Methods for detection of anatoxin-a(s) by liquid chromatography coupled to electrospray ionization-tandem mass spectrometry

Anatoxin-a(s) is a potent irreversible inhibitor of the enzyme acetylcholinesterase with a unique N-hydroxyguanidine methylphosphate ester chemical structure. Determination of this toxin in environmental samples is hampered by the lack of specific methods for its detection. Using the toxic strain of Anabaena lemmermani PH-160 B as positive control, the fragmentation characteristics of anatoxin-a(s) under collision-induced dissociation conditions have been investigated and new LC-MS/MS methods proposed. Recommended ion transitions for correct detection of this toxin are 253 > 58, 253 > 159, 235 > 98 and 235 > 96. Chromatographic separation is better achieved under HILIC conditions employing a ZIC-HILIC column. This method was used to confirm for the first time the production of anatoxin-a(s) by strains of Anabaena oumiana ITEP-025 and ITEP-026. Considering no standard solutions are commercially available, our results will be of significant use for the correct identification of this toxin by LC-MS/MS. (C) 2009 Elsevier Ltd. All rights reserved.

Rapid high-performance liquid chromatographic determination with fluorescence detection of furosemide in human body fluids and its confirmation by gas chromatography-mass spectrometry.

Furosemide (FD: Lasix) is a loop diuretic which strongly increases both urine flow and electrolyte urinary excretion. Healthy volunteers were administered 40 mg orally (dissolved in water) and concentrations of FD were determined in serum and urine for up to 6 h for eight subjects, who absorbed water at a rate of 400 ml/h. Quantification was performed by HPLC with fluorescence detection (excitation at 233 nm, emission at 389 nm) with a limit of detection of 5 ng/ml for a 300-microliters sample. The elution of FD was completed within 4 min using a gradient of acetonitrile concentration rising from 30 to 50% in 0.08 M phosphoric acid. The delay to the peak serum concentration ranged from 60 to 120 min. FD was still easily measurable in the sera from all subjects 6 h after administration. In urine, the excretion rates reached their maximum between 1 and 3 h. The total amount of FD excreted in the urine averaged 11.2 mg (range 7.6-14.0 mg), with a mean urine volume of 3024 ml (range 2620-3596 ml). Moreover, the urine density was lower than 1.010 (recommended as an upper limit in doping analysis to screen diuretics) only for 2 h. An additional volunteer was administered 40 mg of FD and his urine was collected over a longer period. FD was still detectable 48 h after intake. Gas chromatography-mass spectrometry with different types of ionization was used to confirm the occurrence of FD after permethylation of the extract. Negative-ion chemical ionization, with ammonia as reactant gas, was found to be the most sensitive method of detection.

Carbon isotopic ratio analysis by gas chromatography/combustion/isotope ratio mass spectrometry for the detection of gamma-hydroxybutyric acid (GHB) administration to humans.

Since GHB (gamma-hydroxybutyric acid) is naturally produced in the human body, clinical and forensic toxicologists must be able to discriminate between endogenous levels and a concentration resulting from exposure. To suggest an alternative to the use of interpretative concentration cut-offs, the detection of exogenous GHB in urine specimens was investigated by means of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). GHB was isolated from urinary matrix by successive purification on Oasis MCX and Bond Elute SAX solid-phase extraction (SPE) cartridges prior to high-performance liquid chromatography (HPLC) fractioning using an Atlantis dC18 column eluted with a mixture of formic acid and methanol. Subsequent intramolecular esterification of GHB leading to the formation of gamma-butyrolactone (GBL) was carried out to avoid introduction of additional carbon atoms for carbon isotopic ratio analysis. A precision of 0.3 per thousand was determined using this IRMS method for samples at GHB concentrations of 10 mg/L. The (13)C/(12)C ratios of GHB in samples of subjects exposed to the drug ranged from -32.1 to -42.1 per thousand, whereas the results obtained for samples containing GHB of endogenous origin at concentration levels less than 10 mg/L were in the range -23.5 to -27.0 per thousand. Therefore, these preliminary results show that a possible discrimination between endogenous and exogenous GHB can be made using carbon isotopic ratio analyses.

