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Rationale Domoic acid (DA) is a potent neurotoxin that accumulates in shellfish. Routine testing involves homogenization, extraction and chromatographic analysis, with a run time of up to 30 min. Improving throughput using ambient ionization for direct analysis of DA in tissue would result in significant time savings for regulatory testing labs. Methods We assess the suitability of laser ablation electrospray ionization high‐resolution mass spectrometry (LAESI‐HRMS) for high‐throughput screening or quantitation of DA in a variety of shellfish matrices. The method was first optimized for use with HRMS detection. Challenges such as tissue sub‐sampling, isobaric interferences and method calibration were considered and practical solutions developed. Samples included 189 real shellfish samples previously analyzed by regulatory labs as well as mussel matrix certified reference materials. Results Domoic acid was selectively analyzed directly from shellfish tissue homogenates with a run time of 12 s. The limits of detection were between 0.24 and 1.6 mg DA kg−1 tissue, similar to those of LC/UV methods. The precision was between 27 and 44% relative standard deviation (RSD), making the technique more suited to screening than direct quantitation. LAESI‐MS showed good agreement with LC/UV and LC/MS and was capable of identifying samples above and below 5 mg DA kg−1 wet shellfish tissue, one quarter of the regulatory limit. Conclusions These findings demonstrate the suitability of LAESI‐MS for routine, high‐throughput screening of DA. This approach could result in significant time savings for regulatory labs carrying out shellfish safety testing on thousands of samples annually. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.

International audience; Sphingolipids are emerging as important mediators of cellular and developmental processes in plants, and advances in lipidomics have yielded a wealth of information on the composition of plant sphingolipidomes. Studies using Arabidopsis thaliana showed that the dihydroxy long-chain base (LCB) is desaturated at carbon position 8 (d18:1?8). This raised important questions on the role(s) of sphingosine (d18:1?4) and sphingosine-1-phosphate (d18:1?4-P) in plants, as these LCBs appear to be absent in A.similar to thaliana. Here, we surveyed 21 species from various phylogenetic groups to ascertain the position of desaturation of the d18:1 LCB, in order to gain further insights into the prevalence of d18:1?4 and d18:1?8 in plants. Our results showed that d18:1?8 is common in gymnosperms, whereas d18:1?4 is widespread within nonseed land plants and the Poales, suggesting that d18:1?4 is evolutionarily more ancient than d18:1?8 in Viridiplantae. Additionally, phylogenetic analysis indicated that the sphingolipid ?4-desaturases from Viridiplantae form a monophyletic group, with Angiosperm sequences falling into two distinct clades, the Eudicots and the Poales. We propose that efforts to elucidate the role(s) of d18:1?4 and d18:1?4-P should focus on genetically tractable Viridiplantae species where the d18:1 LCB is desaturated at carbon position 4.

The performances of four different mass spectrometers [triple-quadrupole (TQ), time-of-flight (ToF), quadrupole ToF (Q-ToF) and ion trap (IT)] for the detection of the marine lipophilic toxins pectenotoxin-2 (PTX2) and okadaic acid (OA) were investigated. The spectral data obtained with the different mass spectrometric analyzers were used to propose fragmentation schemes for PTX2 in the positive electrospray mode and for OA in the negative electrospray mode. TQ data were used to obtain product ions, while ToF and Q-ToF-MS produced accurate mass data of the precursor ion and product ions, respectively. IT data provided a better understanding of the fragmentation pathways using MS(n) experiments. With respect to analytical performance, all four mass analyzers showed a good linearity (R(2)0.97) and repeatability (CV20%). Detection limits (LoDs) (S/N = 3) were the lowest on triple-quad MS: 12.2 and 2.9 pg on-column for PTX2 and OA, respectively.

Solid-phase microextraction (SPME) has been directly coupled to an ion-trap mass spectrometer (MS) for the determination of the model compound lidocaine in urine, hereby applying MS/MS [fragmentation of [M + H](+) (m/z 235) to a fragment with m/z 86]. The throughput of samples has been increased using non-equilibrium SPME with polydimethylsiloxane (PDMS) fibers. The effect of temperature on the sorption and the desorption was studied. Elevated temperatures during sorption (65degreesC) and desorption (55degreesC) had a considerable influence on the speed of the extraction. The desorption was carried out with a home-made desorption chamber allowing thermostating. Only 1 min sorption and 1 min desorption were performed, after which IMS detection took place, resulting in a total analysis time of 3 min. Detection limits below 1 ng/mL could be obtained despite yields of only 2.1 and 1.5% for a 100- and a 30-mum PDMS-coated fiber, respectively. Furthermore, the determination of lidocaine in urine had acceptable reproducibilities, i.e., relative standard deviations (RSDs) below 10%. A limit of quantitation (RSD

Ion suppression effects were observed during the determination of clenbuterol in urine with solid-phase extraction/multiple-stage ion-trap mass spectrometry (SPE/MS3), despite the use of atmospheric pressure chemical ionisation. During SPE, a polymeric stationary phase (polydivinylbenzene) was applied. Post-cartridge infusion of analyte to the SPE eluate after the extraction of blank urine was performed to obtain a profile of the suppression. Single and multiple-stage NIS were performed to provide insight in the suppressing compounds. The ion suppression was mainly ascribed to two m/z values, but still no identification of the compounds was achieved from the multiple-stage MS data. No ionisable and non-ionisable complexes and/or precipitation of clenbuterol with matrix compounds were observed. A concentration dependence of the percentage of suppression was observed. Up to 70% of the signal was suppressed upon post-cartridge infusion of 0.22 mug/mL (at 5 muL/min) clenbuterol into the eluate, and this decreased to about 4% at infusion of 22 mug/mL clenbuterol. Molecularly imprinted polymers were used to enhance the selectivity of the extraction. Although matrix components were still present after extraction, no interference of these compounds with the analyte was observed. However, the bleeding of the imprint from the polymer (brombuterol) caused significant ion suppression. Copyright (C) 2002 John Wiley Sons, Ltd.

