Determination of oxysterols in oxidatively modified low-density lipoprotein by semi-micro high-performance liquid chromatography with electrochemical detection

A simple method for the determination of oxysterols was developed by semi-micro high-performance liquid chromatography with electrochemical detection (semi-micro HPLC–ECD). Semi-micro HPLC–ECD was established using a C30 microbore column, acetonitrile containing 50 mmol/L LiClO₄ as a mobile phase, and an applied potential at +2.8 V versus Ag/AgCl. The current peak height was linearly related to the amount of sterol injected from 12.5 to 250 pmol (r > 0.999) with a relative standard deviation (RSD) of less than 2.9% (n = 6). This method was applied to the determination of seven oxysterols in oxidatively modified low-density lipoprotein (Ox–LDL). Oxysterols were determined with a recovery of more than 78.0% and an RSD of less than 2.9% (n = 6) except for 7-ketocholesterol. 7-Ketocholesterol was determined as a sum of intact 7-ketocholesterol and its degradation product on saponification, cholesta-3,5-dien-7-one, with a recovery of 98.0% and an RSD of 2.5% (n = 6). From these results, the current method enabled the simultaneous determination of seven oxysterols without any derivatization, providing a useful tool for the assessment of oxysterol contents in Ox–LDL.     

Hyphenated liquid chromatographic techniques in forensic toxicology

The prerequisite of applicability of hyphenated methods in forensic analysis is the achievement of a stage of “final maturity”. In the field of liquid chromatography, HPLC coupled with diode array detection (DAD) seems to fulfill this criterion, whilst the combination with atmospheric pressure ionization mass spectrometry (HPLC–API-MS) is still in a development stage. HPLC–DAD is broadly used as identification tool in forensic and in emergency toxicology. Two main approaches were observed; development of retention index scales for intra-laboratory exchange of data and establishing of databases only for intra-laboratory use. Using these approaches, several databases were established for toxicological relevant substances (illicit and therapeutic drugs and their metabolites, environmental poisons etc.) in biological fluids. Also, complete HPLC–DAD identification systems are commercially available. Further possibility of progress depends on the on-line combination (“triple hyphenation”) with other detection methods, preferably API-MS. HPLC–API-MS, both in electrospray (ESI) and atmospheric pressure chemical ionization (APCI) options, underwent dramatic development in the last decade and is reaching its final shape. The method was broadly applied for various groups of toxicologically relevant substances, a lot of them unaccessible for other techniques, including GC–MS. Particularly important was application of HPLC–API-MS for detection and quantitation of active, polar metabolites of various drugs and for analysis of macromolecules. APCI seems to be more useful for analysis of less polar compounds, whereas ESI is particularly valuable for determination of polar, large molecules (e.g., toxic peptides, polar metabolites etc.) Up to now, HPLC–API-MS has been mainly applied for dedicated analyses, but the introduction of APCI or ESI in systematic toxicological screening may be expected in the near future.     

Simultaneous determination of Ziagen and its phosphorylated metabolites by ion-pairing high-performance liquid chromatography–tandem mass spectrometry

An ion-paring HPLC–MS–MS method with positive ion mode electrospray ionization has been developed to simultaneously quantify Ziagen, carbovir monophosphate, carbovir diphosphate and carbovir triphosphate. N′,N′-Dimethylhexylamine was used as the ion-pairing agent. The presence of this ion-pairing agent allowed the retention and separation of the four compounds on a reversed-phase HPLC column as well as the detection of the nucleotides with positive ion mode electrospray ionization. The limits of detection were found to be better than 25 nM for all the analytes. Calibration curves of the analytes showed excellent linearity over the range of 25 nM to 5 μM. The relative standard deviations and accuracies for replicate analyses of quality control samples were less than 15%. The method has been successfully applied to the analysis of these compounds in human liver cells treated with Ziagen.     

Determination of naphthodianthrones in plant extracts from Hypericum perforatum L. by liquid chromatography–electrospray mass spectrometry

The determination of the naphthodianthrone constituents in extracts of dried blossoms of Hypericum perforatum L. by combined HPLC–electrospray mass spectrometry is described. Hypericin (1), pseudohypericin (2) and their precursor compounds produce intensive negative quasi-molecular ions by deprotonation provided a non-acidic eluent system is used in the HPLC separation. From the [M–H]⁻ ions formed in the electrospray ionization process characteristic daughter ion spectra can be obtained by collisional activation which have been studied by tandem mass spectrometry.     