Detection of Onchocerca volvulus (Nematoda: Onchocercidae) infection in vectors from Amazonian Brazil following mass Mectizan™ distribution

Detection of Onchocerca volvulus in Simulium populations is of primary importance in the assessment of the effectiveness of onchocerciasis control programs. In Brazil, the main focus of onchocerciasis is in the Amazon region, in a Yanomami reserve. The main onchocerciasis control strategy in Brazil is the semi-annually mass distribution of the microfilaricide ivermectin. In accordance with the control strategy for the disease, polymerase chain reaction (PCR) was applied in pools of simuliids from the area to detect the helminth infection in the vectors, as recommended by the Onchocerciasis Elimination Program for the Americas and the World Health Organization. Systematic sampling was performed monthly from September 1998 to October 1999, and a total of 4942 blackflies were collected from two sites (2576 from Balawaú and 2366 from Toototobi). The molecular methodology was found to be highly sensitive and specific for the detection of infected and/or infective blackflies in pools of 50 blackflies. The results from the material collected under field conditions showed that after the sixth cycle of distribution of ivermectin, the prevalence of infected blackflies with O. volvulus had decreased from 8.6 to 0.3% in Balawaú and from 4 to 0.1% in Toototobi.

Seismic detection and characterization of landslides and other mass movements

Seismic methods used in the study of snow avalanches may be employed to detect and characterize landslides and other mass movements, using standard spectrogram/sonogram analysis. For snow avalanches, the spectrogram for a station that is approached by a sliding mass exhibits a triangular time/frequency signature due to an increase over time in the higher-frequency constituents. Recognition of this characteristic footprint in a spectrogram suggests a useful metric for identifying other mass-movement events such as landslides. The 1 June 2005 slide at Laguna Beach, California is examined using data obtained from the Caltech/USGS Regional Seismic Network. This event exhibits the same general spectrogram features observed in studies of Alpine snow avalanches. We propose that these features are due to the systematic relative increase in high-frequency energy transmitted to a seismometer in the path of a mass slide owing to a reduction of distance from the source signal. This phenomenon is related to the path of the waves whose high frequencies are less attenuated as they traverse shorter source-receiver paths. Entrainment of material in the course of the slide may also contribute to the triangular time/frequency signature as a consequence of the increase in the energy involved in the process; in this case the contribution would be a source effect. By applying this commonly observed characteristic to routine monitoring algorithms, along with custom adjustments for local site effects, we seek to contribute to the improvement in automatic detection and monitoring methods of landslides and other mass movements.

Detection of metabolite induction in fungal co-cultures on solid media by high-throughput differential ultra-high pressure liquid chromatography-time-of-flight mass spectrometry fingerprinting.

Access to new biological sources is a key element of natural product research. A particularly large number of biologically active molecules have been found to originate from microorganisms. Very recently, the use of fungal co-culture to activate the silent genes involved in metabolite biosynthesis was found to be a successful method for the induction of new compounds. However, the detection and identification of the induced metabolites in the confrontation zone where fungi interact remain very challenging. To tackle this issue, a high-throughput UHPLC-TOF-MS-based metabolomic approach has been developed for the screening of fungal co-cultures in solid media at the petri dish level. The metabolites that were overexpressed because of fungal interactions were highlighted by comparing the LC-MS data obtained from the co-cultures and their corresponding mono-cultures. This comparison was achieved by subjecting automatically generated peak lists to statistical treatments. This strategy has been applied to more than 600 co-culture experiments that mainly involved fungal strains from the Fusarium genera, although experiments were also completed with a selection of several other filamentous fungi. This strategy was found to provide satisfactory repeatability and was used to detect the biomarkers of fungal induction in a large panel of filamentous fungi. This study demonstrates that co-culture results in consistent induction of potentially new metabolites.

Development and validation of a method for detection and quantification of ochratoxin A in green coffee using liquid chromatography coupled to mass spectrometry

A method using Liquid Chromatography Tanden Mass Spectrometry (LC-MS/MS) with matrix-matched calibration curve was developed and validated for determining ochratoxin A (OTA) in green coffee. Linearity was found between 3.0 and 23.0 ng.g-1. Mean recoveries ranged between 90.45% and 108.81%; the relative standard deviation under repeatability and intermediate precision conditions ranged from 5.39% to 9.94% and from 2.20% to 14.34%, respectively. The limits of detection and quantification were 1.2 ng.g-1 and 3.0 ng.g-¹, respectively. The method developed was suitable and contributed to the field of mycotoxin analysis, and it will be used for future production of the Certified Reference Material (CRM) for OTA in coffee.

