Quantitative analysis of metabolites in complex biological samples using ion-pair reversed-phase liquid chromatography-isotope dilution tandem mass spectrometry

A rapid, sensitive and selective ion-pair reversed-phase liquid chromatography-electrospray ionization isotope dilution tandem mass spectrometry (IP-LC-ESI-ID-MS/MS) was developed for quantitative analysis of free intracellular metabolites in cell cultures. As an application a group of compounds involved in penicillin biosynthesis pathway of Penicillium chrysogenum cells, such as penicillin G (PenG), 6-aminopenicillanic acid (6-APA), benzylpenicilloic acid (PIO), ortho-hydroxyphenyl acetic acid (o-OH-PAA), phenylacetic acid (PAA), 6-oxopipeidine-2-carboxylic acid (OPC), 8-hydroxypenicillic acid (8-HPA), L-alpha-(delta-aminoadipyl)-L-alpha-cystenyl-D-alpha-valine (ACV) and isopenicillin N (IPN) were chosen. (13)C-labeled analogs of the metabolites were added to the sample solutions as internal standards (I.S.). Sample mixtures were analyzed without any sample pretreatment. No extraction recovery check was needed because I.S. was added to the cell samples before extraction process. The method showed excellent precision (relative standard deviation (RSD)相似文献   

Role of the confinement of a root canal on jet impingement during endodontic irrigation

During a root canal treatment the root canal is irrigated with an antimicrobial fluid, commonly performed with a needle and a syringe. Irrigation of a root canal with two different types of needles can be modeled as an impinging axisymmetric or non-axisymmetric jet. These jets are investigated experimentally with high-speed Particle Imaging Velocimetry, inside and outside the confinement (concave surface) of a root canal, and compared to theoretical predictions for these jets. The efficacy of irrigation fluid refreshment with respect to the typical reaction time of the antimicrobial fluid with a biofilm is characterized with a non-dimensional Damk?hler number. The pressure that these jets induce on a wall or at the apex of the root canal is also measured. The axisymmetric jet is found to be stable and its velocity agrees with the theoretical prediction for this type of jet, however, a confinement causes instabilities to the jet. The confinement of the root canal has a pronounced influence on the flow, for both the axisymmetric and non-axisymmetric jet, by reducing the velocities by one order of magnitude and increasing the pressure at the apex. The non-axisymmetric jet inside the confinement shows a cascade of eddies with decreasing velocities, which at the apex does not provide adequate irrigation fluid refreshment.     

Determination of δ‐[L‐α‐aminoadipyl]‐L‐cysteinyl‐D‐valine in cell extracts of Penicillium chrysogenum using ion pair‐RP‐UPLC‐MS/MS

δ‐[L ‐α‐Aminoadipyl]‐L ‐cysteinyl‐D ‐valine (ACV) is a key intermediate in the biosynthesis pathway of penicillins and cephalosporins. Therefore, the accurate quantification of ACV is relevant, e.g. for kinetic studies on the production of these β‐lactam antibiotics. However, accurate quantification of ACV is a challenge, because it is an active thiol compound which, upon exposure to air, can easily react with other thiol compounds to form oxidized disulfides. We have found that, during exposure to air, the oxidation of ACV occurs both in aqueous standard solutions as well as in biological samples. Qualitative and quantitative determinations of ACV and the oxidized dimer bis‐δ‐[L ‐α‐aminoadipyl]‐L ‐cysteinyl‐D ‐valine have been carried out using ion pair reversed‐phase ultra high‐performance liquid chromatography, hyphenated with tandem mass spectrometry (IP‐RP‐UPLC‐MS/MS) as the analytical platform. We show that by application of tris(2‐carboxy‐ethyl)phosphine hydrochloride (TCEP) as the reducing reagent, the total amount of ACV can be determined, while using maleimide as derivatizing reagent enables to quantify the free reduced form only.     

Analysis of glycolytic intermediates in Saccharomyces cerevisiae using anion exchange chromatography and electrospray ionization with tandem mass spectrometric detection

The purpose of this study is to selectively and quantitatively analyze several glycolytic intermediates in cells of Saccharomyces cerevisiae using high-performance anion exchange chromatography (HPAEC) coupled to electrospray ionization tandem mass spectrometry for the analysis. A sodium hydroxide gradient is used to separate the glycolytic compounds and after the column sodium hydroxide is reduced by proton exchange with a membrane device prior to introduction to the mass spectrometer. The detection limits for 10 μl samples are down to the 0.4–5 pmol range. This corresponds for the intracellular metabolites to a range of 2–20 nmol per gram biomass dry weight (DW). Standard addition did reveal some influence of the sample matrix on the measured concentrations. Separation and analysis is hardly affected by the high sulfate and phosphate concentrations (1 mM) in the fermentation medium and by the intracellular matrix. Validation of the glucose-6-phosphosphate LC–MS–MS analysis results with enzymatic analysis showed an excellent agreement between the two methods. The suitability of the method was clearly shown by analyzing a series of steady state S. cerevisiae samples from a carbon limited aerobic chemostat culture.     

