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LC-MS
Terms modified by LC-MS Selected AbstractsThe Bible among Lutherans in America: The ELCA as a Test CaseDIALOG, Issue 1 2006By Erik M. Heen Abstract:, This article describes the biblical hermeneutics that inform the Evangelical Lutheran Church in America by comparing the ELCA's tradition of biblical interpretation with that of the Lutheran Church-Missouri Synod. It sets both against the great social and intellectual challenges of the early twentieth century, including the modernist/fundamentalist controversy. One commonality that surfaces is that both church bodies appropriated pre-modern hermeneutical impulses for "counter modern" biblical apologetics. In this process the LC-MS privileged the period of Lutheran Orthodoxy (17th century) while the ELCA constructed its hermeneutical paradigm through a recovery of the early Reformation (Luther). This observation suggests that both interpretive trajectories need further historical as well as theological review and revision. [source] Historical review of sample preparation for chromatographic bioanalysis: pros and consDRUG DEVELOPMENT RESEARCH, Issue 3 2007Min S. Chang Abstract Sample preparation is a major task in a regulated bioanalytical laboratory. The sample preparation procedure significantly impacts assay throughput, data quality, analysis cost, and employee satisfaction. Therefore, selecting and optimizing an appropriate sample preparation method is essential for successful method development. Because of our recent expertise, this article is focused on sample preparation for high-performance liquid chromatography with mass spectrometric detection. Liquid chromatography with mass spectrometric detection (LC-MS) is the most common detection technique for small molecules used in regulated bioanalytical laboratories. The sample preparation technologies discussed are pre-extraction and post-extraction sample processing, protein precipitation (PPT), liquid,liquid extraction (LLE), offline solid-phase extraction (SPE), and online solid-phase extraction. Since all these techniques were in use for more than two decades, numerous applications and variations exist for each technique. We will not attempt to categorize each variation. Rather, the development history, a brief theoretical background, and selected references are presented. The strengths and the limitations of each method are discussed, including the throughput improvement potential. If available, illustrations from presentations at various meetings by our laboratory are used to clarify our opinion. Drug Dev Res 68:107,133, 2007. ©2007 Wiley-Liss, Inc. [source] The use of in vitro technologies coupled with high resolution accurate mass LC-MS for studying drug metabolism in equine drug surveillanceDRUG TESTING AND ANALYSIS, Issue 1 2010James P. Scarth Abstract The detection of drug abuse in horseracing often requires knowledge of drug metabolism, especially if urine is the matrix of choice. In this study, equine liver/lung microsomes/S9 tissue fractions were used to study the phase I metabolism of eight drugs of relevance to equine drug surveillance (acepromazine, azaperone, celecoxib, fentanyl, fluphenazine, mepivacaine, methylphenidate and tripelennamine). In vitro samples were analyzed qualitatively alongside samples originating from in vivo administrations using LC-MS on a high resolution accurate mass Thermo Orbitrap Discovery instrument and by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap. Using high resolution accurate mass full-scan analysis on the Orbitrap, the in vitro systems were found to generate at least the two most abundant phase I metabolites observed in vitro for all eight drugs studied. In the majority of cases, in vitro experiments were also able to generate the minor in vivo metabolites and sometimes metabolites that were only observed in vitro. More detailed analyses of fentanyl incubates using LC-MS/MS showed that it was possible to generate good quality spectra from the metabolites generated in vitro. These data support the suggestion of using in vitro incubates as metabolite reference material in place of in vivo post-administration samples in accordance with new qualitative identification guidelines in the 2009 International Laboratory Accreditation Cooperation-G7 (ILAC-G7) document. In summary, the in vitro and in vivo phase I metabolism results reported herein compare well and demonstrate the potential of in vitro studies to compliment, refine and reduce the existing equine in vivo paradigm. Copyright © 2010 John Wiley & Sons, Ltd. [source] LC-MS: a powerful tool in workplace drug testingDRUG TESTING AND ANALYSIS, Issue 3 2009E. Gallardo Abstract Workplace drug testing is a well-established application of forensic toxicology and it aims to reduce workplace accidents caused by affected workers. Several classes of abused substances may be involved, such as alcohol, amphetamines, cannabis, cocaine, opiates and also prescription drugs, such as benzodiazepines. The use of alternative biological specimens such as hair, oral fluid or sweat in workplace drug testing presents several advantages over urinalysis,mainly the fact that sample collection can be performed easily without infringing on the examinee's privacy, so the subject is more likely to perform the test. However, drugs are usually present in these alternative specimens at low concentrations and the amount of sample available for analysis is small. The use of highly sensitive techniques is therefore necessary. In fact, the successful interface of liquid chromatography with mass spectrometry (LC-MS) has brought a new light into bioanalytical and forensic sciences as it allows the detection of drugs and metabolites at concentrations that are difficult to analyse using the more commonly adopted GC-MS based techniques. This paper will discuss the importance of LC-MS in supporting workplace drug-testing programmes. The combination of LC-MS with innovative instrumentation such as triple quadrupoles, ion traps and time-of-flight mass spectrometers will also be focused. Copyright © 2009 John Wiley & Sons, Ltd. [source] Comparison of DNA-Reactive Metabolites from Nitrosamine and Styrene Using Voltammetric DNA/Microsomes SensorsELECTROANALYSIS, Issue 9 2009Sadagopan Krishnan Abstract Voltammetric sensors made with films of polyions, double-stranded DNA and liver microsomes adsorbed layer-by-layer onto pyrolytic graphite electrodes were evaluated for reactive metabolite screening. This approach features simple, inexpensive screening without enzyme purification for applications in drug or environmental chemical development. Cytochrome P450 enzymes (CYPs) in the liver microsomes were activated by an NADPH regenerating system or by electrolysis to metabolize model carcinogenic