Detection and quantification of staphylococcus aureus enterotoxin B in food product using isotopic dilution techniques and mass spectrometry

L’entérotoxine B staphylococcique (SEB) est une toxine entérique hautement résistante à la chaleur et est responsable de plus de 50 % des cas d’intoxication d’origine alimentaire par une entérotoxine. L’objectif principal de ce projet de maîtrise est de développer et valider une méthode basée sur des nouvelles stratégies analytiques permettant la détection et la quantification de SEB dans les matrices alimentaires. Une carte de peptides tryptiques a été produite et 3 peptides tryptiques spécifiques ont été sélectionnés pour servir de peptides témoins à partir des 9 fragments protéolytiques identifiés (couverture de 35 % de la séquence). L’anhydride acétique et la forme deutérée furent utilisés afin de synthétiser des peptides standards marqués avec un isotope léger et lourd. La combinaison de mélanges des deux isotopes à des concentrations molaires différentes fut utilisée afin d’établir la linéarité et les résultats ont démontré que les mesures faites par dilution isotopique combinée au CL-SM/SM respectaient les critères généralement reconnus d’épreuves biologiques avec des valeurs de pente près de 1, des valeurs de R2 supérieure à 0,98 et des coefficients de variation (CV%) inférieurs à 8 %. La précision et l’exactitude de la méthode ont été évaluées à l’aide d’échantillons d’homogénat de viande de poulet dans lesquels SEB a été introduite. SEB a été enrichie à 0,2, 1 et 2 pmol/g. Les résultats analytiques révèlent que la méthode procure une plage d’exactitude de 84,9 à 91,1 %. Dans l’ensemble, les résultats présentés dans ce mémoire démontrent que les méthodes protéomiques peuvent être utilisées efficacement pour détecter et quantifier SEB dans les matrices alimentaires. Mots clés : spectrométrie de masse; marquage isotopique; protéomique quantitative; entérotoxines

Highly accurate detection of ovarian cancer using CA125 but limited improvement with serum matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling

Objectives: Our objective was to test the performance of CA125 in classifying serum samples from a cohort of malignant and benign ovarian cancers and age-matched healthy controls and to assess whether combining information from matrix-assisted laser desorption/ionization (MALDI) time-of-flight profiling could improve diagnostic performance. Materials and Methods: Serum samples from women with ovarian neoplasms and healthy volunteers were subjected to CA125 assay and MALDI time-of-flight mass spectrometry (MS) profiling. Models were built from training data sets using discriminatory MALDI MS peaks in combination with CA125 values and tested their ability to classify blinded test samples. These were compared with models using CA125 threshold levels from 193 patients with ovarian cancer, 290 with benign neoplasm, and 2236 postmenopausal healthy controls. Results: Using a CA125 cutoff of 30 U/mL, an overall sensitivity of 94.8% (96.6% specificity) was obtained when comparing malignancies versus healthy postmenopausal controls, whereas a cutoff of 65 U/mL provided a sensitivity of 83.9% (99.6% specificity). High classification accuracies were obtained for early-stage cancers (93.5% sensitivity). Reasons for high accuracies include recruitment bias, restriction to postmenopausal women, and inclusion of only primary invasive epithelial ovarian cancer cases. The combination of MS profiling information with CA125 did not significantly improve the specificity/accuracy compared with classifications on the basis of CA125 alone. Conclusions: We report unexpectedly good performance of serum CA125 using threshold classification in discriminating healthy controls and women with benign masses from those with invasive ovarian cancer. This highlights the dependence of diagnostic tests on the characteristics of the study population and the crucial need for authors to provide sufficient relevant details to allow comparison. Our study also shows that MS profiling information adds little to diagnostic accuracy. This finding is in contrast with other reports and shows the limitations of serum MS profiling for biomarker discovery and as a diagnostic tool

Characterization of the Salmonella typhimurium proteome by semi-automated two dimensional HPLC-mass spectrometry: detection of proteins implicated in multiple antibiotic resistance

The proteome of Salmonella enterica serovar Typhimurium was characterized by 2-dimensional HPLC mass spectrometry to provide a platform for subsequent proteomic investigations of low level multiple antibiotic resistance (MAR). Bacteria (2.15 +/- 0.23 x 10(10) cfu; mean +/- s.d.) were harvested from liquid culture and proteins differentially fractionated, on the basis of solubility, into preparations representative of the cytosol, cell envelope and outer membrane proteins (OMPs). These preparations were digested by treatment with trypsin and peptides separated into fractions (n = 20) by strong cation exchange chromatography (SCX). Tryptic peptides in each SCX fraction were further separated by reversed-phase chromatography and detected by mass spectrometry. Peptides were assigned to proteins and consensus rank listings compiled using SEQUEST. A total of 816 +/- 11 individual proteins were identified which included 371 +/- 33, 565 +/- 15 and 262 +/- 5 from the cytosolic, cell envelope and OMP preparations, respectively. A significant correlation was observed (r(2) = 0.62 +/- 0.10; P < 0.0001) between consensus rank position for duplicate cell preparations and an average of 74 +/- 5% of proteins were common to both replicates. A total of 34 outer membrane proteins were detected, 20 of these from the OMP preparation. A range of proteins (n = 20) previously associated with the mar locus in E. coli were also found including the key MAR effectors AcrA, TolC and OmpF.

