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HPLC-PDA, LC-MS/MS methods were developed for simultaneous determination of a group of oxidative stress biomarkers (OSBs); 2dA, 2dC, 2dU, 3NLT, 5HMU and 8OHdG in 10 simulated artificial body fluids. O-phosphoric acid and methanol composed mobile phases A and B for gradient elution in HPLC-PDA using ODS-2 column. Linearity obtained for 1.0x10(-6)-1.0x10(-4)M range. LODs were 1.73x10(-6), 1.19x10(-6), 2.59x10(-6), 1.40x10(-6), 2.21x10(-6) and 4.07x10(-6)M for 2dU, 8OHdG, 2dA, 2dC, 5HMU and 3NLT, respectively. LOQs were 5.29x10(-6), 4.02x10(-6), 6.82x10(-6), 4.02x10(-6), 6.82x10(-6) and 9.92x10(-6)M. About 10 mM aqueous ammonium acetate solution and methanol containing 0.1% (v/v) formic acid composed mobile phases A and B for gradient elution in LC-MS/MS. Linearity obtained for 1.0x10(-8)-1.0x10(-6)M range. LODs were 2.88x10(-10), 1.01x10(-8), 3.38x10(-9), 1.36x10(-7), 1.81x10(-7) and 1.40x10(-8)M for 2dU, 8OHdG, 2dA, 2dC, 5HMU and 3NLT, respectively. LOQs were 9.37x10(-10), 3.22x10(-8), 1.91x10(-8), 4.53x10(-7), 5.90x10(-7) and 2.18x10(-8)M. Both methods were validated using ICH Q2(R1) guideline. Specificity, linearity, range, accuracy, precision, reproducibility, LOD, LOQ and recovery were achieved. Chemometric analysis was performed on raw PDA and MS data to check their significance for discrimination of OSBs. Sets of single and triple quadrupole fragmentations were evaluated for principle component analysis. Chosen number of PCs successfully distinguished OSBs of interest., Ege University Department of Scientific Research Projects (BAP) [2014-TIP086, 16/ECZ/022], This study was supported by Ege University Department of Scientific Research Projects (BAP, Project numbers: 2014-TIP086 and 16/ECZ/022).

A UPLC-MS/MS assay, employing a reversed-phase separation, has been applied to the analysis of a number of common amino acids and biogenic amines in rat urine. Analytes were derivatised, using 6‑aminoquinolyl‑N‑hydroxysuccinimidyl carbamate (AccQTag Ultra™). Derivatisation with this reagent, by increasing the hydrophobicity of the analytes, enables better retention by improving reversed-phase chromatographic properties and also improves ionisation efficiency to enhance MS-detection. The method allows for the determination of 38 amino compounds in 7.5 min, including baseline resolution of critical isomers. The assay has been validated for the absolute quantification of 29 amino compounds in rat urine, over a concentration range of 0.6–200 μM, for the purpose of exploratory metabolite phenotyping. Acceptable linearity (R2 > 0.995) and intra- and inter-day accuracy (

Being traditionally utilized mainly as appetizers and herbal teas as well as used to ease abdominal pains, colds and gastrointestinal issues, the genusSalviaL. has gained significant consideration owing to its remarkable phytochemicals and industrial importance. The present study aimed to develop and validate an LC–MS/MS method for the qualitative and quantitative investigation of 19 fingerprint phytochemicals in six endemicSalviaspecies. The validation parameters of the developed LC–MS/MS method were repeatability (intermediate precision), recovery (accuracy), limits of detection and quantification, linearity and uncertainty (U% at 95% confidence level (k = 2)). Reversed-phase HPLC separation and mass spectrometry parameters were optimized for each analyte. Ethanol extracts of the studiedSalviaspecies collected in three consecutive years were screened for their fingerprint phytochemicals by using the developed and validated LC–MS/MS method. Moreover, studiedSalviaspecies were subjected to multivariate analysis such as principal component analysis techniques to demonstrate the variabilities in phytochemical contents by years and parts of the samples. Roots, flowers, leaves, branches and whole plant of theSalviaspecies collected in 2015, 2016 and 2017 were used for the analyses. It was observed that the roots and branches ofSalviaspecies were similar in terms of their salvianolic acid A, caffeic acid, and 6,7-dehydroroyleanone components. Accordingly, apigenin, rosmarinic acid, luteolin 7-O-glucoside, caffeic acid, salvianolic acid B, and 6,7-dehydroroyleanone were notable phytochemicals that were present in the studiedSalviaspecies.

Purpose: To explore factors associated with pentosidine accumulation in the human vitreous.Methods: Vitreous samples were obtained during trans pars plana vitrectomy for macular hole or rhegmatogenous retinal detachment. Patient characteristics included age, gender, and diabetes mellitus. Ocular characteristics included pseudophakia, posterior vitreous detachment, and presence of intraocular fibrosis (epiretinal membrane, proliferative vitreoretinopathy, or both). Pentosidine concentration as a measure of accumulation of advanced glycation end products was determined by high performance liquid chromatography.Results: Pentosidine concentrations were measured in 222 vitrectomy samples (118 female and 104 male patients [median age 66 years], treated for macular hole [n = 105] or rhegmatogenous retinal detachment [n = 117]). Pentosidine was found to accumulate significantly with age (P Conclusion: This study confirmed an age-related pentosidine accumulation in the vitreous and found new factors relating to pentosidine levels. Findings support the hypothesis of enzyme-induced vitreous liquefaction and the hypothesis of pentosidine as a pro-fibrotic factor.

Paracetamol (acetaminophen, APAP) is the most frequently used analgesic drug worldwide. However, patients in several specific populations can have an increased exposure to toxic APAP metabolites. Therefore, APAP-protein adducts have been proposed as an alternative marker for the assessment of APAP intoxications and as an effective tool to study and steer APAP treatment in patients with an increased risk of APAP-induced liver damage. These adducts have been determined in plasma or serum as a matrix. Blood microsampling allows the determination of a variety of analytes, including protein adducts, in a drop of blood, facilitating convenient followup of patients in a home-sampling context, as well as repeated sampling of pediatric patients. We therefore evaluated the use of blood-based volumetric microsamples for the quantification of APAP-protein adducts. Quantitative methods for the determination of APAP-protein adducts in dried blood and dried plasma volumetric absorptive microsamples were developed and validated. Also a preliminary evaluation of pediatric patient dried blood microsamples was conducted. Method validation encompassed the evaluation of selectivity, carry over, calibration model, accuracy and precision, matrix effect, recovery and the effect of the hematocrit on the recovery, dilution integrity, and stability. All pre-set acceptance criteria were met, except for stability. Spiking of blank blood with APAP revealed a concentration-dependent ex vivo formation of APAP-protein adducts, resulting in a response for the measurand APAP-Cys, with an apparent role for the red blood cell fraction. Analysis of authentic samples, following intake of APAP at therapeutic dosing, revealed much higher APAP-Cys concentrations in dried blood vs. dried plasma samples, making interpretation of the results in the context of published intervals difficult. In addition, in contrast to what was observed during method validation, the data obtained for the patient samples showed a high and unacceptable variation. We conclude that, for a combination of reasons, dried blood is not a suitable matrix for the quantification of APAP-protein adducts via the measurement of the APAP-Cys digestion product. The collection of plasma or serum, either in the form of a liquid sample or a dried microsample for this purpose is advised.