Motifs specific for the ADR1 NBS–LRR protein family in <Emphasis Type="Italic">Arabidopsis</Emphasis> are conserved among NBS–LRR sequences from both dicotyledonous and monocotyledonous plants

The activated disease resistance (ADR) 1 gene encodes a protein that possesses an N-terminal coiled-coil (CC) motif, nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. ADR1 belongs to a small, atypical Arabidopsis thaliana sub-class containing four CC–NBS–LRR genes. The NBS region of most NBS–LRR proteins possesses numerous conserved motifs. In contrast, the LRR domain, which is subject to positive selection, is highly variable. Surprisingly, sequence analysis revealed that the LRR domain of the ADR1 sub-class was more conserved than the NBS region. Sequence analysis identified two novel conserved motifs, termed TVS and PKAE, specific for this CC–NBS–LRR sub-class. The TVS motif is adjacent to the P-loop, whereas the PKAE motif corresponded to the inter-domain region termed the NBS–LRR linker, which was conserved within the different CC–NBS–LRR classes but varied among classes. These ADR1-specific motifs were employed to identify putative ADR1 homologs in phylogenetically distant and agronomically important plant species. Putative ADR1 homologs were identified in 11 species including rice and in 3 further Poaceae species. The ADR1 sub-class of CC–NBS–LRR proteins is therefore conserved in both monocotyledonous and dicotyledonous plant species.     

Determination of loratadine in human plasma by high-performance liquid chromatographic method with ultraviolet detection

A HPLC–UV determination of loratadine in human plasma is presented. After simple liquid–liquid extraction with 2-methylbutane–hexane (2:1) and evaporation of organic phase the compounds were re-dissolved in 0.01 M HCl, evaporated again and finally separated on a Supelcosil LC-18-DB column. The analyses were done at ambient temperature under isocratic conditions using the mobile phase: CH₃CN–water–0.5 M KH₂PO₄–H₃PO₄ (440:480:80:1, v/v). UV detection was performed at 200 nm with a limit of quantification of 0.5 ng/ml. The precision was found to be satisfactory over the whole range tested (0.5–50 ng/ml) with relative standard deviations of 2.3–6.3 and 5.2–14.1% for intra- and inter-assays, respectively.     

Effects of food rewards offered by ant–plant Macaranga on the colony size of ants

Myrmecophytes (ant–plants) have special hollow structures (domatia) in which obligate ant partners nest. As the ants live only on the plants and feed exclusively on plant food bodies, sap-sucking homopterans in the domatia, and/or the homopterans honeydew, they are suitable for the study of colony size regulation by food. We examined factors regulating ant colony size in four myrmecophytic Macaranga species, which have strictly species-specific association with Crematogaster symbiont ants. Intra- and interspecific comparison of the plants showed that the ant biomass per unit food biomass was constant irrespective of plant developmental stage and plant species, suggesting that the ant colony size is limited by food supply. The primary food offered by the plants to the ants was different among Macaranga species. Ants in Macaranga beccariana and Macaranga bancana relied on homopterans rather than food bodies, and appeared to regulate the homopteran biomass and, as a consequence, regulate the ants own biomass. In contrast, ants in Macaranga winkleri and Macaranga trachyphylla relied primarily on food bodies rather than homopterans, and the plants appeared to manipulate the ant colony size. Per capita plant investment in ants (ant dry weight plant dry weight^–1) was different among the four Macaranga species. The homoptera-dependent M. beccariana and M. bancana harbored lower biomass of ants than the food-body dependent M. winkleri, suggesting that energy loss is involved in the homoptera-interposing symbiotic system which has one additional trophic level. The plants investment ratio to the ants generally decreased as plants grew. The evolution of the plant reward-offering system in ant–plant–homopteran symbioses is discussed with an emphasis on the role of homopterans.     