compounds nitrosamine and styrene. Reactive metabolites formed in the films were trapped as adducts with nucleobases on DNA. The DNA damage was detected by square-wave voltammetry (SWV) using Ru(bpy) as a DNA-oxidation catalyst. These sensors showed a larger rate of increase in signal vs. reaction time for a highly toxic nitrosamine than for the moderately toxic styrene due to more rapid reactive metabolite-DNA adduct formation. Results were consistent with reported in vivo TD50 data for the formation of liver tumors in rats. Analogous polyion/ liver microsome films prepared on 500,nm silica nanoparticles (nanoreactors) and reacted with nitrosamine or styrene, provided LC-MS or GC analyses of metabolite formation rates that correlated well with sensor response. [source] Analysis of urinary metabolites for metabolomic study by pressurized CECELECTROPHORESIS, Issue 23 2007Guoxiang Xie Abstract A new approach for the metabolomic study of urinary samples using pressurized CEC (pCEC) with gradient elution is proposed as an alternative chromatographic separation tool with higher degree of resolution, selectivity, sensitivity, and efficiency. The pCEC separation of urinary samples was performed on a RP column packed with C18, 5,,m particles with an ACN/water mobile phase containing TFA. The effects of the acid modifiers, applied voltage, mobile phase, and detection wavelength were systematically evaluated using eight spiked standards, as well as urine samples. A typical analytical trial of urine samples from Sprague Dawley (S.D.) rats exposed to high-energy diet was carried out following sample pretreatment. Significant differences in urinary metabolic profiles were observed between the high energy diet-induced obesity rats and the healthy control rats at the 6th,wk postdose. Multivariate statistical analysis revealed the differential metabolites in response to the diet, which were partially validated with the putative standards. This work suggests that such a pCEC-based separation and analysis method may provide a new and cost-effective platform for metabolomic study uniquely positioned between the conventional chromatographic tools such as HPLC, and hyphenated analytical techniques such as LC-MS. [source] On-line sample stacking and short-end injection CE for the determination of fluoxetine and norfluoxetine in plasma: Method development and validation using experimental designsELECTROPHORESIS, Issue 18 2007Chia-Chia Lu Abstract A short-end injection CE method combining field-amplified sample stacking (FASS) is presented for the analysis of fluoxetine (FL) and norfluoxetine in plasma. In this study, FASS enhanced the sensitivity about 1100-fold, while short-end injection reduced the analysis time to less than 4,min. Parameters involved in the separations were investigated using a central composite design (CCD) and response surface methodology to optimize the separation conditions in a total of only 32 runs. Samples injected into the capillary for 99.9,s at a voltage of ,5,kV were stacked in a water plug (0.5,psi, 9,s). Baseline resolution of FL and its major metabolite was achieved using a BGE formulation consisting of phosphate,triethanolamine at low pH, and a separation voltage of ,10,kV. Five percent methanol was added as organic modifier to enhance selectivity and resolution. The linear range was between 10 and 500,ng/mL (r >0.9946), covering the expected plasma therapeutic ranges. The LOD in plasma were 4,ng/mL (S/N,=,3), a value comparable to that obtained using LC-MS, showing the success of the on-line stacking technique. Our method was also successfully validated in quantification and pharmacokinetic studies with three volunteer plasma samples and could be applied to pharmacogenetic studies. [source] Enantioselective analysis of ketamine and its metabolites in equine plasma and urine by CE with multiple isomer sulfated ,-CDELECTROPHORESIS, Issue 15 2007Regula Theurillat Abstract CE with multiple isomer sulfated ,-CD as the chiral selector was assessed for the simultaneous analysis of the enantiomers of ketamine and metabolites in extracts of equine plasma and urine. Different lots of the commercial chiral selector provided significant changes in enantiomeric ketamine separability, a fact that can be related to the manufacturing variability. A mixture of two lots was found to provide high-resolution separations and interference-free detection of the enantiomers of ketamine, norketamine, dehydronorketamine, and an incompletely identified hydroxylated metabolite of norketamine in liquid/liquid extracts of the two body fluids. Ketamine, norketamine, and dehydronorketamine could be unambiguously identified via HPLC fractionation of urinary extracts and using LC-MS and LC-MS/MS with 1,mmu mass discrimination. The CE assay was used to characterize the stereoselectivity of the compounds' enantiomers in the samples of five ponies anesthetized with isoflurane in oxygen and treated with intravenous continuous infusion of racemic ketamine. The concentrations of the ketamine enantiomers in plasma are equal, whereas the urinary amount of R -ketamine is larger than that of S -ketamine. Plasma and urine contain higher S - than R -norketamine levels and the mean S -/R -enantiomer ratios of dehydronorketamine in plasma and urine are lower than unity and similar. [source] Determination of tobacco-specific N -nitrosamines in rabbit serum by capillary zone electrophoresis and capillary electrophoresis-electrospray ionization-mass spectrometry with solid-phase extractionELECTROPHORESIS, Issue 11 2006Chenchen Li Abstract In this paper, we propose a new strategy for separation and determination of tobacco-specific N -nitrosamines (TSNAs), a group of strong carcinogens found only in tobacco products, by using CZE and CE-MS associated with SPE. Six TSNAs: N'-nitrosonornicotine, N'-nitrosoanatabine, N'-nitrosoanabasine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, and 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol were simultaneously separated by either of two CZE methods, one of which worked with ammonium formate buffer (pH,2.5) and another with citrate buffer (pH,2.4), as well as a CE-MS method. The CZE conditions including pH and concentration of running buffer, capillary length, applied voltage, and capillary temperature were systematically optimized. For CE-MS method, an optimized sheath liquid consisted of methanol,water was used at a flow rate of 10,,L/min. With SPE procedure, our proposed CE-MS method was successfully applied to determine TSNAs after 15,min metabolism in rabbits. A comparison study between CZE and CE-MS methods for quantitative purposes was carried out, showing that both methods provided similar separation efficiency, selectivity, repeatability, linearity, and recovery. However, CE-MS method was better