Liquid chromatography (LC) hyphenated to mass spectrometry is currently the most widely used means of determining metabolic phenotypes via both untargeted and targeted analysis. At present a range of analytical separations, including reversed-phase, hydrophilic interaction and ion-pair LC are employed to maximise metabolome coverage with ultra (high) performance liquid chromatography (UHPLC) increasingly displacing conventional high performance liquid chromatography because of the need for short analysis times and high peak capacity in such applications. However, it is widely recognized that these methodologies do not entirely solve the problems facing researchers trying to perform comprehensive metabolic phenotyping and in addition to these "routine" approaches there are continuing investigations of alternative separation methods including 2-dimensional/multi column approaches. These involve either new stationary phases or multidimensional combinations of the more conventional materials currently used, as well as application of miniaturization or "new" approaches such as supercritical HP and UHP- chromatographic separations. There is also a considerable amount of interest in the combination of chromatographic and ion mobility separations, with the latter providing both an increase in resolution and the potential to provide additional structural information via the determination of molecular collision cross section data. However, key problems remain to be solved including ensuring quality, comparability across different laboratories and the ever present difficulty of identifying unknowns.

We report on an experimental approach to measure the pressure stability and mechanical strength of monolithic silica capillary columns with different diameters (50 and 100 mu m i.d.) and considering two different domain sizes, typical for the second generation monoliths or smaller. The approach consists of exposing the capillaries to ultra-high pressures (gradually stepwise increased from 20 to 80 MPa), with intermediate measurements of the column efficiency, permeability and retention factors to check the mechanical stability of the bed. It was observed that all tested columns withstood the imposed pressure stress, i.e., all the tested parameters remained unaffected up till the maximal test pressure of 80 MPa. The applied pressure gradient corresponded to 320 MPa/m. The two 100 mu m i.d.-capillary columns were also exposed to pressures between 80 and 90 MPa for a prolonged time (8 h), and this did not cause any damage either. (C) 2016 Elsevier B.V. All rights reserved.

The aim of this study was to compare different analytical methods that are currently in use in the Netherlands for the measurement of whole blood vitamin B6.This method comparison study consisted of two separate parts. (1) Four laboratories participated in a pilot study in which the commercial Chromsystems and INstruchemie method, and a laboratory developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and HPLC method were compared. Sixty-nine frozen whole blood samples and six lyophilized whole blood samples were used for comparison. (2) In the nationwide part of the study, 49 laboratories participated in the analysis of three identical sets of two whole blood samples of which one set was freshly analyzed, one set was analyzed after storage at −20 °C and one set was analyzed after lyophilization.In both parts of the study, the HPLC and LC-MS/MS methods showed equivalent results for all sample types tested. The Chromsystems method showed a positive bias of 45% (pilot study) and 30% (nationwide study) towards the LC-MS/MS method when fresh or frozen samples were used. The measurement of lyophilized samples showed no differences between the methods. The results of the INstruchemie method were inconclusive due to the low number of participants.The different analytical methods for measuring vitamin B6 produce different results when whole blood patient samples are measured. The recognition of a reference method or the development of suitable reference materials and quality control materials might serve as a first step towards improved standardization or harmonization of the whole blood vitamin B6 assay.

International audience; Haemochromatosis is defined as systemic iron overload of genetic origin, caused by a reduction in the concentration of the iron regulatory hormone hepcidin, or a reduction in hepcidin-ferroportin binding. Hepcidin regulates the activity of ferroportin, which is the only identified cellular iron exporter. The most common form of haemochromatosis is due to homozygous mutations (specifically, the C282Y mutation) in HFE, which encodes hereditary haemochromatosis protein. Non-HFE forms of haemochromatosis due to mutations in HAMP, HJV or TFR2 are much rarer. Mutations in SLC40A1 (also known as FPN1; encoding ferroportin) that prevent hepcidin-ferroportin binding also cause haemochromatosis. Cellular iron excess in HFE and non-HFE forms of haemochromatosis is caused by increased concentrations of plasma iron, which can lead to the accumulation of iron in parenchymal cells, particularly hepatocytes, pancreatic cells and cardiomyocytes. Diagnosis is noninvasive and includes clinical examination, assessment of plasma iron parameters, imaging and genetic testing. The mainstay therapy is phlebotomy, although iron chelation can be used in some patients. Hepcidin supplementation might be an innovative future approach.

Background: Given the growing interest in the health benefits of vitamin K, there is great need for development of new high-throughput methods for quantitative determination of vitamin K in plasma. We describe a simple and rapid method for measurement of plasma vitamin K1 (phylloquinone [PK]) and K2 (menaquinones [MK]-4 and -7). Furthermore, we investigated the association of fasting plasma vitamin K with functional vitamin K insufficiency in renal transplant recipients (RTR). Methods: We used HPLC-tandem mass spectrometry with atmospheric pressure chemical ionization for measurement of plasma PK, MK-4, and MK-7. Solid-phase extraction was used for sample clean-up. Mass spectrometric detection was performed in multiple reaction monitoring mode. Functional vitamin K insufficiency was defined as plasma desphospho-uncarboxylated matrix Gla protein (dp-ucMGP) >500 pmol/L. Results: Lower limits of quantitation were 0.14 nmol/L for PK and MK-4 and 4.40 nmol/L for MK-7. Linearity up to 15 nmol/L was excellent. Mean recoveries were >92%. Fasting plasma PK concentration was associated with recent PK intake (ρ=0.41, p=0.002) and with plasma MK-4 (ρ=0.49, p Conclusions: The developed rapid and easy-to-use LC-MS/MS method for quantitative determination of PK, MK-4, and MK-7 in human plasma may be a good alternative for the labor-intensive and time-consuming LC-MS/MS methods and enables a higher sample throughput.

International audience; A composite 30-cm capillary was prepared. The head of the capillary was a 1.5-cm original and miniaturized aptamer-based monolithic affinity support that was in-line coupled to the end of the capillary used for capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection. The device was used for the preconcentration, separation and quantification of ochratoxin A (OTA) as a test solute. The 1.5-cm preconcentration unit consists of a fritless affinity monolithic bonded with 5'-SH-modified oligonucleotide aptamers. A vinyl spacer was used for thiol-ene photoclick chemistry with a 5 min irradiation at 365 nm. Photografting allowed to confine the binding reaction to the desired silica monolithic segment, upstream the empty section of the CE capillary using an UV mask. The photografting procedure was optimized preparing 10-cm capillary monoliths for nano-LC. The retention factors of cationic solutes in ion-exchange nano-LC allowed to follow the aptamer binding on the monolith. The reproducibility of the photografting process was satisfactory with inter-capillary variation lower than 10%. The aptamer bonding density can be increased by successive graftings of 100 mu M aptamer concentration solution (5 pmol/cm/grafting). The optimal conditions to successfully perform the in-line coupling (preconcentration, elution and separation of OTA) with the composite capillary were adjusted depending on individual requirements of each step but also insuring compatibility. Under optimized conditions, OTA was successfully preconcentrated and quantified down to 0.1 pg (percolation of 2.65 mu L of a 40 ng/L OTA solution). A quantitative recovery of OTA (93 +/- 2%) was achieved in a single elution of 30 pg percolated OTA amount. The reproducibility of the overall process was satisfactory with a relative standard deviation lower than 10% with negligible non-specific adsorption. This device was applied for the preconcentration and analysis of OTA in beer and wine at the ppb level within a total analysis time of 30 min.

