Determination of sultopride and tiapride in serum by gas chromatography using a surface ionisation detector

A sensitive and selective method has been developed for the determination of sultopride and tiapride in serum using gas chromatography with a surface ionisation detector. No interfering peaks from endogenous substances were observed. The method showed good reproducibility and accuracy, and the standard curve was linear up to 2 μg/ml with a correlation coefficient of 0.999. This method is applicable to pharmacokinetic studies and therapeutic drug monitoring of sultopride and tiapride.     

Lichen substance concentrations in the lichen Hypogymnia physodes are correlated with heavy metal concentrations in the substratum

Lichen substances (i.e. lichen-specific carbon-based secondary compounds) are known to be involved in the uptake and immobilization of metal ions, though the biochemical mechanisms of this interaction are largely unexplained. Previous research on potential effects of lichen substances on heavy metal uptake and tolerance mostly focused on lichens in heavily polluted areas with exceptionally high metal concentrations. In the present study, we aimed at gathering information as to whether lichen substances might be involved in the fine-tuning of metal uptake even at not or low-polluted sites. Therefore, we studied lichen substance concentrations in the epiphytic lichen Hypogymnia physodes and metal concentrations in its substratum in a montane spruce forest of Germany. H. physodes produces two depsides and five depsidones, which had been shown to be involved in metal homeostasis, namely in Cu and Mn uptake, in previous laboratory experiments. The amount of lichen substances increased with increasing heavy metal concentration in the substratum, though the latter varied only in the range of a few μmol g⁻¹ between the sample trees. Variability of lichen substance concentrations in H. physodes within the individual trees was low. Among the different lichen substances of H. physodes, the amount of the depsidone physodalic acid relative to the total of lichen substances was most closely correlated to the concentrations of Cu and Mn in the substratum, whereas the amount of the depsidone 3-hydroxyphysodic acid decreased both with increasing concentrations of these two metals and physodalic acid. Thus, our data suggest that lichen substances contribute to metal homeostasis not only in heavy metal-rich habitats, but also at not or low-polluted sites where the lichen substances apparently help to maintain constant intracellular metal concentrations despite of spatially varying availabilities of metal ions.     

Miniaturized mass-selective detector with atmospheric pressure chemical ionization

The miniaturized mass-spectrometric detector with atmospheric pressure chemical ionization (APCI) is described. The analyzer employed in this instrument is the monopole with rod 54 mm in length and 2 mm in radius, which retains its efficiency up to 0.13 Pa ( approximately 10(-3)Torr). Together with the ion source, channeltron ion detector, radio frequency power supply and preamplifier, it is packed into a case with dimension of 185 mm x 100 mm x 70 mm. Mass spectrometer with the whole vacuum system weighs about 20 kg. In spite of low power of the vacuum system, the limit of detection at ppt level is achieved. The "strong" fragmentation mode is suggested for high-specific detection of phosphor-containing substances. The detector conforms to multicapillary column attachment.     

Characterization of a biological detector cell for quantitation of inositol 1,4,5-trisphosphate

Prior strategies to measure inositol 1,4,5-trisphosphate (IP(3)) in single cells either have been qualitative or have had a limited spatial resolution. Capillary electrophoresis combined with a biological detector cell has been used to quantitate IP(3) in small regions of a Xenopus oocyte. To improve the detection limits of this method, we elucidated the experimental parameters which influenced the sensitivity and reliability of the IP(3)-detector cell coupled to capillary electrophoresis. The variables which influenced the detector cell were the magnitude of the voltage drop across the detector cell, the duration of this voltage drop, the direction of fluid flow in the capillary, the concentration of free Ca(2+) around the detector cell, and the presence of protease inhibitors during permeabilization of the detector cell. For the sample volumes imposed by the capillary diameter, the detector cell acted primarily as an IP(3) mass detector rather than a concentration detector. Characterization of the experimental variables influencing the sensitivity and reliability of this detector cell has the potential to enhance other analyte measurements performed by mating capillary electrophoresis with a biological detector cell.     

