensable vapours.Several examples are given, illustrating the versatility and resolution of the systems. For instance, it allowed to detect, for the first time, a distinct substep at the top of the nitrogen/graphite and argon/graphite isotherms at 77 K. This is now explained by a sudden localization or “freezing” of the monolayer near its completion.