Establishment of callus and cell suspension cultures from Gypsophila paniculata leaf segments and study of the attachment of host cells by Erwinia herbicola pv. gypsophilae

Callus and cell suspension cultures were initiated from leaf segments of G. paniculata. Fresh and dry weights measurements of callus showed that callus growth was optimal on MS medium supplemented with 1.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg l^–1 benzyladenin (BA). Calli cultured on this medium, showed a two-fold increase in fresh weight by the fourth week of incubation. The initiated hard green callus was repeatedly subcultured on MS medium containing increasing concentrations of 2,4-D in order to increase its friability. The friable callus was then used for establishment of a cell suspension culture. Maximum growth of the suspension culture was on medium supplemented with 1.0 mg l^–1 2,4-D and 0.2 mg l^–1 BA.The suspension culture was used for studying plant host attachment in both electron and light microscopy. Upon infection with E. herbicola, plant cells showed aggregate formation within 24 h of infection. In the presence of the pathogenic Ehg,the number of aggregates formed was 342 aggregates ml^–1, in the presence of the non-pathogenic Ehg154 aggregates ml^–1 and in the control 115 aggregates ml^–1. These results show that the pathogenic strain causes formation of cell aggregates 5.8 times greater than the non-pathogenic one. Based on these results, it can be hypothesized that bacterial cells of the pathogenic strains bind to the plant cells and may form a bridge for attachment of plant cells to one another. Observations by electron microscope show that bacterial cells do attach to plant cells and that this attachment might be via formation of a bridge between the bacteria and the plant cell.     

Liquid chromatographic–electrospray tandem mass spectrometric determination of novel antifungal agents in plasma

A rapid, selective, sensitive and reproducible HPLC–electrospray tandem mass spectrometric method has been developed for the analysis of novel triazole antifungal agents, SYN-2869 and its derivatives (SYN-2836, SYN-2903 and SYN-2921), in rat plasma using SYN-2506 as an internal standard. Isolation of these compounds from plasma and sample desalting were performed by a simple extraction procedure involving protein precipitation, vacuum-drying and reconstitution with acetonitrile. For all the agents, linearity was observed over the range of 10–10 000 ng/ml (r≥0.996) and the limit of quantitation was 10 ng/ml using a 100-μl plasma volume. A measurement rate of 400–500 samples/day/instrument could be achieved using this method.     

Production of enokipodins A, B, C, and D: a new group of antimicrobial metabolites from mycelial culture of Flammulina velutipes

Antimicrobial compounds enokipodins A, B, C, and D were originally isolated from the culture filtrates of Flammulina velutipes mycelial culture. Analysis of antibacterial activity by the paper disk method and quantification of enokipodins A–D by high performance liquid chromatography (HPLC) showed that F. velutipes mycelia produced enokipodins A–D in their late growing phase. Great genetic variability in production of these compounds was observed among ten strains of F. velutipes in analyses of antimicrobial activity by the hole-plate diffusion method and quantification by HPLC. Enokipodins A–D demonstrated antimicrobial activity mainly against the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus. Evaluation of minimum inhibitory doses (MIDs) showed that MIDs of enokipodins A and C for B. subtilis were as low as that of the penicillin G antibiotic.     

Excitotoxin-induced changes in transglutaminase during differentiation of cerebellar granule cells

Summary. Excitotoxicity induced by NMDA receptor stimulation is able to increase the activity of many enzymes involved in neuronal cell death. Primary cultures of rat cerebellar granule cells were used to elucidate the role of transglutaminase reaction in the excitotoxic cell response, and to evaluate the role of glutamate receptors in cell survival and degeneration. Granule neurons, maintained in vitro for two weeks, were exposed to NMDA at different stages of differentiation. Following NMDA receptor activation, increases in transglutaminase activity were observed in cell cultures. The levels of enzyme activity were higher in cells at 5 days in vitro than in those at 8–9 or 13–14 days in vitro. Moreover, NMDA exposure up-regulated tTG expression in neurons as young as 5 days in vitro. These cultures also exhibited morphological changes with clear apoptotic features. Results obtained demonstrate that susceptibility of granule cells to excitotoxicity depends on the developmental stage of neurons.     