This study has been aimed at providing a qualitative and quantitative evaluation of selected phytochemicals such as phenolic acids, flavonoids, oleuropein, fatty acids profile, and volatile oil compounds, present in wild olive leaves harvested in Portugal, as well as at determining their antioxidant and cytotoxic potential against human melanoma HTB-140 and WM793, prostate cancer DU-145 and PC-3, hepatocellular carcinoma Hep G2 cell lines, as well as normal human skin fibroblasts BJ and prostate epithelial cells PNT2. Gallic, protocatechuic, p-hydroxybenzoic, vanillic acids, apigenin 7-O-glucoside, luteolin 7-O-glucoside, and rutin were identified in olive leaves. The amount of oleuropein was equal to 22.64 g/kg dry weight. (E)-Anethole (32.35%), fenchone (11.89%), and (Z)-3-nonen-1-ol (8%) were found to be the main constituents of the oil volatile fraction, whereas palmitic, oleic, and alpha-linolenic acid were determined to be dominating fatty acids. Olive leaves methanol extract was observed to exerted a significant, selective cytotoxic effect on DU-145 and PC-3 cell lines. Except the essential oil composition, evaluated wild olive leaves, with regard to their quantitative and qualitative composition, do not substantially differ from the leaves of other cultivars grown for industrial purposes and they reveal considerable antioxidant and cytotoxic properties. Thus, the wild species may prove to be suitable for use in traditional medicine as cancer chemoprevention. Polish Ministry of Science and Higher Education [K/ZDS/004122, K/ZDS/006239, K/ZDS/006228] info:eu-repo/semantics/publishedVersion

Beta2-adrenergic agonists, or β2-agonists, are considered essential bronchodilator drugs in the treatment of bronchial asthma, both as symptom-relievers and, in combination with inhaled corticosteroids, as disease-controllers. The use of β2-agonists is prohibited in sports by the World Anti-Doping Agency (WADA) due to claimed anabolic effects, and also, is prohibited as growth promoters in cattle fattening in the European Union. This paper reviews the last seven-year (2006-2012) literature concerning the development of novel β2-agonists molecules either by modifying the molecule of known β2-agonists or by introducing moieties producing indole-, adamantyl- or phenyl urea derivatives. New emerging β2-agonists molecules for future therapeutic use are also presented, intending to emphasize their potential use for doping purposes or as growth promoters in the near future.

The present study provides experimental evidence for the fact that the peak deconvolution method can be applied to accurately measure the column-only dispersion of the current generation of high speed and high efficiency columns. Unlike the conventional variance difference method, it furthermore preserves any prevailing asymmetry of the column-only peak. This has been demonstrated by testing the same column on three different system configurations, with different extra-column volumes, and showing that, after deconvolution, the resulting column-only peaks coincide very well and produce very similar column-only plate height values (typical relative standard deviation comprising all runs on three different system configurations is 2-2.5%). Extensively studying a large set of theoretically produced peaks (with exactly known variance and asymmetry), it could be shown that the main criterion for the validity of the deconvolution method is that the variance of the system-only peak is minimum 1.5 times smaller than the variance of the column + system peak. The need to add a radial mixer unit to accurately assess the system only contributions has been demonstrated as well. To illustrate its use and merits, the deconvolution method has been used to establish so-called multiple van Deemter curves, wherein plate height curves relating to different peak width definitions are shown in the same plot. These plots can give new insights in the intrinsic asymmetry of the column -only dispersion. (C) 2016 Elsevier B.V. All rights reserved.

Monolithic silica columns have been used as a separation medium because of the advantages which are (1) high column permeability and (2) high column efficiency, due to the characteristic structure possessing relatively large through pores and thin skeletons. For the preparation of monolithic silica, polyethylene glycol (PEG) is dissolved in the feed solution to induce a phase separation via spinodal decomposition, resulting in formation of the co-continuous structure. Regarding development of monolithic silica column, it is still challenging to provide desirable column performance with a column with small domain size, compared to that of a packed column with sub-2 µm particles or fused-core shell ones. Therefore, in this study we examined the effect of PEGs with different molecular weight on resulting monolithic structure and performance of a monolithic silica capillary column. Fabrication of monolithic silica rods was conducted under various preparation conditions using PEGs with different molecular weight. The resulting monolith rods were used to examine the mesoporosity with argon physisorption technique and the macroporosity with mercury intrusion porosimetry. In addition, the chromatographic performance of a monolithic silica capillary column, prepared under the similar preparation conditions for a monolith rod, was characterized after the octadecylsilylation of the silica surface. The results obtained with the monolith rods showed the following important findings: (1) without relation to the difference in molecular weight (MW) of the PEG, it is feasible to fabricate monolith rods possessing macropore size of 0.5‒1.4 µm by tuning the amount of PEGs in the feed solutions, whereas the macropore volume and the mesoporosity remain similar. (2) Considering the preparation conditions producing the smallest domain and macropore size (0.5 µm), the rod prepared with the PEG of MW = 20,000 g/mol provided a narrower macropore size distribution compared to that prepared with PEG of MW = 10,000 g/mol, which is commonly used. The column prepared with PEG of MW = 20,000 g/mol gave a plate height number of H = 4.0 µm for hexylbenzene at an optimal linear velocity of u0 = 2.6 mm/s while maintaining a higher column permeability than that of a packed column with 3 µm silica particles. Comparing the kinetic performance at 30 MPa showed that this monolithic silica column outperforms the second-generation monolithic silica columns developed up till now in the practically most relevant range of applications requiring N  40,000 plates.

Here, we report on 7-nitro-4-(phenylthio)benzofurazan (NBF-SPh), the most potent derivative among a set of patented anticancer 7-nitrobenzofurazans (NBFs), which have been suggested to function by perturbing protein-protein interactions. We demonstrate that NBF-SPh participates in toxic redox-cycling, rapidly generating reactive oxygen species (ROS) in the presence of molecular oxygen, and this is the first report to detail ROS production for any of the anticancer NBFs. Oxygraph studies showed that NBF-SPh consumes molecular oxygen at a substantial rate, rivaling even plumbagin, menadione, and juglone. Biochemical and enzymatic assays identified superoxide and hydrogen peroxide as products of its redox-cycling activity, and the rapid rate of ROS production appears to be sufficient to account for some of the toxicity of NBF-SPh (LC50 = 12.1 mu M), possibly explaining why tumor cells exhibit a sharp threshold for tolerating the compound. In cell cultures, lipid peroxidation was enhanced after treatment with NBF-SPh, as measured by 2-thiobarbituric acid-reactive substances, indicating a significant accumulation of ROS. Thioglycerol rescued cell death and increased survival by 15-fold to 20-fold, but pyruvate and uric acid were ineffective protectants. We also observed that the redox-cycling activity of NBF-SPh became exhausted after an average of approximately 19 cycles per NBF-SPh molecule. Electrochemical and computational analyses suggest that partial reduction of NBF-SPh enhances electrophilicity, which appears to encourage scavenging activity and contribute to electrophilic toxicity.

A simple and rapid semi-automated solid-phase (SPE) extraction method has been developed for the analysis of ochratoxin A in aqueous matrices related to biodegradation experiments (namely digestive contents and faecal excreta), with a view of using this method to follow OTA biodegradation by human intestinal microbiota. Influence of extraction parameters that could affect semi-automated SPE efficiency was studied, using C18-silica as the sorbent and water as the simplest matrix, being further applied to the matrices of interest. Conditions finally retained were as follows: 5-mL aqueous samples (pH 3) containing an organic modifier (20% ACN) were applied on 100-mg cartridges. After drying (9 mL of air), the cartridge was rinsed with 5-mL H2O/ACN (80:20, v/v), before eluting the compounds with 3 x 1 mL of MeOH/THF (10:90, v/v). Acceptable recoveries and limits of quantification could be obtained considering the complexity of the investigated matrices and the low volumes sampled: this method was also suitable for the analysis of ochratoxin B in faecal extracts. Applicability of the method is illustrated by preliminary results of ochratoxin A biodegradation studies by human intestinal microbiota under simple in vitro conditions. Interestingly, partial degradation of ochratoxin A was observed, with efficiencies ranging from 14% to 47% after 72 h incubation. In addition, three phase I metabolites could be identified using high resolution mass spectrometry, namely ochratoxin alpha, open ochratoxin A and ochratoxin B. (C) 2012 Elsevier B.V. All rights reserved.