suited for the analysis of TSNAs in complicated biological samples for its sensitivity and extra information on molecular structure. Having good accordance with our previous work by using LC-MS, the new CE-MS method is expected to be an alternative to the LC-MS method and applied to study the metabolism of TSNAs. [source] Anatoxin-a toxin in the cyanobacterium Planktothrix rubescens from a fishing pond in northern ItalyENVIRONMENTAL TOXICOLOGY, Issue 3 2004Emanuela Viaggiu Abstract A heavy algal bloom occurring in a fishing pond in northern Italy full of Salmo trutta was examined for algae taxonomy and toxic production. The dominant algal species (98%) was identified as the cyanobacterium Planktothrix rubescens (D.C. ex GOMONT) Komarek Anagnostidis, based on morphological examination, and it was revealed to be toxic in mouse and Vibrio fischeri bioassays. The toxin was identified as anatoxin-a using high-performance liquid chromatography and confirmed using liquid chromatography,mass spectrometry (LC-MS). The mouse bioassay gave signs of poisoning, as previously reported for anatoxin-a. The LC-MS confirmed the presence of an anatoxin-a peak at m/z 166 (M+H+). The content of toxin in the field population was estimated at 12.13 ,g/g of fresh cells. The bloom was sustained by the very high N/P ratio in the water. This is the first report in Italy of an anatoxin-a-producing Planktothrix rubescens population. © 2004 Wiley Periodicals, Inc. Environ Toxicol 19: 191,197, 2004. [source] Dynamics, stability and iron-binding activity of frataxin clinical mutantsFEBS JOURNAL, Issue 14 2008Ana R. Correia Friedreich's ataxia results from a deficiency in the mitochondrial protein frataxin, which carries single point mutations in some patients. In the present study, we analysed the consequences of different disease-related mutations in vitro on the stability and dynamics of human frataxin. Two of the mutations, G130V and D122Y, were investigated for the first time. Analysis by CD spectroscopy demonstrated a substantial decrease in the thermodynamic stability of the variants during chemical and thermal unfolding (wild-type > W155R > I154F > D122Y > G130V), which was reversible in all cases. Protein dynamics was studied in detail and revealed that the mutants have distinct propensities towards aggregation. It was observed that the mutants have increased correlation times and different relative ratios between soluble and insoluble/aggregated protein. NMR showed that the clinical mutants retained a compact and relatively rigid globular core despite their decreased stabilities. Limited proteolysis assays coupled with LC-MS allowed the identification of particularly flexible regions in the mutants; interestingly, these regions included those involved in iron-binding. In agreement, the iron metallochaperone activity of the Friedreich's ataxia mutants was affected: some mutants precipitate upon iron binding (I154F and W155R) and others have a lower binding stoichiometry (G130V and D122Y). Our results suggest that, in heterozygous patients, the development of Friedreich's ataxia may result from a combination of reduced efficiency of protein folding and accelerated degradation in vivo, leading to lower than normal concentrations of frataxin. This hypothesis also suggests that, although quite different from other neurodegenerative diseases involving toxic aggregation, Friedreich's ataxia could also be linked to a process of protein misfolding due to specific destabilization of frataxin. [source] IPSE/alpha-1, a major secretory glycoprotein antigen from schistosome eggs, expresses the Lewis X motif on core-difucosylated N-glycansFEBS JOURNAL, Issue 10 2006Manfred Wuhrer Schistosomes are parasitic flatworms that infect millions of people in (sub)tropical areas around the world. Glycoconjugates of schistosomes play a critical role in the interaction of the different developmental stages of the parasite with the host. In particular, glycosylated components of the eggs produced by the adult worm pairs living in the bloodstream are strongly immunogenic. We have investigated the glycosylation of interleukin-4-inducing factor from schistosome eggs (IPSE/alpha-1), a major secretory egg antigen from Schistosoma mansoni that triggers interleukin-4 production in human basophils, by MS analysis of tryptic glycopeptides. Nanoscale LC-MS(/MS) and MALDI-TOF(/TOF)-MS studies combined with enzymatic degradations showed that monomeric IPSE/alpha-1 contains two N-glycosylation sites, which are each occupied for a large proportion with core-difucosylated diantennary glycans that carry one or more Lewis X motifs. Lewis X has been reported as a major immunogenic glycan element of schistosomes. This is the first report both on the expression of Lewis X on a specific schistosome egg protein and on a protein-specific glycosylation analysis of schistosome eggs. [source] Folding of epidermal growth factor-like repeats from human tenascin studied through a sequence frame-shift approachFEBS JOURNAL, Issue 21 2004Francesco Zanuttin In order to investigate the factors that determine the correct folding of epidermal growth factor-like (EGF) repeats within a multidomain protein, we prepared a series of six peptides that, taken together, span the sequence of two EGF repeats of human tenascin, a large protein from the extracellular matrix. The peptides were selected by sliding a window of the average length of tenascin EGF repeats over the sequence of EGF repeats 13 and 14. We thus obtained six peptides, EGF-f1 to EGF-f6, that are 33 residues long, contain six cysteines each, and bear a partial overlap in the sequence. While EGF-f1 corresponds to the native EGF-14 repeat, the others are frame-shifted EGF repeats. We carried out the oxidative folding of these peptides in vitro, analyzed the reaction mixtures by acid trapping followed by LC-MS, and isolated some of the resulting products. The oxidative folding of the native EGF-14 peptide is fast, produces a single three-disulfide species with an EGF-like disulfide topology and a marked difference in the RP-HPLC retention time compared with the starting product. On the contrary, frame-shifted peptides fold more slowly and give mixtures of three-disulfide species displaying RP-HPLC retention times that are closer to those of the reduced peptides. In contrast to the native EGF-14, the three-disulfide products that could be isolated are mainly unstructured, as determined by CD and NMR spectroscopy. We conclude that both kinetics and thermodynamics drive the correct pairing of cysteines, and speculate about how cysteine mispairing could trigger disulfide reshuffling in vivo. [source] Pharmacokinetics after an intravenous single dose of the opioid ketobemidone in childrenACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 4 