High-throughput comprehensive analysis of D- and L-amino acids using ultra-high performance liquid chromatography with a circular dichroism (CD) detector and its application to food samples

A rapid and comprehensive analytical method for D- and L-enantiomers of proteinogenic amino acids was developed using ultra-high performance liquid chromatography (UHPLC) equipped with a circular dichroism (CD) detector. Pre-column derivatization reagents were examined for enhanced sensitivity and selectivity for UV and CD detection: 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was selected. The method, using a CD detector, does not require separation of optical isomers on a column to calculate the enantio ratio (%D) using the g-factor value and produces a simple chromatogram in comparison to other reported methods. Using this advantage, combined with UHPLC technology, analysis time for the derivatized proteinogenic amino acids was within 5.5 min. The UV detection limit was 4.9-23 pmol/injection and the CD detection limit was 11-64 pmol/injection. The method was applied to the analysis of D- and L-amino acids in food samples. D-Ala, D-Asp, D-Glu and D-Ser were detected at high concentrations in some Japanese black vinegars, fermented milks and yogurts. The results were identical to the results determined by the OPA method. We suggest the UHPLC-CD method would be useful in screening the D-amino acid content of foods and in helping to clarify the importance and reason for the presence of D-amino acids in foods.     

Does secondary chemistry enable lichens to grow on iron-rich substrates?

Lichen substances are shown to increase or to inhibit the adsorption of Fe at cation exchange sites. The influence on the adsorption strongly differs between individual lichen substances and is different for Fe²⁺ and Fe³⁺. These results add a new biological role to the known functions of lichen secondary metabolites. In an experiment with cellulose filters, which were soaked with acetone solutions of lichen substances and were then incubated with micromolar solutions of FeCl₂ or FeCl₃, many lichen substances were found to increase Fe³⁺ adsorption, whereas others had no effect. Most lichen substances had no effect on Fe²⁺ adsorption, but two were found to reduce and one to increase the level of adsorption. Lichens of Fe-poor and -rich sites contain lichen substances with different adsorption behavior towards Fe²⁺ and Fe³⁺. All the studied lichen substances, which only occur in lichens of Fe-poor sites, turned out to be effective Fe³⁺ adsorbents. Lichens of Fe-bearing rock and slag, however, were found to lack lichen substances, or to contain substances that did not adsorb Fe³⁺ and had no effect on Fe²⁺ adsorption, or thirdly, to contain substances that increased Fe³⁺ adsorption, but decreased Fe²⁺ adsorption. These results suggest that lichen substances do play a significant role in Fe adsorption in lichens and determine their tolerance to excess concentrations of Fe. Notwithstanding the strong correlation between the secondary chemistry of lichen species and their preference for Fe-rich or Fe-poor substrates, the postulated mechanism of temporary Fe adsorption by lichen substances has to be subject of future biochemical research.     

High performance thin layer chromatography (HPTLC), an improved technique for screening lichen substances

Abstract: High performance thin layer chromatography (HPTLC) is a method that can be used for screening lichen substances. It is as simple to use as standard TLC, but has many advantages: It is more sensitive, it is possible to run more samples in a shorter period of time, and the amount of solvent used is much smaller. The material needed and the methods used are described in detail. Horizontal chromatogram development was used. Since two of the solvents used in system B have been substituted, and since the properties of the HPTLC plates are slightly different, our results are not entirely in accordance with the standardized TLC method. A revised table for the identification of 69 lichen substances (obtained from 62 taxa) is accordingly presented.     

Lichen chemistry and selective grazing by the coleopteran Lasioderma serricorne

Part of the Lichenological Herbarium of the University of Trieste (TSB) was infested by Lasioderma serricorne, a polyphagus coleopteran. 1440 samples of 50 lichen species with different chemistry (Parmelia s.lat., Physcia s.lat.) were analyzed in order to assess whether the lichen diet of the insect was species- and especially lichen-chemistry-selective. Multivariate analysis indicates a significant negative correlation between the presence of some lichen substances and grazing phenomena. Atranorin, calcium oxalate, fumarprotocetraric acid, gyrophoric acid, lecanoric acid, skyrin, usnic acid and zeorin play an antiherbivorous role.     