Microorganisms depend on their ability to modulate their metabolic composition according to specific circumstances, such as different phases of the growth cycle and circadian rhythms, fluctuations in environmental conditions, as well as experimental perturbations. A thorough understanding of these metabolic adaptations requires the ability to comprehensively identify and quantify the metabolome of bacterial cells in different states. In this review, we present an overview of the diverse metabolomics approaches recently adopted to explore the metabolism of a wide variety of microorganisms. Focusing on a selection of illustrative case studies, we assess the different experimental designs used and explore the major achievements and remaining challenges in the field. We conclude by discussing the important complementary information provided by computational methods such as genome-scale metabolic modeling, which enable an integrated analysis of metabolic state changes in the context of overall cellular physiology.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Cranberry proanthocyanidins (PACs) were characterised by HPLC (after thiolysis) and LC-ESI-MS analysis after fractionation by normal-phase chromatography. According to the HPLC retention time and mass spectra of compounds released after depolymerisation reaction, PACs are based on epicatechin and dimer A2 units along with catechin (minor component) and epigallocatechin (trace amounts). Fractionation at semi-preparative scale on normal phase allowed to remove other phenolic compounds than PACs (flavonols, phenolic acids and anthocyanins) and to separate the PACs according to their polymerisation degrees. Six fractions were eluted. PAC concentration, mean degree of polymerisation and percentage of A-type bonds were calculated for these six fractions by HPLC after thiolysis. Whereas the three first fractions contained phenolic acids and flavonols, the three latest fractions were enriched in PACs. Genuine PACs (A and B-types) from DP2 to DP16 were detected by LC-DAD-ESI-MS analysis. Fragmentation on ion trap spectrometer allowed us to determine the position of A-type bonds. Derived PACs were also observed: anthocyanin ethyl-bridged PACs (monomer to tetramer) in the three last fractions, flavonol-ethyl-PACs (monomer and dimer) in the third fraction and pyranoanthocyanin derivatives in the last fraction. (C) 2011 Elsevier Ltd. All rights reserved.

A generic method based on LC with full-scan high-resolution (Orbitrap) mass spectrometry (MS) was systematically investigated for the simultaneous detection of a wide range of plant toxins in a variety of food and feed matrices. For a selection of 150 substances, representing various chemical classes, the limit of detection was established using fixed LC-MS conditions. Ion suppression effects and selectivity were evaluated using generic extracts from representative and relevant matrices (food supplement, honey, silage, compound feed). The majority of the substances could be measured as positive ions after electrospray ionisation (ESI(+)). Using a mass resolving power of 100,000 a reliable high mass accuracy was obtained despite the high abundance of co-extractants in the sample extracts. This enabled the use of ±5¿ppm mass extraction windows, which in turn resulted in a high degree of selectivity. On the other hand, except for honey, strong ion suppression effects were frequently observed which adversely affected the detection limits. Nevertheless, for the majority of the substances the detection limits were in the range 0.01-0.05¿mg¿kg(-1). Since non-selective sample preparation and non-targeted data acquisition were performed, the presence of plant toxins initially not targeted for during data review can be subsequently investigated, which is a very useful option because for many known toxins no analytical reference standards are yet available. The applicability of the method was demonstrated by analysis of a variety of real-life samples purchased on the market or from cases of intoxication. These included honey, herbal tea, food supplements, poppy seeds, traditional Chinese medicines, compound feed, silage and herb-based feed additives. Plant toxins that were detected included various pyrrolizidine alkaloids, grayanotoxins, opium alkaloids, strychnine, ricinine (a marker for ricin), aconitine, aristolochic acid and cardiac glycosides (e.g. digitoxin, digoxin).

Many pathophysiological conditions are associated with increased gastrointestinal permeability, reflecting an elevated risk of endotoxaemia, inflammation, and sepsis. Permeability tests are increasingly used in clinical practice to obtain information on gastrointestinal functioning, but tests are often restricted to the small intestine, and require large oral sugar doses. Therefore, a novel multi-sugar assay was developed, allowing assessment of whole gut permeability changes in urinary and plasma samples collected at regular intervals from 10 healthy volunteers at baseline and after intake of monosaccharides (rhamnose and erythritol) and disaccharides (sucrose, lactulose, and sucralose). Samples were analyzed by isocratic cation-exchange LC-MS. Sample preparation and detection conditions were optimized. After centrifugation, chromatographic separation was achieved on an IOA-1000 column set at 30 degrees C. Column effluent was mixed with ammonia for sugar-ammonium adduct formation. The lower limit of detection was 0.05mumol/L for disaccharides and 0.1mumol/L for monosaccharides. Linearity for each probe was between 1 and 1000mumol/L (R(2): 0.9987-0.9999). Coefficients of variation were

International audience; Food supplements can contain polycyclic aromatic hydrocarbons (PAH). The European Food Safety Authority (EFSA) has defined 16 priority PAH that are both genotoxic and carcinogenic and identified 8 priority PAH (PAH8) or 4 of these (PAH4) as good indicators of the toxicity and occurrence of PAH in food. The current study aims to determine benzo[a]pyrene and other EFSA priority PAH in different categories of food supplements containing botanicals and other ingredients. In 2003 up to 2008, benzo[a]pyrene exceeded the limit of quantification (LOQ) in 553 (44%) of 1258 supplements with a lower bound mean of 3.37 µg kg-1. In 2008 and 2009, benzo[a]pyrene and 12 other EFSA priority PAH were determined in 333 food supplements. Benzo[a]pyrene exceeded the LOQ in 210 (63%) food supplements with a lower bound mean of 5.26 µg kg-1. Lower bound mean levels for PAH4 and PAH8 (-indeno[1,2,3-cd]pyrene) were 33.5 µg kg-1 and 40.5 µg kg-1, respectively. Supplements containing resveratrol, Ginkgo biloba, St. John's wort and propolis showed relatively high PAH4 levels in 2008 and 2009. Before 2008, supplements with these ingredients and also dong quai, green tea or valerian contained relatively high benzo[a]pyrene levels. On average, PAH4 intake resulting from food supplement use will be at the lower end of the range of contributions of main food groups to PAH4 exposure, although individual food supplements can contribute significantly to PAH4 exposure. Regular control of EFSA indicator PAH levels in food supplements may prove a way forward to further reduce the intake of PAH from food.

In the past decades, there has been a renewed interest in the use of natural dye plants for textile dyeing, e.g. Reseda luteola (weld). Its main yellow dye constituents are the flavones luteolin-7,3'-O-diglucoside, luteolin-7-O-glucoside and luteolin. The aim of this work was to develop a simple validated industrially usable quantitative method to assess the flavone content of R. luteola samples. The flavones were overnight extracted from the dried and ground aerial parts of the plant at room temperature via maceration with methanol-water 8:2. Afterwards, they were quantified through internal standardisation against chrysin by RP-HPLC-UV at 345 nm. The efficiency of the one-step extraction was 95%. The limits of detection (LOD) and quantitation (LOQ) were ≤ 1 ng and ≤ 3 ng, respectively, providing ample sensitivity for the purpose. The precision expressed as relative standard deviation of the entire method was