2010S. LUNDEBERG Background: Ketobemidone is often used as an alternative to morphine in children in the Scandinavian countries. The aim of this clinical trial was to explore the pharmacokinetics of ketobemidone in children because these properties have not been reported previously. Methods: Thirty children, newborn to 10 years, scheduled for elective surgery were included in the trial. Ketobemidone hydrochloride was administered as a single intravenous bolus dose and ketobemidone and norketobemidone concentrations were measured by LC-MS over 8 h. Pharmacokinetic parameters were determined using compartmental methods. Results: Six children were excluded from pharmacokinetic analysis because of incomplete blood sampling. The values of ketobemidone clearance (l/h/kg) given as median (range) were 0.84 (0.29,3.0) in Group A (0,90 days), 0.89 (0.55,1.35) in Group B (1,2.5 years) and 0.74 (0.50,0.99) in Group C (7,10 years). The corresponding values for apparent volume of distribution (l/kg) were 4.4 (3.7,6.9) (Group A), 2.6 (2.0,5.6) (Group B) and 3.9 (2.7,5.0 (Group C), and for elimination half-life (h) 3.0 (1.4,8.9) (Group A), 2.0 (1.2,4.7) (Group B) and 3.7 (2.4,6.9) (Group C), respectively. In the two neonates the elimination half-life was almost 9 h. The metabolite norketobemidone did not reach levels above the limit of quantification (0.07 ng/ml) in any of the patients. Conclusion: The pharmacokinetic parameters of ketobemidone in children older than 1 month appear to be similar to those in adults. Because of the large interindividual variability of the pharmacokinetics in neonates, further studies especially in this age group are warranted. [source] Functional, genetic and chemical characterization of biosurfactants produced by plant growth-promoting Pseudomonas putida 267JOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2009Marco Kruijt Abstract Aims:, Plant growth-promoting Pseudomonas putida strain 267, originally isolated from the rhizosphere of black pepper, produces biosurfactants that cause lysis of zoospores of the oomycete pathogen Phytophthora capsici. The biosurfactants were characterized, the biosynthesis gene(s) partially identified, and their role in control of Phytophthora damping-off of cucumber evaluated. Methods and Results:, The biosurfactants were shown to lyse zoospores of Phy. capsici and inhibit growth of the fungal pathogens Botrytis cinerea and Rhizoctonia solani. In vitro assays further showed that the biosurfactants of strain 267 are essential in swarming motility and biofilm formation. In spite of the zoosporicidal activity, the biosurfactants did not play a significant role in control of Phytophthora damping-off of cucumber, since both wild type strain 267 and its biosurfactant-deficient mutant were equally effective, and addition of the biosurfactants did not provide control. Genetic characterization revealed that surfactant biosynthesis in strain 267 is governed by homologues of PsoA and PsoB, two nonribosomal peptide synthetases involved in production of the cyclic lipopeptides (CLPs) putisolvin I and II. The structural relatedness of the biosurfactants of strain 267 to putisolvins I and II was supported by LC-MS and MS-MS analyses. Conclusions:, The biosurfactants produced by Ps. putida 267 were identified as putisolvin-like CLPs; they are essential in swarming motility and biofilm formation, and have zoosporicidal and antifungal activities. Strain 267 provides excellent biocontrol activity against Phytophthora damping-off of cucumber, but the lipopeptide surfactants are not involved in disease suppression. Significance and Impact of the Study:,Pseudomonas putida 267 suppresses Phy. capsici damping-off of cucumber and provides a potential supplementary strategy to control this economically important oomycete pathogen. The putisolvin-like biosurfactants exhibit zoosporicidal and antifungal activities, yet they do not contribute to biocontrol of Phy. capsici and colonization of cucumber roots by Ps. putida 267. These results suggest that Ps. putida 267 employs other, yet uncharacterized, mechanisms to suppress Phy. capsici. [source] Cyanobacteria from benthic mats of Antarctic lakes as a source of new bioactivitiesJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2008N. Biondi Abstract Aims:, To exploit the cyanobacterial diversity of microbial mats growing in the benthic environment of Antarctic lakes for the discovery of novel antibiotic and antitumour activities. Methods and results:, In all, 51 Antarctic cyanobacteria isolated from benthic mats were cultivated in the laboratory by optimizing temperature, irradiance and mixing. Productivity was generally very low (,60 mg l,1 d,1) with growth rates (,) in the range of 0·02,0·44 d,1. Growth rates were limited by photosensitivity, sensitivity to air bubbling, polysaccharide production or cell aggregation. Despite this, 126 extracts were prepared from 48 strains and screened for antimicrobial and cytotoxic activities. Seventeen cyanobacteria showed antimicrobial activity (against the Gram-positive Staphylococcus aureus, the filamentous fungus Aspergillus fumigatus or the yeast Cryptococcus neoformans), and 25 were cytotoxic. The bioactivities were not in accordance with the phylogenetic grouping, but rather strain-specific. One active strain was cultivated in a 10-l photobioreactor. Conclusions:, Isolation and mass cultivation of Antarctic cyanobacteria and LC-MS (liquid chromatography/mass spectrometry) fractionation of extracts from a subset of those strains (hits) that exhibited relatively potent antibacterial and/or antifungal activities, evidenced a chemical novelty worthy of further investigation. Significance and impact of the study:, Development of isolation, cultivation and screening methods for Antarctic cyanobacteria has led to the discovery of strains endowed with interesting antimicrobial and antitumour activities. [source] Indirect identification of isoprenoid quinones in Escherichia coli by LC-MS with atmospheric pressure chemical ionization in negative modeJOURNAL OF BASIC MICROBIOLOGY, Issue 6 2004Mengchun Gao Dr. A novel analytical method was applied for identification of isoprenoid quinones in Escherichia coli by liquid chromatography atmospheric press chemical ionization mass spectrometry in negative mode (LC-NI-APCI-MS). Extraction and clean-up of sample were carried out on Sep-Pak Plus Silica solid-phase extraction cartridges. Ubiquinone-7 (UQ-7), Ubiquinone-8 (UQ-8) and Mequinone-8 (MK-8) were determined directly using combined information on retention time, molecular ion mass, fragment ion masses and UV characteristic spectrometry without any standard reagent. It was found that UQ-8 was the major component of isoprenoid quinones in Escherichia coli under aerobic condition. Compared with UQ-8, the relative abundance of UQ-7 and MK-8 is only 15% and 14%, respectively. The average recoveries of UQ-6, UQ-10 and vitamin K1 in Escherichia coli were investigated by standard spiking experiment. The recoveries were achieved in the range from 94 to 106%, and the relative standard deviations (RSD) of the triplicate analysis of the spiked samples (UQ-6, UQ-10 and vitamin K1) ranged from 3 to 8%. The detection limits of LC-NI-APCI-MS were estimated to be 5, 40 and 0.8 ,g/g dry cell for UQ-6, UQ-10 and vitamin K1, respectively. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Protein profile study of breast-tissue homogenates by HPLC-LIFJOURNAL OF BIOPHOTONICS, Issue 5 2009K. Kalyan Kumar Abstract Proteomics is a promising approach for molecular understanding of neoplastic processes including response to treatment. Widely used 2D-gel electrophoresis/Liquid chromatography coupled with mass spectrometry (LC-MS) are time consuming and not cost effective. We have developed a high-sensitivity (femto/subfemtomoles of protein/20 ,l) High Performance Liquid Chromatography-Laser Induced Fluorescence HPLC-LIF instrument for studying protein profiles of biological samples. In this study, we have explored the feasibility of classifying breast tissues by multivariate analysis of chromatographic data. We have analyzed 13 normal, 17 malignant, 5 benign and 4 post-treatment breast-tissue homogenates. Data was analyzed by Principal Component Analysis PCA in both unsupervised and supervised modes on derivative and baseline-corrected chromatograms. Our findings suggest that PCA of derivative chromatograms gives better classification. Thus, the HPLC-LIF instrument is not only suitable for generation of chromatographic data using femto/subfemto moles of proteins but the data can also be used for objective diagnosis via multivariate analysis. Prospectively, identified fractions can be collected and analyzed by biochemical and/or MS methods. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Methotrexate induced differentiation in colon cancer cells is primarily due to purine deprivationJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2006R. Singh Abstract The folate antagonist methotrexate (MTX) inhibits synthesis of tetrahydrofolate (THF), pyrimidines and purines, and induces differentiation in several cell types. At 1 µM, MTX reduced proliferation and induced differentiation in HT29 colon cancer cells; the latter effect was augmented (P,<,0.001) by thymidine (100 µM) but was reversed (P,<,0.001) by the purines, hypoxanthine (Hx; 100 µM) and adenosine (100 µM). In contrast 5-fluoro-uracil (5-FU), a specific thymidylate synthase (TS) inhibitor, had no effect on differentiation, suggesting that MTX-induced differentiation is not due to a reduction in thymidine but to the inhibition of purine biosynthesis. Inhibition of cyclic AMP (cAMP) by RpcAMP (25 µM) further enhanced (P,<,0.001) MTX induced differentiation, whereas the cAMP activator forskolin (10 µM) reversed (P,<,0.001) MTX induced differentiation. These observations implicate a central role of adenosine and cAMP in MTX induced differentiation. By combining Western blot analysis with liquid chromatography-mass spectrometry (LC-MS)and HPLC analyses we also reveal both the expression and activity of key enzymes (i.e. methionine synthase (MS), s-adenosylhomocysteinase, cystathionine ,-synthase and ornithine decarboxylase) regulating methyl cycle, transsulfuration and polyamine pathways in HT29 colon cancer cells. At 1 µM, MTX induced differentiation was associated with a marked reduction in the intracellular concentrations of adenosine and, consequently, S-adenosylmethionine (SAM), S-adenosylhomocysteine, polyamines and glutathione (GSH). Importantly, the marked reduction in methionine that accompanied MS inhibition following MTX treatment was non-limiting with respect to SAM synthesis. Collectively, these findings indicate that the effects of MTX on cellular differentiation and single carbon metabolism are primarily due to the intracellular depletion of purines. J. Cell. Biochem. © 2006 Wiley-Liss, Inc. [source] Oxidative Degradation of Bisphenol A by Fruit HomogenatesJOURNAL OF FOOD SCIENCE, Issue 9 2005Masaaki Imanaka ABSTRACT: The oxidative degradation of bisphenol A (BPA) by several fruit homogenates was investigated. Their homogenates were incubated with BPA at 25 °C for 0 to 120 min, and the acetone extracts were analyzed by high-performance liquid chromatography (HPLC) with a photodiode array detector (200 to 650 nm). The 2 degradation products (UK-1 and UK-2) from BPA were detected on HPLC chromatograms (280 nm). UK-1 and UK-2 were identified to be 2-(3,4-dihydroxyphenyl)-2-(4-ydroxyphenyl) propane, (3-OH-BPA) and 4-[1-(3,4-dihydroxyphenyl)-isopropyl]benzene-1,2-diol, (3,3,-diOH-BPA), respectively, by HPLC-MassPectrometry (LC-MS). In the process of incubation, the peak of 3-OH-BPA attained the maximum value in the 1st 20 min, and that of 3,3,-diOH-BPA increased more slowly, attaining the maximum in 50 min. On the other hand, incubation of 3-OH-BPA (instead of BPA) with grape homogenates gave the maximum peak of 3,3,-diOH-BPA in only 10 min. 3, 3,-diOH-BPA was a polyphenol compound that contained 4 hydroxyl groups. These results suggested that BPA would be degraded (converted) to brown pigments through the compounds of 3-OH-BPA and 3, 3,-diOH-BPA in some fruit homogenates. [source] The synthesis of multiply 13C-labelled plant and mammalian lignans as internal standards for LC-MS and GC-MS analysisJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 13 2005Tara Fryatt Abstract The syntheses of multiply 13C-labelled derivatives of the two mammalian lignans, enterolactone and enterodiol, and two of their plant lignan precursors, secoisolariciresinol and matairesinol, are described. Three 13C atoms were incorporated into each lignan using potassium [13C]cyanide as the source for all of the 13C atoms. The compounds were prepared for use as internal standards in the LC-MS and GC-MS analysis of lignans. Copyright © 2005 John Wiley & Sons, Ltd. [source] Studies on the metabolism of the ,9-tetrahydrocannabinol precursor ,9-tetrahydrocannabinolic acid A (,9-THCA-A) in rat using LC-MS/MS, LC-QTOF MS and GC-MS techniquesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 10 2009Julia Jung Abstract In Cannabis sativa, ,9-Tetrahydrocannabinolic acid-A (,9-THCA-A) is the non-psychoactive precursor of ,9-tetrahydrocannabinol (,9-THC). In fresh plant material, about 90% of the total ,9-THC is available as ,9-THCA-A. When heated (smoked or baked), ,9-THCA-A is only partially converted to ,9-THC and therefore, ,9-THCA-A can be detected in serum and urine of cannabis consumers. The aim of the presented study was to identify the metabolites of ,9-THCA-A and to examine particularly whether oral intake