Dissociation and metal-binding characteristics of yellow lichen substances suggest a relationship with site preferences of lichens

Background and Aims: Many species of lichen-forming fungi contain yellow or orangeextracellular pigments belonging to the dibenzofurans (usnicacid), anthraquinones (e.g. parietin) or pulvinic acid group.These pigments are all equally efficient light screens, leadingus to question the potential ecological and evolutionary significanceof diversity in yellow and orange lichen substances. Here thehypothesis is tested that the different pigments differ in metal-bindingcharacteristics, which suggest that they may contribute to adaptationto sites differing in pH and metal availability. Methods: UV spectroscopy was used to study the dissociation and the pHdependence of the metal-binding behaviour of seven isolatedlichen substances in methanol. Metals applied were selectedmacro- and micro-nutrients (Cu²⁺, Fe²⁺, Fe³⁺, Mg²⁺, Mn²⁺ andZn²⁺). Key Results: All the pigments studied are strong to moderate acids with pK_a1values between 2·8 and 4·5. Metal complexationis common in the lichen substances studied. Complexation takesplace under acidic conditions with usnic acid, but under alkalineconditions with parietin and most compounds of the pulvinicacid group. The pulvinic acid derivative rhizocarpic acid formsmetal complexes both in the acidic and the alkaline range. Conclusions: Metal complexation by lichen substances could be a prerequisitefor lichen substance-mediated control of metal uptake. Assumingsuch an effect at pH values where the affinity of the metalfor the lichen substance is intermediate would explain the strongpreference of lichens with usnic or rhizocarpic acids to acidicsubstrata. Moreover, it would explain the preference of lichenswith parietin and some lichens with compounds of the pulvinicacid group either for nutrient-rich substrata at low pH or forcalcareous substrata.     

Small-volume electrochemical detector for microcolumn liquid chromatography

A submicroliter electrochemical detector for liquid chromatography has been designed, using pressure-annealed pyrolytic graphite technology. The analytical performance of this detector was studied in connection with a reversed-phase packed microcapillary column at very low flow-rates. Although the miniaturized version of the electrochemical detector is less sensitive, a direct analysis of a number of urinary metabolites in 0.1–1.0 μl samples is feasible.     

Determination of the surface dose of a water phantom using a semiconductor detector for diagnostic kilovoltage x-ray beams

PurposeTo determine the surface dose of a water phantom using a semiconductor detector for diagnostic kilovoltage x-ray beams.MethodsAn AGMS-DM+ semiconductor detector was calibrated in terms of air kerma measured with an ionization chamber. Air kerma was measured for 20 x-ray beams with tube voltages of 50–140 kVp and a half-value layer (HVL) of 2.2–9.7 mm Al for given quality index (QI) values of 0.4, 0.5, and 0.6, and converted to the surface dose. Finally, the air kerma and HVL measured by the AGMS-DM+ detector were expressed as a ratio of the surface dose for 10 × 10 and 20 × 20 cm² fields. The ratio of both was represented as a function of HVL for the given QI values and verified by comparing it with that calculated using the Monte Carlo method.ResultsThe air kerma calibration factor, CF, for the AGMS-DM+ detector ranged from 0.986 to 1.016 (0.9% in k = 1). The CF values were almost independent of the x-ray fluence spectra for the given QI values. The ratio of the surface dose to the air kerma determined by the PTW 30,013 chamber and the AGMS-DM+ detector was less than 1.8% for the values calculated using the Monte Carlo method, and showed a good correlation with the HVL for the given QI values.ConclusionIt is possible to determine the surface dose of a water phantom from the air kerma and HVL measured by a semiconductor detector for given QI values.     

Investigations of the performance of a high-performance liquid chromatography system with an electrochemical detector

A procedure for the determination of norepinephrine and dopamine, based on high-performance liquid chromatography, is evaluated using an electrochemical detector system. The use of an inorganic mobile phase to provide resolution of low retention amines and extend column life is discussed. A high degree of correlation between estimations of endogenous catecholamine levels is reported using both electrochemical and fluorometric detector systems. Inter-assay reproducibility of the extraction method, and sensitivity and linearity of response of the electrochemical detector system are shown to be consistent across trials. The system described is determined to be accurate, sensitive, and reliable over time.     