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is rapidly gaining ground in special clinical chemistry laboratories. It significantly increases the analytic potential in clinical chemistry, especially in the field of low molecular weight biomarker analysis. This review overviews current LC-MS/MS methods used for the quantification of biogenic amines and their metabolites. New possibilities offered by this technique are illustrated by recently developed assays for biogenic amines. Major shortcomings of conventional chromatographic techniques, such as labor-intensive sample preparation, long analysis times and often the relatively low specificity, are circumvented by using LC-MS/MS. In addition, LC-MS/MS has broad analyte compatibility and high analytical performance. In the last 5 years introduction of LC-MS/MS in routine diagnostics has resulted in improved assays for diagnosis and follow-up of neuroendocrine tumors characterized by the secretion of biogenic amines. Due to their labile nature and low concentration ranges biogenic amines require extensive and careful sample preparation. Introduction of new sophisticated techniques such as selective sorbents adsorption is evolving. This enables not only more specific analyte selection, but also automation of the complicated clean-up procedure. Automated sample clean-up can be directly coupled to LC-MS/MS, which facilitates reproducible and efficient handling of the growing number of samples to be analyzed in laboratories.. (C) 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Analysis of catecholamines (epinephrine, norepinephrine and dopamine) in plasma and urine is used for diagnosis and treatment of catecholamine-producing tumors Current analytical techniques for catecholamine quantification are laborious, time-consuming and technically demanding Our aim was to develop an automated on-line solid phase extraction method coupled to high performance liquid chromatography-tandem mass spectrometry (XLC-MS/MS) for the quantification of free catecholamines in urine Five microlitie urine equivalent was pre-purified by automated on-line solid phase extraction, using phenylbotonic acid complexation. Reversed phase (pentafluorophenylpropyl column) chromatography was applied Mass spectrometric detection was operated in multiple reaction monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization Urinary reference intervals wet e set in 24-h urine collections of 120 healthy subjects XLC-MS/MS was compared with liquid chromatography with electrochemical detection (HPLC-ECD). Total run-time was 14 min. Intra- and inter-assay analytical variations were 0.99) Quantification limits were 1 47 nmol/L, 15 8 nmol/L and 11 7 nmol/L for epinephrine. norepinephrine and dopamine, respectively XLC-MS/MS correlated well with HPLC-ECD (correlation coefficient >0 98) Reference intervals were 1-10 mu mol/mol, 10-50 mu mol/mol and 60-225 mu mol/mol creatinine for epinephrine, norepinephrine and dopamine, respectively Advantages of the XLC-MS/MS catecholamme method include its high analytical performance by selective PBA affinity and high specificity and sensitivity by unique MS/MS fragmentation (C) 2010 Elsevier B V. All rights reserved.

In this review we will give an overview of the issues related to biomarker discovery studies with a focus on liquid chromatography-mass spectrometry (LC-MS) methods. Biomarker discovery is based on a close collaboration between clinicians, analytical scientists and chemometritians/statisticians. It is critical to define the final purpose of a biomarker or biomarker pattern at the onset of the study and to select case and control samples accordingly. This is followed by designing the experiment, starting with the sampling strategy, sample collection, storage and separation protocols, choice and validation of the quantitative profiling platform followed by data processing, statistical analysis and validation workflows. Biomarker candidates that result after statistical validation should be submitted for further validation and, ideally, be connected to the disease mechanism after their identification. Since most discovery studies work with a relatively small number of samples, it is necessary to assess the specificity and sensitivity of a given biomarker-based assay in a larger set of independent samples, preferably analyzed at another clinical center. Targeted analytical methods of higher throughput than the original discovery method are needed at this point and LC-tandem mass spectrometry is gaining acceptance in this field. Throughout this review, we will focus on possible sources of variance and how they can be assessed and reduced in order to avoid false positives and to reduce the number of false negatives in biomarker discovery research.

This review deals with the measurement of the volatile Sulfur compounds hydrogen sulfide, methanethiol and dimethyl sulfide in various biological matrices of rats and humans (blood, serum, tissues, urine, breath, feces and flatus). Hydrogen sulfide and methanethiol both contain the active thiol (-SH) group and appear in the free gaseous form, in the acid-labile form and in the dithiothreitol-labile form. Dimethyl sulfide is a neutral molecule and exists only in the free form. The foul odor of these sulfur volatiles is a striking characteristic and plays a major role in bad breath, feces and flatus. Because sulfur is a biologically active element, the biological significance of the sulfur volatiles are also highlighted. Despite its highly toxic properties, hydrogen sulfide has been lately recommended to become the third gasotransmitter, next to nitric oxide and carbon monoxide, based on high concentration found in healthy tissues, such as blood and brain. However, there is much doubt about the reliability of the assay methods used. Many artifacts in the sulfide assays exist. The methods to detect the various forms of hydrogen sulfide are critically reviewed and compared with findings of our group. Recent findings that free gaseous hydrogen sulfide is absent in whole blood urged the need to revisit its role as a blood-borne signaling molecule. (C) 2009 Elsevier B.V. All rights reserved.

A thermophilic spore-forming bacterium (strain AMP) was isolated from a thermophilic methanogenic bio- reactor that was fed with cobalt-deprived synthetic medium containing methanol as substrate. 16S rRNA gene analysis revealed that strain AMP was closely related to the aceto- genic bacterium Moorella thermoacetica DSM 521 T (98.3% sequence similarity). DNA-DNA hybridization showed 75.2 § 4.7% similarity to M. thermoacetica DSM 521 T , suggesting that strain AMP is a M. thermoacetica strain. Strain AMP has a unique one-carbon metabolism compared to other Moorella species. In media without cobalt growth of strain AMP on methanol was only sus- tained in coculture with a hydrogen-consuming methano- gen, while in media with cobalt it grew acetogenically in the absence of the methanogen. Addition of thiosulfate led to sulWde formation and less acetate formation. Growth of strain AMP with CO resulted in the formation of hydrogen as the main product, while other CO-utilizing Moorella strains produce acetate as product. Formate supported growth only in the presence of thiosulfate or in coculture with the methanogen. Strain AMP did not grow with H2/ CO 2 , unlike M. thermoacetica (DSM 521 T ). The lack of growth with H2/CO2 likely is due to the absence of cyto- chrome b in strain AMP.

Auteur de correspondance: harry.gruppen@wur.nl; International audience; In order to fully explore the biofunctional potential of proanthocyanidins (PA), isolated and well-characterised PA dimers are of great importance. Current methods to obtain pure A- and B-type dimers are laborious, because they comprise multiple chromatographic steps, often yielding only one or two specific dimers. In the current study, an efficient isolation procedure is described, to isolate a large variety of A-type dimers from peanut skins and B-type dimers from grape seeds. Yields increased 20-400 times for A-type dimers and about 10 times for B-type dimers compared to other methods with a lesser number of chromatographic steps. Dinners isolated from peanut skins were identified as; epicatechin-(2-O-7, 4-8)-catechin (A1), epicatechin-(2-O-7, 4-8)-epicatechin (A2), epicatechin-(2-O-7, 4-6)-catechin, epicatechin-(2-O-7, 4-8)-entcatechin, isolated from peanut skins for the first time, and epicatechin-(4-6)-catechin (B7). Dimers from grape seeds were identified as; epicatechin-(4-8)-catechin (B1), epicatechin-(4-8)-epicatechin (132), catechin-(4-8)-catechin (B3) and catechin-(4-8)-epicatechin (B4)

Podosphaera fusca is the main causal agent of cucurbit powdery mildew in Spain. Four Bacillus subtilis strains, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, with proven ability to suppress the disease on melon in detached leaf and seedling assays, were subjected to further analyses to elucidate the mode of action involved in their biocontrol performance. Cell-free supernatants showed antifungal activities very close to those previously reported for vegetative cells. Identification of three lipopeptide antibiotics, surfactin, fengycin, and iturin A or bacillomycin, in butanolic extracts from cell-free culture filtrates of these B. subtilis strains pointed out that antibiosis could be a major factor involved in their biocontrol ability. The strong inhibitory effect of purified lipopeptide fractions corresponding to bacillomycin, fengycin, and iturin A on P. fusca conidia germination, as well as the in situ detection of these lipopeptides in bacterial-treated melon leaves, provided interesting evidence of their putative involvement in the antagonistic activity. Those results were definitively supported by site-directed mutagenesis analysis, targeted to suppress the biosynthesis of the different lipopeptides. Taken together, our data have allowed us to conclude that the iturin and fengycin families of lipopep-tides have a major role in the antagonism of B. subtilis toward P. fusca.