of ,9-THCA-A leads to in vivo formation of ,9-THC in a rat model. After oral application of pure ,9-THCA-A to rats (15 mg/kg body mass), urine samples were collected and metabolites were isolated and identified by liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high resolution LC-MS using time of flight-mass spectrometry (TOF-MS) for accurate mass measurement. For detection of ,9-THC and its metabolites, urine extracts were analyzed by gas chromatography-mass spectrometry (GC-MS). The identified metabolites show that ,9-THCA-A undergoes a hydroxylation in position 11 to 11-hydroxy-,9-tetrahydrocannabinolic acid-A (11-OH-,9-THCA-A), which is further oxidized via the intermediate aldehyde 11-oxo-,9-THCA-A to 11-nor-9-carboxy-,9-tetrahydrocannabinolic acid-A (,9-THCA-A-COOH). Glucuronides of the parent compound and both main metabolites were identified in the rat urine as well. Furthermore, ,9-THCA-A undergoes hydroxylation in position 8 to 8-alpha- and 8-beta-hydroxy-,9-tetrahydrocannabinolic acid-A, respectively, (8,-Hydroxy-,9-THCA-A and 8,-Hydroxy-,9-THCA-A, respectively) followed by dehydration. Both monohydroxylated metabolites were further oxidized to their bishydroxylated forms. Several glucuronidation conjugates of these metabolites were identified. In vivo conversion of ,9-THCA-A to ,9-THC was not observed. Copyright © 2009 John Wiley & Sons, Ltd. [source] Modern MALDI time-of-flight mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2009Marvin L. Vestal Abstract This paper focuses on development of time-of-flight (TOF) mass spectrometry in response to the invention of matrix-assisted laser desorption/ionization (MALDI). Before this breakthrough ionization technique for nonvolatile molecules, TOF was generally considered as a useful tool for exotic studies of ion properties but was not widely applied to analytical problems. Improved TOF instruments and software that allow the full potential power of MALDI to be applied to difficult biological applications are described. A theoretical approach to the design and optimization of MALDI-TOF instruments for particular applications is presented. Experimental data are provided that are in excellent agreement with theoretical predictions of resolving power and mass accuracy. Data on sensitivity and dynamic range using kilohertz laser rates are also summarized. These results indicate that combinations of high-performance MALDI-TOF and TOF-TOF with off-line high-capacity separations may ultimately provide throughput and dynamic range several orders of magnitude greater than those currently available with electrospray LC-MS and MS-MS. Copyright © 2009 John Wiley & Sons, Ltd. [source] Investigating the presence of pesticide transformation products in water by using liquid chromatography-mass spectrometry with different mass analyzers,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2008Félix Hernández Abstract Many pesticide transformation products (TPs) can reach environmental waters as a consequence of their normally having a higher polarity than their parent pesticides. This makes the development of analytical methodology for reliable identification and subsequent quantification at the sub-microgram per liter levels necessary, as required under current legislation. In this paper we report the photodegradation of several pesticides frequently detected in environmental waters from the Spanish Mediterranean region using the high-resolution and exact-mass capabilities of hybrid quadrupole time-of-flight mass spectrometry (QTOF MS) hyphenated to liquid chromatography (LC). Once the main photodegradation/hydrolysis products formed in aqueous media were identified, analytical methodology for their simultaneous quantification and reliable identification in real water samples was developed using on-line solid-phase extraction (SPE)-LC-tandem MS with a triple-quadrupole (QqQ) analyzer. The methodology was validated in both ground and surface water samples spiked at the limit of quantification (LOQ) and 10 × LOQ levels, i.e. 50 and 500 ng/l, obtaining satisfactory recoveries and precision for all compounds. Subsequent analysis of ground and surface water samples resulted in the detection of a number of TPs higher than parent pesticides. Additionally, several soil-interstitial water samples collected from the unsaturated zone were analyzed to explore the degradation/transformation of some pesticides in the field using experimental plots equipped with lisimeters. Several TPs were found in these samples, with most of them having also been detected in ground and surface water from the same area. This paper illustrates the extraordinary potential of LC-MS(/MS) with QTOF and QqQ analyzers for qualitative/structural and quantitative analysis, respectively, offering analytical chemists one of the most powerful tools available at present to investigate the presence of pesticide TPs in water. Copyright © 2007 John Wiley & Sons, Ltd. [source] Accessible proteomics space and its implications for peak capacity for zero-, one- and two-dimensional separations coupled with FT-ICR and TOF mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2006Jennifer L. Frahm The number and wide dynamic range of components found in biological matrixes present several challenges for global proteomics. In this perspective, we will examine the potential of zero-dimensional (0D), one-dimensional (1D), and two-dimensional (2D) separations coupled with Fourier-transform ion cyclotron resonance (FT-ICR) and time-of-flight (TOF) mass spectrometry (MS) for the analysis of complex mixtures. We describe and further develop previous reports on the space occupied by peptides, to calculate the theoretical peak capacity available to each separations-mass spectrometry method examined. Briefly, the peak capacity attainable by each of the mass analyzers was determined from the mass resolving power (RP) and the m/z space occupied by peptides considered from the mass distribution of tryptic peptides from National Center for Biotechnology Information's (NCBI's) nonredundant database. Our results indicate that reverse-phase-nanoHPLC (RP-nHPLC) separation coupled with FT-ICR MS offers an order of magnitude improvement in peak capacity over RP-nHPLC separation coupled with TOF MS. The addition of an orthogonal separation method, strong cation exchange (SCX), for 2D LC-MS demonstrates an additional 10-fold improvement in peak capacity over 1D LC-MS methods. Peak capacity calculations for 0D LC, two different 1D RP-HPLC methods, and 2D LC (with various numbers of SCX fractions) for both RP-HPLC methods coupled to FT-ICR and TOF MS are examined in detail. Peak capacity production rates, which take into account the total analysis time, are also considered for each of the methods. Furthermore, the significance of the