Determination of the pseudoephedrine content in pharmaceutical formulations and in biological fluids using a microbore HPLC system interfaced to a microfluidic chemiluminescence detector

A novel automated precolumn derivatization followed by separation using liquid chromatography for the determination of pseudoephedrine (PSE) by a microfluidic chemiluminescence detector has been developed. An on‐line derivatization procedure was utilized by converting PSE into a highly light emitting species in a Ru(bipy)₃²⁺‐peroxydisulphate chemiluminescence (CL) system by derivatizing it with a 1.0 M formaldehyde solution. The derivatized analyte was directly injected into a microbore high‐performance liquid chromatography (HPLC) system coupled to an on‐chip chemiluminescence detector. The newly developed highly selective, sensitive and fast HPLC‐CL method was validated and successfully applied for the analysis of PSE in pharmaceutical formulations and a human urine sample. The selectivity of the method is not only due to the HPLC separation but is also due to the highly selective detection principle of the Ru(bipy)₃²⁺‐peroxydisulphate CL system used. There was no interference observed from the common preservatives and excipients used in pharmaceutical preparations, which did not show any significant CL signal. The retention time of PSE was less than 3 min, and the detection limits and quantification limits were found to be 5.7 and 26.0 µg L^–1, respectively. Copyright © 2015 John Wiley & Sons, Ltd.     

粗叶悬钩子叶中多酚类与三萜类化合物的研究

采用高效液相色谱-串联质谱（HPLC-MS/MS）联用技术,对中国南方民间用于治疗肝损伤和癌症的药材粗叶悬钩子（Rubus aleaefolius Poir.）叶中的多酚类和三萜类成分进行了分析。通过与文献数据和部分对照品的紫外光谱、负离子模式下的一级和二级质谱信息进行比较,从粗叶悬钩子叶中共分析了18个化合物,鉴定了其中2个多酚类化合物,推测了9个多酚类和7个三萜类化合物的可能结构,并对三萜类化合物的质谱裂解途径进行了解析。基于对三萜类化合物裂解规律的研究,推测了1个三萜,即3-对香豆酰氧基-19α-羟基熊果酸。芦丁、绿原酸和3-对香豆酰氧基-19α-羟基熊果酸在本植物中属首次报道。     

Determination of 3-nitrotyrosine by high-pressure liquid chromatography with a dual-mode electrochemical detector

3-Nitrotyrosine, a product of tyrosine nitration, is useful as a marker for the generation of reactive nitrogen oxide species with short half-lives such as peroxynitrite. A reverse-phase high-pressure liquid chromatographic method using a dual-mode electrochemical detector in series with a photodiode array detector has been developed to determine the levels of 3-nitrotyrosine in biological samples. The principle of this method involves reduction of 3-nitrotyrosine at an upstream gold amalgam electrode and oxidation of the resulting product(s) at a downstream glassy carbon electrode. 3-Nitrotyrosine is quantified by the amount of the current generated at the downstream electrode, and a femtomole detection level can be achieved. The disappearance of the corresponding peak when the electrochemical detector is used only in the single oxidative mode provides additional evidence for the identity of 3-nitrotyrosine in the sample. Tyrosine from the same sample is determined by its UV absorption at 280 nm, thus eliminating the need for an internal standard. With this method a dose-dependent increase of 3- to 10-fold in the levels of protein 3-nitrotyrosine was observed in the blood plasma, and a 2- to 4-fold increase in the lung cytosols, of rats treated with the lung carcinogen and nitrating agent tetranitromethane.     

Depressing synapse as a detector of frequency change

In this article we discuss the short-term synaptic depression using a mathematical model. We derive the model of synaptic depression caused by the depletion of synaptic vesicles for the case of infinitely short stimulation time and show that the analytical formulas for the postsynaptic potential (PSP) and kinetic functions take simple closed form. A solution in this form allows an analysis of the characteristics of depression as a function of the models parameters and the derivation of analytic formulas for measures of short time synaptic depression commonly used in experimental studies. Those formulas are used to validate the model by fitting it to two types of synapses described in the literature. Given the fitted parameters we discuss the behavior of the synapse in situations involving frequency change. We also indicate a possible role of depressing synapses in information processing as not only a filter of high frequency input but as a detector of the return from high frequency stimulation to the stimulation within frequency band specific for a given synapse.     

Correction for missed events based on a realistic model of a detector.