Concentrations of trans-resveratrol, catechin and epicatechin were analyzed in musts and wines produced from seven red and four white grape cultivars from various wine growing regions of Turkey. Phenolics were quantified using an HPLC method optimized for the separation of wine phenolics. Wine samples contained higher phenolics levels than the corresponding musts. With the exception of Semillion, white wines and musts contained lower concentrations of phenolics than red wines and musts. However, the white cultivar Semillion had the highest concentrations of catechin and epicatechin among all wine and must samples. Semillion wine catechin and epicatechin were 13.7 and 11.8 mg/L, respectively. The highest level of trans-resveratrol among the white cultivars was found in Narince wine (1.93 mg/L). Within the red wine and must cultivars, Bogazkere, Okuzgozu, and Cabernet contained the highest concentrations of flavan-3-ols and trans-resveratrol. Catechin was the major phenolic in all wines and most musts. Epicatechin was the major phenolic in 6 of the 11 must samples, but none of the wine samples. trans-Resveratrol was generally found in lowest concentrations in both wines and musts.

The amphipathic character of soyasaponins and their consequent biological and technological properties are well-recognised. However, mainly due to the absence of purified compounds, no data are available on the amphiphilic surfactant properties of individual soyasaponins. In this study we developed a preparative method for the purification of the main soyasaponins species from soy germ. Reversed-phase chromatography (Source 15 RPC) gave a good resolution of the various soyasaponins, and was used for the purification of non- and fully-acetylated soyasaponin Ab, DDMP-conjugated and unconjugated soyasaponins Ba and Bb. For these compounds, the critical micelle concentration (CMC), the minimal attainable surface tension (γCMC) and the surface density (Γmax) were determined using the Wilhelmy plate method. The order of CMC values was as follows: soyasaponin Bb < Ba < βg < αg < Ab < non-acetylated Ab, and the CMC was found to range from 0.56 and to 3.2 g/L. It was concluded that the number of sugar side chains, the presence of acetyl groups and the presence of a DDMP-group are the main factors influencing the CMC of soyasaponins.

We have investigated which of the different existing peak variance read-out methods (including the effect of a deconvolution pre-treatment method) are most suited to eliminate the system contribution from the total observed band broadening observed in LC systems. Emphasis is put on the most demanding case, i.e., the measurement of non-retained component peaks, which typically are very narrow and tailed. The problem with such peaks is that the method that is generally considered to be the only mathematically correct method (i.e., the method of moments) leads to peak variance values that are so strongly dominated by the tail of the peak that they become highly exaggerated and practically meaningless (i.e., they are dominated by the peak width at 10 or 12 sigma(t), which corresponds to resolutions and peak purities that are so high they are never pursued in practice). Interestingly, filtering away the extra-column contribution from the entire peak shape using peak deconvolution (wherein not only the second ordermoment is corrected but also all other moments) produces corrected 4 sigma(t)- and half height peak widths that are physically meaningful, i.e., the corrected values allow to make sufficiently accurate predictions of how the peak width at 4 sigma(t) and at half height changes when the column length changes. This result now allows to navigate away from the classical method of moments to define the column plate height, and resort to plate heights based on the practically much more relevant 4 sigma(t)- and 5 sigma(t)-widths, provided theses are corrected via peak deconvolution. (C) 2016 Elsevier B.V. All rights reserved.

In this work, a new sulfonated polystyrene based porous layer was synthesized on the wall of a capillary by a single step in situ polymerization process. To obtain a capillary suited for electrochromatography, vinylsulfonic acid (VSA) was, next to divinylbenzene (DVB), copolymerized to induce charges for the electroosmotic flow (EOF) generation. The VSA ratio in the monomer mixture and the polymerization time were optimized while the chromatographic characteristics of the obtained open tubular columns were investigated in electrochromatography. To allow unambiguous study of only chromatographic processes, evaluations were performed with a mixture of sufficiently retained and electrophoretically neutral parabens. Comparison of SEM pictures and chromatograms revealed that the polymerization time had a great influence on the polymer layer morphology and on the chromatographic performance. An increase in the VSA ratio, led to an increase in the mobile phase velocity but simultaneously lowered paraben retention. The novel optimized stationary phase could generate a stable and significant electro-osmotic flow (EOF) of 1.1 mm/s over a wide pH range which could be produced in a reproducible manner. Minimal plate heights of 10 μm, equivalent to the capillary internal diameter, were obtained. The open-tubular character of this optimized porous layer column allowed successful analyses at elevated temperature, resulting in a maximum efficiency of 85,500 plates for a 75 cm capillary and linear velocities up to 1.4 mm/s. Finally, a thermal gradient was successfully applied, leading to artificial sharpened peaks with a peak capacity of 55 in a 20 min time span.

DDMP saponin can be converted to saponin B by the loss of its DDMP group (2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one). The stability of DDMP saponin from pea was investigated under various conditions (temperature, ethanol concentration, pH). DDMP saponin in water was observed to be unstable at acidic and alkaline pHs, and to have an optimal stability around pH 7. In water, DDMP saponin became unstable at temperatures >30 °C. The presence of ethanol, however, had a stabilizing effect on the DDMP group. The loss of the DDMP group at 65 °C could be completely prevented at >30% (v/v) ethanol. The breakdown reaction of DDMP saponin and the subsequent formation of saponin B was modelled using a multi-response modelling approach and was found to be best described by a first-order reaction. The activation energy was estimated to be 49 kJ/mol, indicating a chemical reaction with moderate temperature dependence. A mechanism of DDMP saponin decomposition is proposed, consisting of a fast protonation or deprotonation, followed by a rate-determining step in which maltol is the leaving group.

Virgin females of the minute parasitoid wasp Trichogramma turkestanica produce about 2 pg/h of two putative sex pheromonal compounds. These compounds could be successfully sampled during 20-50 h with SPME from 1.8 mL vials, containing 50-110 wasps and analysed by GC-MS. Accurate mass measurements at the 1 ng scale were possible with an internal standard of sulphur. One compound with MW 236 was identified as a C17H32 hydrocarbon while the other compound was the corresponding allylic alcohol with composition C17H32O. The alcohol could be silylated on-fibre and its mass spectrum suggested the presence of a conjugated 2,4-diene moiety. A miniaturised solvent extraction system for SPME needles, using 5 microL of acetone in a microtube was developed. After reaction of the extracted volatiles with the dienophile 4-methyl-1,2,4-triazoline-3,5-dione (MTAD), GC-MS of the MTAD adducts confirmed the presence of a diene. Interpretation of the combined mass spectral data, in combination with retention indexes of both compounds on non-polar and polar columns, suggested 2,6,8,12-tetramethyltrideca-2,4-diene and 2,6,8,12-tetramethyltrideca-2,4-dien-1-ol as most probable structures. These compounds have not been described previously. Biogenetically, they are most likely polyketides made up of a C4 starter unit that has been elongated with C2 and C3 units. Further biological and synthetic studies are necessary to prove their role as sex pheromone, confirm the proposed structures and determine the correct stereochemistry of the double bonds and the methyl groups.

The aim of this study was to investigate the chemopreventive effects of widely consumed cruciferous vegetables, namely Brussels sprouts and red cabbage towards 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced preneoplastic lesions [liver glutathione-S-transferase placental positive (GST-P(+)) foci and colonic aberrant crypt foci (ACF)]. Male F344 rats were treated with IQ (100 mg/kg bw/g) on 10 alternating days and received drinking water supplemented with Brussels sprouts and red cabbage juices (5% v/v) before and during the carcinogen treatment. From each vegetable two different cultivars were tested. Brussels sprouts reduced the frequency of IQ-induced aberrant foci in both organs (41-52% in the colon and 27-67% in the liver). Also, Brussels sprouts drastically diminished (85-91%) the size of liver GST-P(+) foci, but no such effect was seen in the colon. With red cabbage, the size of liver GST-P(+) foci was markedly reduced (41-83%) whereas the foci frequency was only moderately decreased (19-50%). No protection was seen in the colon after treatment with red cabbage. Cooking (10 min, 100 degrees C) of the vegetables had no influence on their protective effects. The stronger chemoprotective effects of Brussels sprouts may be due to the fact that the overall glucosinolate contents were substantially (2-3-fold) higher than those of the cabbage cultivars, but it was not possible to attribute the reduction of preneoplastic lesions to specific glucosinolates. The activities of hepatic UDP-glucuronosyltransferase form 2 (UDPGT-2) and cytochrome P4501A2 were increased by both vegetables. The induction effect of Brussels sprouts on the activity of UDPGT-2 was more marked than that of the red cabbage cultivars, suggesting that increased glucuronidation of IQ may account for the reduction of the preneoplastic lesions. Our findings support the assumption that Brassica vegetables protect against the carcinogenic effects of heterocyclic amines.