Identification and quantification of five macrolide antibiotics in several tissues, eggs and milk by liquid chromatography–electrospray tandem mass spectrometry

We present an electrospray high-performance liquid chromatographic tandem mass spectrometric (HPLC–MS–MS) method capable of determining in several tissues (muscle, kidney, liver), eggs and milk the following five macrolides: tylosin, tilmicosin, spiramycin, josamycin, erythromycin. Roxithromycin was used as an internal standard. The method uses extraction in a Tris buffer at pH 10.5, followed by protein precipitation with sodium tungstate and clean-up on an Oasis solid-phase extraction column. The HPLC separation was performed on a Purospher C₁₈ column (125×3 mm I.D.) protected by a guard column, with a gradient of aqueous 0.1 M ammonium acetate–acetonitrile as the mobile phase at a flow-rate of 0.7 ml min⁻¹. Protonated molecules served as precursor ions for electrospray ionisation in the positive ion mode and four product ions were chosen for each analyte for multiple reaction monitoring (MRM). A validation study was conducted to confirm the five macrolides by MRM HPLC–MS–MS analysis of a negative control and fortified samples. All of the samples analysed were confirmed with four ions. The ion ratio reproducibility limit ranged from 2.4 to 15%. All compounds could be detected and quantified at half-maximum residue limits (MRLs). The method is specific, quantitative and reproducible enough to conform to European Union recommendations within the concentration range 0.5 MRL–2 MRL (accuracy: 80 to 110%, relative standard deviation: 2 to 13%). This whole method allows extraction and analysis of up to 50 samples per day.     

The Synthesis of New Porphyrin–Quinone Dyad Systems

New porphyrin–quinone dyad systems containing spacer groups of various lengths and structures and sterically hindered 5,10,15,20-tetrakis(3,5-di-tert-butylphenyl)porphyrin as an electron donor were synthesized. These compounds seem to be promising models for studying the photoinduced electron transfer.     

Biochemical properties of new synthetic carnosine analogues containing the residue of 2,3-diaminopropionic acid: the effect of N-acetylation

Summary. Three novel carnosine analogues 7–9 containing the residue of L(+)2,3-diaminopropionic acid with different degree of N-acetylation instead of -alanine have been synthesized and characterized. Comparative analysis of hydrolysis by carnosinase revealed that the mono- and bis-acetylated compounds 8 and 9 are resistant to enzymatic hydrolysis and act as competitive inhibitors of this enzyme. The hydroxyl radical scavenging potential of the three analogues was evaluated by their ability to inhibit iron/H₂O₂-induced degradation of deoxyribose. The second-order rate constants of the reaction of compounds 7–9 with hydroxyl radical were almost identical to that of carnosine. These compounds were also found to act as protective agents against peroxynitrite-dependent damage as assessed by their ability to prevent nitration of free tyrosine induced by this species.     

Simultaneous determination of fentanyl and midazolam using high-performance liquid chromatography with ultraviolet detection

When measuring fentanyl and midazolam simultaneously in the same plasma sample with standard high-performance liquid chromatography–ultraviolet (HPLC–UV) detection, overlap of the fentanyl peak by the midazolam peak occurs, which makes fentanyl determination impossible. We tested the hypothesis that by acidifying the methanol mobile phase with 0.02% perchloric acid, 70%, it would be possible to separate both peaks. The UV detector was set at 200 nm. Calibration curves for fentanyl (range 0–2000 pg/ml) and midazolam (range 0–400 ng/ml) were linear (r>0.99). The detection limits were 200 pg/ml (fentanyl) and 10 ng/ml (midazolam). Precision and accuracy for intra- and inter-assay variability as well as in-line validation with quality control samples (QCS) were acceptable (< 15 and 20%, respectively), except for fentanyl QCS of 200 pg/ml (17.8% precision). Although less sensitive than gas chromatography–mass spectrometry (GC–MS), reliable measurements of fentanyl, simultaneously with midazolam, can be performed with this HPLC–UV system.     

Plant-microbe interactions to probe regulation of plant carbon metabolism

Plant growth and development is dependent on coordinated assimilate production, distribution and allocation. Application of biochemical and molecular techniques substantially contributed to a better understanding of these processes, although the underlying regulatory mechanisms are still not fully elucidated and attempts to improve crop yield by modulating carbon partitioning were only partially successful. Plant pathogens also interfere with source–sink interaction. To this end they have evolved a wide range of sophisticated strategies to allow their systemic spread, suppression of plant defence and induction of sink function to support nutrient acquisition for their growth. Studying compatible interactions of plants and pathogens like viruses, bacteria and fungi can be exploited to investigate different levels of source–sink regulation. The identification of microbial factors and their host targets involved in regulation of plant primary metabolism may allow developing novel strategies to increase crop yield. Here we will discuss recent studies on plant–microbe interactions aimed at elucidating mechanisms of compatibility.     