Observation of spin-polarized surface states on ultrathin bct Mn(001) films by spin-polarized scanning tunneling spectroscopy

We report the observation of a magnetic contrast of up to 20% in the scanning tunneling spectroscopy dI/dV maps obtained with Fe-coated tips on Mn(001) layers grown on an Fe(001) whisker at 370 K. These nanometer resolution microscopy results show that the layers couple antiferromagnetically. By normalizing the dI/dV curves by tunneling probability functions, we found a spin-dependent peak on the body-centered-tetragonal (bct) Mn(001) surface at +0.8 V, whose high spin polarization gives rise to the dI/dV map contrast. Band structure calculations allow one to identify the +0.8 V peak as due to two spin-polarized d(z(2)) surface states.     

Prevalence and Relative Risk of Dysphonia in Rheumatoid Arthritis

SUMMARY: Laryngeal involvement in rheumatoid arthritis is not uncommon and may include cricoarytenoid arthritis or vocal fold lesions such as vocal fold rheumatoid nodules or bamboo nodes. Dysphonia or voicing problems can be the result of such laryngeal involvement. This cohort study investigates the prevalence and the relative risk of dysphonia when suffering from rheumatoid arthritis compared to that of healthy subjects. One hundred and sixty-six subjects with rheumatic arthritis and 148 healthy control subjects completed two quality-of-life questionnaires: the Voice Handicap Index and a three-item outcome scale. Both instruments measure the quality of the voice itself and the extent of impairment resulting from dysphonia as experienced by the patient in social and occupational settings. Patients proved to have statistically significant higher prevalence and relative risk of dysphonia. Depending on the questionnaire being used, prevalence data of dysphonia in patients varied between 12% and 27%, whereas the healthy subjects showed prevalence data varying from about 3% to 8%. A patient's relative risk varied from about 3 to 4 when compared to healthy subjects. Patients suffering from rheumatoid arthritis have a clearly higher risk of dysphonia compared to healthy subjects.     

Murahashi Cross-Coupling at −78 °C: A One-Pot Procedure for Sequential C−C/C−C,C−C/C−N,and C−C/C−S Cross-Coupling of Bromo-Chloro-Arenes

The coupling of organolithium reagents, including strongly hindered examples, at cryogenic temperatures (as low as −78 °C) has been achieved with high-reactivity Pd-NHC catalysts. A temperature-dependent chemoselectivity trigger has been developed for the selective coupling of aryl bromides in the presence of chlorides. Building on this, a one-pot, sequential coupling strategy is presented for the rapid construction of advanced building blocks. Importantly, one-shot addition of alkyllithium compounds to Pd cross-coupling reactions has been achieved, eliminating the need for slow addition by syringe pump.     

How to estimate the Value at Risk under incomplete information

A key problem in financial and actuarial research, and particularly in the field of risk management, is the choice of models so as to avoid systematic biases in the measurement of risk. An alternative consists of relaxing the assumption that the probability distribution is completely known, leading to interval estimates instead of point estimates. In the present contribution, we show how this is possible for the Value at Risk, by fixing only a small number of parameters of the underlying probability distribution. We start by deriving bounds on tail probabilities, and we show how a conversion leads to bounds for the Value at Risk. It will turn out that with a maximum of three given parameters, the best estimates are always realized in the case of a unimodal random variable for which two moments and the mode are given. It will also be shown that a lognormal model results in estimates for the Value at Risk that are much closer to the upper bound than to the lower bound.     

Nickel‐Catalyzed Cross‐Coupling of Organolithium Reagents with (Hetero)Aryl Electrophiles

Nickel‐catalyzed selective cross‐coupling of aromatic electrophiles (bromides, chlorides, fluorides and methyl ethers) with organolithium reagents is presented. The use of a commercially available nickel N‐heterocyclic carbene (NHC) complex allows the reaction with a variety of (hetero)aryllithium compounds, including those prepared via metal‐halogen exchange or direct metallation, whereas a commercially available electron‐rich nickel‐bisphosphine complex smoothly converts alkyllithium species into the corresponding coupled product. These reactions proceed rapidly (1 h) under mild conditions (room temperature) while avoiding the undesired formation of reduced or homocoupled products.