Two edible plants in Southeast Asia, Gynura bicolor and G.divaricata, are not only known to be nutritive but also useful as medicinal herbs. Previous phytochemical investigation of Gynura species showed the presence of hepatotoxic pyrrolizidine alkaloids (PAs), indicating the toxic risk of using these two plants. The present study was designed to analyze the distribution of PA components and tried to evaluate the preliminary toxicity of these two Gynura species. Eight samples of G.bicolor and G.divaricata from five different Chinese locations were collected and their specific PAs were qualitatively characterized by applying an UPLC/MS/MS spectrometry method. Using a pre-column derivatization HPLC method, the total retronecine ester-type PAs in their alkaloids extracts were quantitatively estimated as well. Finally, their genotoxicity was investigated with an effective high-throughput screening method referred to as Vitotox test and their potential cytotoxicity was tested on HepG2 cells. It was found that different types of PAs were widely present in Gynura species collected from south of China. Among them, no significant genotoxic effects were detected with serial concentrations through the present invitro assay. However, the cytotoxicity assay of Gynura plants collected from Jiangsu displayed weak activity at the concentration of 100mg/ml. It is important to note that this research validates in part the indication that the use of Gynura species requires caution.

A mathematical procedure using the Matlab (R) PDE toolbox to calculate the numerical constant appearing in the general Taylor-Aris expression for the dispersion in a laminar flow through open-tubular conduits with a variety of quasi-rectangular cross-sectional shapes is described. The procedure has been applied to assess the effect of some of the most frequently occurring etching imperfections (linear or curved tapering of the inter-pillar distance along the depth coordinate, occurrence of local notches) in etched pillar array columns. In addition, covering a broad range of possible geometries, a number of new shapes and optimal geometries to minimize the dispersion in open-tubular microchannels and pillar array columns have been proposed. Making a full shape-sensitivity study, it was also found that, whereas the proposed designs can theoretically reduce the dispersion up to a factor of 8, relatively small deviations from this ideal shape can however again dramatically increase the dispersion. Designers should therefore be very careful before implementing an optimized shape and should first aim at solving the etching imperfection problems. (C) 2014 Elsevier B.V. All rights reserved.

The study of radial and axial temperature profiles always has been an area interest both in liquid chromatography (LC) and supercritical fluid chromatography (SFC). Whereas in LC always an increase in temperature is observed due to the dominance of viscous heating, in SFC, especially for low modifier content, a decrease in temperature is found due to the much larger decompression cooling. However, for higher modifier content and higher operating pressure, the temperature effects become a trade-off between viscous heating and decompression cooling, since in SFC the latter is a strong function of operating pressure and mobile phase composition. At a temperature of 40 °C and for neat CO2, the effect of decompression cooling and viscous heating cancel each other out at a pressure 450 bar. This pressure decreases almost linearly with volume fraction of methanol to 150 bar at 25 vol%. As a result, both cooling and heating effects can be observed when operating at high back pressure, large column pressure drops or high modifier content. For example at a back pressure of 150 bar and a column pressure drop of 270 bar decompression cooling is observed throughout the column. However at 300 bar back pressure and the same pressure drop, the mobile phase heats up in the first part of the column due to viscous heating and then cools in the second part due to decompression cooling. When coupling columns (2.1 mm × 150 mm, 1.8 μm fully porous particles) at very high operating pressure (e.g. 750 bar for 8 vol%), the situation is even more complex. E.g. at a back pressure of 150 bar and using 8 vol% methanol, viscous heating is only observed in the first column whereas only decompression cooling in the second. Further increasing the inlet pressure up to 1050 bar resulted in no excessive temperature differences along the column. This implies that the inlet pressure of SFC instrumentation could be expanded above 600 bar without additional band broadening caused by excessive radial temperature differences.

We report on a large scale in silica comparison study of so-called chromatographic response functions (CRFs). These are single number descriptors of the separation quality that can be used to guide search-based optimizations for chromatographic separations. A comprehensive set of literature and new CRFs were compared for their ability to guide a search based on first order chromatographic data (i.e., no spectral information available) and for cases where the number of sample compounds is not known beforehand. The results are discussed based on the available separation power. It was found that CRFs increasing monotonically with the number of observed peaks perform significantly better than those that do not possess this property. CRFs based on the discrimination factor or the peak-to-valley ratio can better cope with peak asymmetry than CRFs based on Snyder resolution R. Unfortunately, the former lose their advantage as soon as the noise level becomes significant. Most CRFs perform best when the search is conducted on a column offering just, or, even better, a bit less than the required efficiency to baseline separate the sample. The best results over the entire range of possible efficiencies are obtained with a CRF giving preference to the number of observed compounds before further ranking the conditions based on the achieved separation resolution or the required analysis time. When the search is conducted on columns with an insufficient efficiency, even the best possible CRFs suffer from the incomplete information about the sample, and deviating searches cannot be avoided without resorting to spectral information of the sample. (C) 2014 Elsevier B.V. All rights reserved.

Over the past two years low levels of prednisolone have been reported in bovine urine by a number of laboratories in European Union member states. Concentrations vary, but are reported to be below approximately 3 µg l(-1). Forty per cent of bovine urine samples from the Dutch national control plan had concentrations of prednisolone between 0.11 and 2.04 µg l(-1). In this study the mechanism of formation of prednisolone was investigated. In vitro conversion of cortisol by bacteria from faeces and soil, bovine liver enzymes and stability at elevated temperatures were studied. In vitro bovine liver S9 incubation experiments showed a significant 20% decrease of cortisol within 6 h, and formation of prednisolone was observed from 0.2 g l(-1) at t = 0 to 0.5 g l(-1) at t = 6. Under the influence of faeces, the stability of cortisol in urine is reduced and cortisol breaks down within 50 h. Prednisolone is formed up to 4 µg l(-1) at 70°C after 15 h. However, this decreases again to zero after 50 h. With soil bacteria, a slower decrease of cortisol was observed, but slightly higher overall formation of prednisolone, up to 7 µg l(-1) at 20°C. As opposed to incurred urine, in fortified urine incubated with faeces or soil bacteria no prednisolone was detected. This difference may be explained by the presence of natural corticosteroids in the incurred sample. With UPLC-QToF-MS experiments, in urine and water samples incubated with faeces, metabolites known from the literature could be (tentatively) identified as 20β-hydroxy-prednisolone, cortisol-21-sulfate, oxydianiline, tetrahydrocortisone-3-glucuronide and cortexolone, but for all compounds except 20β-hydroxy-prednisolone no standards were available for confirmation. Based on the results of this study and literature data, for regulatory purposes a threshold of 5 µg l(-1) for prednisolone in bovine urine is proposed. Findings of prednisolone in concentrations up to 5 µg l(-1) in bovine urine can, most likely, originate from other sources than illegal treatment with growth promoters.