space occupied by peptides is discussed. Copyright © 2006 John Wiley & Sons, Ltd. [source] Microwave-assisted TFA cleavage of peptides from Merrifield resinJOURNAL OF PEPTIDE SCIENCE, Issue 1 2010Alicja Kluczyk Abstract Microwave-assisted (MW) reactions are of special interest to the chemical community due to faster reaction times, cleaner reactions and higher product yields. The adaptation of MW to solid phase peptide synthesis resulted in spectacular syntheses of difficult peptides. In the case of Merrifield support, used frequently in synthesis of special peptides, the conditions used in product cleavage are not compatible with off-resin monitoring of the reaction progress. The application of MW irradiation in product removal from Merrifield resin using trifluoroacetic acid (TFA) was investigated using model tetrapeptides and the effects were compared with standard trifluoromethanesulphonic acid (TFMSA) cleavage using elemental analysis as well as chromatographic (HPLC) and spectroscopic (IR) methods. The deprotection of benzyloxycarbonyl and benzyl groups in synthetic bioactive peptides was analyzed using LC-MS and MS/MS experiments. In a 5 min microwave-assisted TFA reaction at low temperature, the majority of product is released from the resin, making the analytical scale MW-assisted procedure a method of choice in monitoring the reactions carried out on Merrifield resin due to the short reaction time and compatibility with HPLC and ESI-MS conditions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. [source] Synthesis of the C -terminal domain of the tissue inhibitor of metalloproteinases-1(TIMP-1)JOURNAL OF PEPTIDE SCIENCE, Issue 7 2003József Bódi Abstract According to recent investigations, the C -terminal domain of the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) is responsible for some biological effects that are independent of the enzyme-inhibiting effect of the N -terminal domain of the molecule. The C -terminal domain has been prepared for structure,biological activity investigations. After the chemical synthesis and the folding of the linear peptide, LC-MS and MALDI-MS analysis revealed that two isomers with different disulphide bond arrangements were formed. Since more than 30 folding experiments resulted in products with a very similar HPLC-profile, it was concluded that in the absence of the TIMP-1 N -terminal domain no entirely correct folding of the C -terminal domain occurred. Furthermore, it was observed that, in spite of several purification steps, mercury(II) ions were bound to the 6SH-linear peptide; it was demonstrated,using disulphide bonded TIMP-1(Cys145 -Cys166) as a model,that mercury(II) ions can cause peptide degradation at pH 7.8 as well as in 0.1% trifluoroacetic acid. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd. [source] Pharmaceutical impurity identification: A case study using a multidisciplinary approachJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 9 2004Karen M. Alsante Abstract A multidisciplinary team approach to identify pharmaceutical impurities is presented in this article. It includes a representative example of the methodology. The first step is to analyze the sample by LC-MS. If the structure of the unknown impurity cannot be conclusively determined by LC-MS, LC-NMR is employed. If the sample is unsuitable for LC-NMR, the impurity needs to be isolated for conventional NMR characterization. Although the technique of choice for isolation is preparative HPLC, enrichment is often necessary to improve preparative efficiency. One such technique is solid-phase extraction. For complete verification, synthesis may be necessary to compare spectroscopic characteristics to those observed in the original sample. Although not widely practiced, an effective means of getting valuable structural information is to conduct a degradation study on the purified impurity itself. This systematic strategy was successfully applied to the identification of an impurity in the active pharmaceutical ingredient 1-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)-3-[4-(1-hydroxy-1-methyl-ethyl)-furan-2-sulphonylurea. Identification required the use of all of the previously mentioned techniques. The instability of the impurity under acidic chromatographic conditions presented an additional challenge to purification and identification. However, we turned this acidic instability to an advantage, conducting a degradation study of the impurity, which provided extensive and useful information about its structure. The following discussion describes how the information gained from each analytical technique was brought together in a complementary fashion to elucidate a final structure. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:2296,2309, 2004 [source] Oxidative metabolism by Thalassiosira weissflogii (Bacillariophyceae) of a diol-ester of okadaic acid, the diarrhetic shellfish poisoningJOURNAL OF PHYCOLOGY, Issue 2 2000Anthony J. Windust Previous investigations into the comparative toxicity of the diarrhetic shellfish poisoning (DSP) toxins to Thalassiosira weissflogii (Grun.) Fryxell et Hasle found that this diatom oxidatively metabolized okadaic acid diol-ester (OA diol-ester) to a more water-soluble product. This oxidative transformation of OA diol-ester by the diatom is significant for two reasons. First, it is known that dinophysistoxin-4 (DTX-4), the primary DSP toxin produced by the dinoflagellate Exuviaella lima (Ehr.) Butschli, will be hydrolyzed to the diol-ester following cell rupture (e.g. ingestion by a predator). Second, it implies that the ester, an uncharged, lipophilic intermediate, can easily enter cells and therefore may play an important role in the uptake and transfer of DSP toxins through the food web. It has been suggested that the water soluble DTX-4 may also be the form in which DSP toxins are excreted from the producing cell. Therefore, the stability of DTX-4 was examined when incubated either in fresh seawater medium into which washed cells of E. lima were introduced or in seawater medium conditioned by E. lima cells. Rapid hydrolysis of DTX-4 to the diol-ester took place in both cases. Thus, regardless of the route by which DTX-4 is liberated from the cell, either by cell disruption or excretion, the diol-ester will be the dominant form of the toxin to challenge associated organisms. To examine the metabolism of OA diol-ester by T. weissflogii in more detail, serial cultures of the diatom were challenged with OA diol-ester at a concentration of 2.0 ,g·mL,1. The metabolism and fate of the diol-ester in both cellular and medium fractions were monitored over 3 days using liquid chromatography with either ultraviolet (LC-UV) or mass spectrometric (LC-MS) detection. During the course of the experiment, all of the diol-ester was metabolized. LC-MS