Are endophyte-mediated effects on herbivores conditional on soil nutrients?

Neotyphodium endophytes are assumed to have mutualistic relationship with their grass hosts, mainly resulting from mycotoxin production increasing plant resistance to herbivores by the fungus that subsists on the plant. To study importance of often ignored environmental effects on these associations, we performed a greenhouse experiment to examine the significance of endophyte infection and nutrient availability for bird-cherry aphid (Rhopalosiphum padi) performance on meadow fescue (Lolium pratense). Naturally endophyte-infected (E+), uninfected (E–), or manipulatively endophyte-free (ME–) half-sib families of meadow fescue were grown on two soil nutrient levels. Endophyte infection reduced aphid performance in general. However, to our knowledge, this is the first study to demonstrate experimentally that herbivore performance decreases on E+ host plants with increasing availability of nutrients in soils. Potential improvement in herbivore performance in high nutrient soils and decreased plant performance in low nutrient soils in ME– plants, compared to E– and E+ plants, suggests that loss of endophyte infection after long coevolutionary relationship may be critical to plant fitness.     

Determination of a novel selective inhibitor of type 1 5α-reductase in human plasma by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

A sensitive and specific assay of human plasma for the determination of (5α,7β,16β)-16[(4-chlorophenyl)oxy]-4,7-dimethyl-4-aza-andronstan-3-one (I), a selective inhibitor of human type 1 5α-reductase, has been developed. The method is based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS–MS) detection. The analyte (I) and internal standard, Proscar (II), were isolated from the basified biological matrix using a liquid–liquid extraction with methyl-tert.-butyl ether (MTBE). The organic extract was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The MS–MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer using a heated nebulizer interface. Multiple reaction monitoring using the precursor→product ion combinations of m/z 430→114 and 373→305 was used to quantify I and internal standard (II), respectively. The assay was validated in the concentration range of 0.5 to 500 ng/ml in human plasma. The precision of the assay, expressed as coefficient of variation (C.V.), was less than 7% over the entire concentration range, with adequate assay specificity and accuracy. The HPLC–MS–MS method provided sufficient sensitivity to completely map the 24 h pharmacokinetic time-course following a single 0.5 mg dose of I.     

Investigation of the in vitro biotransformation of R-(+)-thalidomide by HPLC, nano-HPLC, CEC and HPLC–APCI-MS

High-performance liquid chromatography (HPLC), nano-HPLC, capillary electrochromatography (CEC) and on-line HPLC–atmospheric pressure chemical ionization mass spectrometry (APCI-MS) techniques were used for the identification and detailed characterization of two new metabolites of the former sedative drug thalidomide (TD). The advantages of nano-HPLC and CEC are higher peak efficiency and a drastic decrease in the analysis time, which, together with lower sample dilution during the analyses, allowed to obtain a detection sensitivity that was comparable to HPLC with common-sized columns. Both, nano-HPLC and CEC could be realized in the commercially available capillary electrophoresis system HP^3D. On-line HPLC–APCI-MS coupling is a very useful technique for the rapid identification of metabolites without any need for reference compounds.     

The efficiency of evapotranspiration of landfill leachate in the soil–plant system with willow Salix amygdalina L.

The application of a specific species of willow—Salix amygdalina L., marked by high transpiration ability—is a cheap and effective method of landfill leachate disposal. A 2-year study examined the effectiveness of leachate evapotranspiration from soil–plant systems with willow species S. amygdalina L. Evapotranspiration from soil–plant systems planted with willow was from 1.28 up to 5.12 times higher than evaporation from soil surface barren of vegetation. This proves the usefulness of soil–plant systems with willow in landfill leachate treatment through vaporization. Evapotranspiration efficiency, as opposed to total amount of water added into the lysimeter, was not strong enough to vaporize all input of the landfill leachate in the lysimeters. This may indicate that the ground water requires isolation when soil systems remain under landfill leachate irrigation. Linear dependence between willow biomass growth and transpiration was observed to be significant (p < 0.05). Additionally, the research showed that the application of sewage sludge into the soil caused an increase in vaporization efficiency.