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.

Capillary electrokinetic separation techniques offer high efficiency and peak capacity, and can be very useful for the analysis of samples containing a large variety of (unknown) compounds. Such samples are frequently met in impurity profiling of drugs (detection of potential impurities in a pharmaceutical substance or product) and in general sample profiling (determination of differences or similarities between samples). In this paper, the potential, merits, and limitations of electrokinetic separation techniques for profiling purposes are evaluated using examples from literature. A distinction is made between impurity profiling, forensic profiling and profiling of natural products, and the application of capillary zone electrophoresis, micellar electrokinetic chromatography, and capillary electrochromatography in these fields is discussed. Attention is devoted to important aspects such as selectivity, resolution enhancement, applicability, detection, and compound confirmation and quantification. The specific properties of the various electrokinetic techniques are discussed and compared with more conventional techniques as liquid chromatography.

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.

Objectives. Liquorice abuse can lead to severe clinical complications, caused by its active compound 18 beta-glycyrrhetinic acid (18 beta GA). 18 beta GA inhibits dehydrogenase activity of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). This enzyme catalyses the interconversion between cortisol and cortisone and normally protects the mineralocorticoid receptor from being activated by cortisol. Diagnosing liquorice abuse can be notoriously difficult. The aim of our study was to develop an accurate and clinically applicable 18 beta GA urinary assay.Design. We developed a urinary 18 beta GA assay based on gas chromatography and mass spectrometry (GCMS) with sufficient sensitivity to detect 18 beta GA at low concentrations. The assay was validated in four volunteers consuming different amounts of liquorice. We applied its use in two patients with hypokalaemic hypertension and suppressed plasma renin activity and serum aldosterone, who were suspected of liquorice abuse.Results. The detection limit for 18 beta GA of the GC assay was 10 mu g L-1, which was lowered to 3 mu g L-1 by subsequent application of MS. In all volunteers, urinary 18 beta GA was detected during liquorice intake. Urinary 18 beta GA remained detectable until 5 days after stopping continued liquorice intake and until at least 51 h after ingestion of a single large amount. Urinary 18 beta GA was demonstrated in both patients, establishing a diagnosis of liquorice abuse. One patient showed changes in urinary cortisol metabolites, consistent with 11 beta HSD inhibition. Changes in cortisol metabolites were less pronounced in the other patient.Conclusion. Liquorice abuse can result in hypokalaemic hypertension with prolonged suppression of plasma renin activity and aldosterone concentration. This is caused by 18 beta GA-mediated inhibition of 11 beta HSD, resulting in activation of the renal mineralocorticoid receptor by cortisol. Urinary 18 beta GA measurement by GCMS is a useful aid in establishing liquorice abuse.

The direct enantiomeric separation of 32 racemic 2-amidotetralins on the commercially available tris-(3,5-dimethylphenylcarbamate) derivative of cellulose, coated on silica gel (Chiralcel OD), is presented. To date, the selection of a column for the chiral separation of a racemic mixture is done empirically. Studying the impact of small changes in the chemical structure of a series of amidotetralins on the separation behavior may help to give an insight in the chiral recognition mechanism. The amidotetralins differed structurally in three of their substituents, which were never directly located on the chiral carbon atom. The enantiomers of 24 out of 32 amidotetralins could be resolved with a resolution >1.5. Hydrogen bonding and pi-pi interactions are supposed to be the major analyte-chiral stationary phase (CSP) interactions. However, the spatial arrangement of the enantiomers may play an important role too. Increasing the bulkiness of the acyl substituent led to an increase in the resolution (R(s)), whereas a more bulky substituent on the aromatic ring resulted in a very low resolution. The introduction of a chlorine atom into the acyl substituent additionally increased the resolving power. (C) 1997 Wiley-Liss, Inc.

In order to obtain milligram amounts of the enantiomers of a series of compounds to be tested for binding to the melatonin binding site, a system for semipreparative enantiomeric separation was set up using tri-acetylcellulose as the chiral stationary phase. Interactions of this class of compounds with tri-acetylcellulose were examined on an analytical scale with a series of 20 compounds. Apparently, both steric and electrostatic interactions determine retention behavior on tri-acetylcellulose. Semipreparative separations were carried out for a subset of seven compounds. The purity of the first eluting enantiomer usually was around 99%, whereas the purity of the second eluting enantiomer was slightly less. The system described is easy to use and has the major advantage that a series of compounds can be separated with one technique. The purities obtained are sufficient for a first screen of their affinity. (C) 1994 Wiley-Liss, Inc.

Direct enantiomeric separations of 17 chiral amidotetralins by means of high performance liquid chromatography were performed on stationary phases composed of tris(3,5-dimethylphenylcarbamate) derivatives of cellulose and amylose, coated on silica gel. The enantiomers of 15 out of 17 amidotetralins were resolved with a resolution of more than 1.5 by at least one of the chiral stationary phases. The stationary phases showed complementary results with regard to the separation of the amidotetralins, that is, pairs that did not separate on the cellulose-type column were well separated on the amylose-type column, and vice versa. There was no significant correlation between the chromatographic properties of the chiral stationary phases. (C) 1993 Wiley-Liss, Inc.