The coupling of high performance liquid chromatography with nuclear magnetic resonance spectroscopy (LC-NMR) is one of the most powerful methods for the separation and structural elucidation of unknown compounds in mixtures. The recent progress in pulse field gradients and solvent suppression, the improvement in probe technology, and the construction of high held magnets have given a new stimulus to this technique, which has emerged since the mid 1990s as a very efficient method for the on-line identification of organic molecules. LC-NMR thus represents a potentially interesting complementary technique to LC-UV-MS in phytochemical analysis for the detailed on-line structural analysis of natural products. Recent applications have fully demonstrated the usefulness of this technique. A brief review of the applications of LC-NMR in natural product chemistry is presented in this paper, and a summary of the basic principles and modes of operation of LC-NMR is provided. Selected examples of LC-NMR analyses of plant metabolites in crude extracts or in enriched fractions are outlined and used to illustrate the different strategies for employing the technique. The practical possibilities and limitations of LC-MMR in its application to the analysis of crude plant extracts are discussed by means of several examples. Analytical strategies involving LC multi-coupled (hyphenated) techniques for the chemical screening and dereplication of crude plant extracts are presented. An analysis of the future development of the technique with respect to its application in photochemical analysis is also given. Copyright (C) 2001 John Wiley & Sons, Ltd.

The capillary electrochromatographic (CEC) analysis of basic compounds on octadecyl-silica stationary phases (Hypersil ODS and Spherisorb ODS I) was studied. A basic drug (fluvoxamine) and one of its possible impurities were used as test compounds. With an eluent of acetonitrile-phosphate buffer (pH 7.0), the compounds could be baseline-separated; however, broad and tailing peaks were obtained. To minimise detrimental interactions with residual silanol groups, the pH of the mobile phase was lowered to 2.5, but the plate numbers were still quite low (

In this study, we analyzed 22 strawberry and soil samples after their collection over the course of 2 years to compare the residue profiles from organic farming to integrated pest management practices in Portugal. For sample preparation, we used the citrate-buffered version of the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method. We applied three different methods for analysis: (i) 27 pesticides were targeted using LC-MS/MS; (ii) 143 were targeted using low pressure GC - tandem mass spectrometry (LP-GC-MS/MS); and (iii) More than > 600 pesticides were screened in a targeted and untargeted approach using comprehensive, two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOF-MS). Comparison was made of the analyses using the different methods for the shared samples. The results were similar, thereby providing satisfactory confirmation of both similarly positive and negative findings. No pesticides were found in the organic-farmed samples. In samples from integrated pest management practices, 9 pesticides were determined and confirmed to be present ranging from 2 µg kg-1 for fluazifop-p-butyl to 50 µg kg-1 for fenpropathrin. Concentrations of residues in strawberries were less than European maximum residue limits.

The development of a novel methodology for extraction and preconcentration of the most commonly used anionic surface active agents (SAAs), linear alkylbenzene sulfonates (LAS), is presented herein. The present method, based on the use of silica-magnetite nanoparticles modified with cationic surfactant aggregates, was developed for determination of C-10-C-13 LAS homologues. The proposed methodology allowed quantitative recoveries of C-10-C-13 LAS homologues by using a reduced amount of magnetic nanoparticles. Limits of detection were in the range 0.8-1.9 mu g L-1 for C-10-C-13 LAS homologues, while the repeatability, expressed as relative standard deviation (RSD), ranged from 2.0 to 3.9% (N = 6). Finally, the proposed method was successfully applied to the analysis of a variety of natural water samples. (C) 2013 Elsevier B.V. All rights reserved.

Phloroglucinol is an ubiquitous secondary metabolite encountered in a free state or polymerised as phlorotannins in brown macroalgae, and present in higher plants. FRAP and TEAC assays measured the antioxidant properties of phloroglucinol in non-biological conditions. Additionally, the biological effects of phloroglucinol (4-400 μM) were scrutinised using cellular oxidative stress markers, such as the generation of ROS, antioxidant defences (concentration of GSH and activities of GPx, GR and GST), and levels of MDA as a marker for lipid peroxidation. The direct effect was assessed immediately after an incubation period, whereas for the protective effect, the incubation period was followed by 3-h treatment with the pro-oxidant t-BOOH. The results indicated that despite having a higher radical scavenging capacity than Trolox after 30. min, phloroglucinol was not a suitable antioxidant standard for phlorotannins. Regarding the biological effects, phloroglucinol had no impact on cell viability, reduced levels of ROS and increased antioxidant defences in the direct treatment for most concentrations. The results of the protective effect were mitigated as phloroglucinol failed to protect from ROS generation but evoked a significant recovery of the stress-altered cellular antioxidant defences to restful conditions. Additionally, MDA levels were greatly reduced, preventing a radical chain oxidation., This work was supported by the Grant AGL2007-64042/ALI and Project CSD 2007-00063 from Programa Consolider-Ingenio from the Spanish Ministry of Science and Innovation (CICYT), an EU Erasmus Grant under the Lifelong Learning Programme and a Mobility/Training Grant from the Sea Change Networking Initiative of the Marine Institute, Ireland.

International audience; The commercial C18 columns packed with sub-3 μm shell particles were tested and compared to a reference UHPLC column, in terms of kinetic performance as well as selectivity, retention capability, peak shape and loading capacity. For this purpose, a set of pharmaceutically relevant molecules was selected, including acidic, neutral and basic drugs. Regarding kinetic performance, h(opt) values for the shell particles were found between 1.7 and 2, while the UHPLC column provided a value of approximately 2.5. However, this impressive performance should be considered with caution, particularly for the construction of kinetic plots since h(opt) values were sometimes related to the column dimensions, depending on the provider (h(opt) comprised between 1.8 and 2.6 for longer columns of 150 mm packed with shell particles). Despite the non-porous inner core of the shell particles representing between 25 and 36% of the particle, we demonstrated that the decrease in retention was on the maximum equal to 15% for Ascentis column while Acquity and Poroshell were strictly equivalent in terms of retention. Concerning loading capacity, it remains comparable to that of fully porous sub-2 μm particles and always more pronounced with 0.1% formic acid vs. phosphate buffer. The loading capacity of the different columns was found to be better correlated to the pore volume or surface coverage than the shell thickness. Experimentally, the most pronounced overloading was observed with the Poroshell. Finally, the selectivity and peak shape were evaluated using a mixture of basic and acidic drugs. It appears that results were very similar between sub-3 μm shell particles and fully porous sub-2-μm particles for our mixture of compounds, showing the ability to transfer existing methods to shell particles, with only limited adjustments. This study confirms the potential of columns packed with shell particles and demonstrates the interest of such column technology with pharmaceutical compounds.

Oxidative stress is of importance in the pathophysiology of sickle cell disease (SCD). In this open label randomized pilot study the effects of oral N-acetylcysteine (NAC) on phosphatidylserine (PS) expression as marker of cellular oxidative damage (primary end point), and markers of hemolysis, coagulation and endothelial activation and NAC tolerability (secondary end points) were studied. Eleven consecutive patients (ten homozygous [HbSS] sickle cell patients, one HbS beta(0)-thalassemia patient) were randomly assigned to treatment with either 1,200 or 2,400 mg NAC daily during 6 weeks. The data indicate an increment in whole blood glutathione levels and a decrease in erythrocyte outer membrane phosphatidylserine exposure, plasma levels of advanced glycation end-products (AGEs) and cell-free hemoglobin after 6 weeks of NAC treatment in both dose groups. One patient did not tolerate the 2,400 mg dose and continued with the 1,200 mg dose. During the study period, none of the patients experienced painful crises or other significant SCD or NAC related complications. These data indicate that N-acetylcysteine treatment of sickle cell patients may reduce SCD related oxidative stress.

In such a complicated field as proteomic analysis, scientists are more and more challenged in implementing separation systems capable to provide enhanced separation power, as well as sensitivity of detection for adequate identification and, to a lesser extent, quantification of the separated compounds. To address such issues, several combinations of different separation modes have been investigated in comprehensive liquid chromatographic platforms, in which the entire sample eluted from the first dimension is subjected to a secondary chromatographic separation. The different applications exploited for comprehensive LC analysis of intact or digested proteins are the focus of this review, in which advantages and disadvantages of the different columns combinations, interfaces, and operating modes are pointed out. The combination with mass spectrometry as part of the total system is stressed, and illustrated in more detail. Theoretical concerns and practical requirements will be briefly discussed, as well.