Quantitative patch-clamp analysis based on dwell-time histograms has to deal with the problem of missed events. The correction of the evaluated time constants has to take into account the characteristics of the detector used for the reconstruction of the time series. In previous approaches a simple model of the detector has been used, which is based on the assumption that all events shorter than the temporal resolution tres were missed, irrespective of the preceding events. Rather than the standard assumption of a fixed dead time, we introduce a more realistic model of a detector by a continuous-time version of the Hinkley detector. The combined state of the channel and the detector obeys a Markov model, which is governed by a Fokker-Planck-Kolmogorov partial differential equation. The steady-state solution leads to the determination of the apparent time constants tau o and tau c depending on the true rate constants koc and kco and the temporal resolution tres of the detector. Simulations with different kinds of detectors, including the Bessel filter with half-amplitude threshold detection, are performed. They show that our new equation predicts the dependence of tau c and tau o on koc, kco, and tres better than the standard equation used until now.     

The phenolic compounds in <Emphasis Type="Italic">Cladonia</Emphasis> lichens are not antimicrobial in soils

According to classic text books on lichen biology, the phenolic secondary chemicals in lichens have antibiotic effects on soil microorganisms and mycorrhizal fungi in ecosystems. However, the experimental evidence for this under natural conditions is still relatively scarce. We examined some of the assumptions behind the concept of antimicrobial effects of lichen secondary substances: (1) the secondary substances of Cladonia stellaris, usnic and perlatolic acids, are leached out from the lichens by rainwater; (2) these substances inhibit the microbial activity of soil, and; (3) since they are extremely resistant to microbial decomposition, the soil underneath a continuous lichen mat is enriched in usnic and perlatolic acids. Our results did not support any of these assumptions. The evidence for the antimicrobial activity of lichen secondary substances seems to be weak in comparison to other suggested functions such as light filtering and herbivore protection. We suggest that it is time to re-evaluate the evidence for the antimicrobial ecological role of lichen secondary substances in natural systems.     

Lichens as a potential natural source of bioactive compounds: a review

Biological activity of material whether known in folk medicine or observed in planned screening program has been the starting point in the drug research. The general pattern is the isolation of active principles, elucidation their structures, followed by attempts for modulation of its activity potential by chemical modification. Lichens are valuable plant resources and are used as medicine, food, fodder, perfume, spice, dyes and for miscellaneous purposes throughout the world. Lichens are well known for the diversity of secondary metabolites that they produce. Compounds isolated from various lichen species have been reported to display diverse biological activities. Here we review the medicinal efficacy of lichen substances, which intends to explore the pharmaceutical potential of lichen substances.     

Extremotolerance and Resistance of Lichens: Comparative Studies on Five Species Used in Astrobiological Research II. Secondary Lichen Compounds

Lichens, which are symbioses of a fungus and one or two photoautotrophs, frequently tolerate extreme environmental conditions. This makes them valuable model systems in astrobiological research to fathom the limits and limitations of eukaryotic symbioses. Various studies demonstrated the high resistance of selected extremotolerant lichens towards extreme, non-terrestrial abiotic factors including space exposure, hypervelocity impact simulations as well as space and Martian parameter simulations. This study focusses on the diverse set of secondary lichen compounds (SLCs) that act as photo- and UVR-protective substances. Five lichen species used in present-day astrobiological research were compared: Buellia frigida, Circinaria gyrosa, Rhizocarpon geographicum, Xanthoria elegans, and Pleopsidium chlorophanum. Detailed investigation of secondary substances including photosynthetic pigments was performed for whole lichen thalli but also for axenically cultivated mycobionts and photobionts by methods of UV/VIS-spectrophotometry and two types of high performance liquid chromatography (HPLC). Additionally, a set of chemical tests is presented to confirm the formation of melanic compounds in lichen and mycobiont samples. All investigated lichens reveal various sets of SLCs, except C. gyrosa where only melanin was putatively identified. Such studies will help to assess the contribution of SLCs on lichen extremotolerance, to understand the adaptation of lichens to prevalent abiotic stressors of the respective habitat, and to form a basis for interpreting recent and future astrobiological experiments. As most of the identified SLCs demonstrated a high capacity in absorbing UVR, they may also explain the high resistance of lichens towards non-terrestrial UVR.