analysis revealed the presence of multiple oxidative products of OA diol-ester in the medium fraction, including a carboxylic acid derivative. The major metabolites were isolated in sufficient quantity to permit structural elucidation by NMR and MS. All the metabolites identified resulted from oxidation of the diol-ester side chain with the primary sites of attack at the terminal, subterminal, and unsaturated carbons. OA was found in both cellular and medium fractions, and its production was directly correlated with the metabolism of the diol-ester. The relative partitioning of both OA diol-ester and its oxidation products between cells and medium supports the contention that OA diol-ester can readily enter cells, be metabolized, and then excreted in more water-soluble forms. [source] Pharmaceutical antibiotic compounds in soils , a reviewJOURNAL OF PLANT NUTRITION AND SOIL SCIENCE, Issue 2 2003Sören Thiele-Bruhn Antibiotics are highly effective, bioactive substances. As a result of their consumption, excretion, and persistence, they are disseminated mostly via excrements and enter the soils and other environmental compartments. Resulting residual concentrations in soils range from a few ,g upto g kg,1 and correspond to those found for pesticides. Numerous antibiotic molecules comprise of a non-polar core combined with polar functional moieties. Many antibiotics are amphiphilic or amphoteric and ionize. However, physicochemical properties vary widely among compounds from the various structural classes. Existing analytical methods for environmental samples often combine an extraction with acidic buffered solvents and the use of LC-MS for determination. In soils, adsorption of antibiotics to the organic and mineral exchange sites is mostly due to charge transfer and ion interactions and not to hydrophobic partitioning. Sorption is strongly influenced by the pH of the medium and governs the mobility and transport of the antibiotics. In particular for the strongly adsorbed antibiotics, fast leaching through soils by macropore or preferential transport facilitated by dissolved soil colloids seems to be the major transport process. Antibiotics of numerous classes are photodegraded. However, on soil surfaces this process if of minor influence. Compared to this, biotransformation yields a more effective degradation and inactivation of antibiotics. However, some metabolites still comprise of an antibiotic potency. Degradation of antibiotics is hampered by fixation to the soil matrix; persisting antibiotics were already determined in soils. Effects on soil organisms are very diverse, although all antibiotics are highly bioactive. The absence of effects might in parts be due to a lack of suitable test methods. However, dose and persistence time related effects especially on soil microorganisms are often observed that might cause shifts of the microbial community. Significant effects on soil fauna were only determined for anthelmintics. Due to the antibiotic effect, resistance in soil microorganisms can be provoked by antibiotics. Additionally, the administration of antibiotics mostly causes the formation of resistant microorganisms within the treated body. Hence, resistant microorganisms reach directly the soils with contaminated excrements. When pathogens are resistant or acquire resistance from commensal microorganisms via gene transfer, humans and animals are endangered to suffer from infections that cannot be treated with pharmacotherapy. The uptake into plants even of mobile antibiotics is small. However, effects on plant growth were determined for some species and antibiotics. Pharmazeutische Antibiotika in Böden , ein Überblick Antibiotika sind hochgradig wirksame, bioaktive Substanzen. Infolge ihrer Anwendung, Ausscheidung und Persistenz werden sie meist über die Exkremente in Böden und andere Umweltkompartimente eingetragen. Die resultierenden Rückstandskonzentrationen in Böden im Bereich von wenigen ,g bis zu g kg,1 entsprechen in etwa denen von Pflanzenschutzmitteln. Die Molekülstruktur von Antibiotika besteht häufig aus einem unpolaren Kern und polaren Randgruppen. Viele Antibiotika sind amphiphil oder amphoter und bilden Ionen, jedoch weisen die zahlreichen Antibiotika unterschiedlicher Strukturklassen stark divergierende physikochemische Eigenschaften auf. In den vorliegenden Nachweis"methoden für Umweltproben werden häufig sauer gepufferte Lösungsmittel zur Extraktion und eine Bestimmung mittels LC-MS kombiniert. Die Adsorption der Antibiotika an den organischen als auch an den mineralischen Bodenaustauschern erfolgt zumeist durch Ladungs- und Ionenwechselwirkungen und weniger durch hydrophobe Bindungen. Das Verteilungsverhalten hängt dabei entscheidend vom pH-Wert des Mediums ab und beeinflusst die Mobilität und Verlagerung der Antibiotika. Bei vielen, insbesondere stark adsorbierten Antibiotika sind v.,a. schnelle Fließvorgänge wie durch präferenziellen und Makroporenfluss sowie der Cotransport mit gelösten Bodenkolloiden von besonderer Bedeutung. Antibiotika vieler Strukturklassen können durch Licht abgebaut werden. Dieser Abbaupfad spielt auf Bodenoberflächen jedoch nur eine untergeordnete Rolle. Hingegen kommt es insbesondere durch biologische Transformationsprozesse zu einer intensiven Degradation und Inaktivierung der Antibiotika. Verschiedene Metaboliten weisen jedoch ebenfalls ein antibiotisches Potential auf. Der Abbau der Antibiotika wird durch die Festlegung in Böden gehemmt; dementsprechend wurde eine Persistenz verschiedener Antibiotika nachgewiesen. Trotz der starken bioaktiven Wirkung aller Antibiotika sind die festgestellten Effekte auf Bodenorganismen sehr unterschiedlich. Dies liegt nicht zuletzt an einem Mangel an geeigneten Testmethoden. In der Regel sind jedoch von Dosis und Wirkungsdauer abhängige Effekte insbesondere auf Mikroorganismen festzustellen, die zu Veränderungen der Mikroorganismenpopulation führen können. Lediglich durch Anthelmintika wurden deutliche Wirkungen auf Vertreter der Bodenfauna hervorgerufen. Infolge der antibiotischen Wirkung der Pharmazeutika kann eine Resistenzbildung bei Bodenorganismen ausgelöst werden. Zudem hat die Medikation von Antibiotika die Bildung resistenter Mikroorganismen bereits im behandelten Organismus zur Folge. Durch deren anschließende Ausscheidung gelangen resistente Keime auch direkt in die Böden. Handelt es sich um resistente Pathogene oder kommt es zur Übertragung der Resistenzgene zwischen kommensalen und pathogenen Mikroorganismen, so besteht das erhebliche Risiko einer nicht therapierbaren Infektion von Mensch und Tier. Die Aufnahme selbst mobiler Antibiotika in die Pflanzen ist sehr gering. Dennoch wurden bei einigen Pflanzenarten Wirkungen von Antibiotika auf das Wachstum nachgewiesen. [source] | ||||||||||||||||||||||||||||||||||