Detection and Characterization of Cholesterol-Oxidized Products Using HPLC Coupled to Dopant Assisted Atmospheric Pressure Photoionization Tandem Mass Spectrometry

Oxidation of cholesterol (Ch) by a variety of reactive oxygen species gives rise mainly to hydroperoxides and aldehydes. Despite the growing interest in Ch-oxidized products, the detection and characterization of these products is still a matter of concern. In this work, the main Ch-oxidized products, namely, 3 beta-hydroxycholest-5-ene-7 alpha-hydroperoxide (7 alpha-OOH), 3 beta-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 alpha-hydroperoxide (6 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 beta-hydroperoxide (6 beta-OOH), and 3 beta-hydroxy-5 beta-hydroxy-B-norcholestane-6 beta-carboxaldehyde (ChAld), were detected in the same analysis using high-performance liquid chromatography (HPLC) coupled to dopant assisted atmospheric pressure photoionization tandem mass spectrometry. The use of selected reaction monitoring mode (SRM) allowed a sensitive detection of each oxidized product, while the enhanced product ion mode (EPI) helped to improve the confidence of the analyses. Isotopic labeling experiments enabled one to elucidate mechanistic features during fragmentation processes. The characteristic fragmentation pattern of Ch-oxidized products is the consecutive loss of 1120 molecules, yielding cationic fragments at m/z 401, 383, and 365. Homolytic scissions of the peroxide bond are also seen. With (18)O-labeling approach, it was possible to establish a fragmentation order for each isomer. The SRM transitions ratio along with EPI and (18)O-labeled experiments give detailed information about differences for water elimination, allowing a proper discrimination between the isomers:Phis is of special interest considering the emerging role of Ch-oxidized products in the development of diseases.

Comparative analysis of the volatile fraction from Annona cherimola Mill. cultivars by solid-phase microextraction and gas chromatography–quadrupole mass spectrometry detection

The analysis of volatile compounds in Funchal, Madeira, Mateus and Perry Vidal cultivars of Annona cherimola Mill. (cherimoya) was carried out by headspace solid-phase microextraction (HS-SPME) combined with gas chromatography–quadrupole mass spectrometry detection (GC–qMSD). HS-SPME technique was optimized in terms of fibre selection, extraction time, extraction temperature and sample amount to reach the best extraction efficiency. The best result was obtained with 2 g of sample, using a divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fibre for 30 min at 30 °C under constant magnetic stirring (800 rpm). After optimization of the extraction methodology, all the cherimoya samples were analysed with the best conditions that allowed to identify about 60 volatile compounds. The major compounds identified in the four cherimoya cultivars were methyl butanoate, butyl butanoate, 3-methylbutyl butanoate, 3-methylbutyl 3-methylbutanoate and 5-hydroxymethyl-2-furfural. These compounds represent 69.08 ± 5.22%, 56.56 ± 15.36%, 56.69 ± 9.28% and 71.82 ± 1.29% of the total volatiles for Funchal, Madeira, Mateus and Perry Vidal cultivars, respectively. This study showed that each cherimoya cultivars have 40 common compounds, corresponding to different chemical families, namely terpenes, esters, alcohols, fatty acids and carbonyl compounds and using PCA, the volatile composition in terms of average peak areas, provided a suitable tool to differentiate among the cherimoya cultivars.

Solid phase microextraction, mass spectrometry and metabolomic approaches for detection of potential urinary cancer biomarkers: a powerful strategy for breast cancer diagnosis

A sensitive assay to identify volatile organic metabolites (VOMs) as biomarkers that can accurately diagnose the onset of breast cancer using non-invasively collected clinical specimens is ideal for early detection. Therefore the aim of this study was to establish the urinary metabolomic profile of breast cancer patients and healthy individuals (control group) and to explore the VOMs as potential biomarkers in breast cancer diagnosis at early stage. Solid-phase microextraction (SPME) using CAR/PDMS sorbent combined with gas chromatography–mass spectrometry was applied to obtain metabolomic information patterns of 26 breast cancer patients and 21 healthy individuals (controls). A total of seventy-nine VOMs, belonging to distinct chemical classes, were detected and identified in control and breast cancer groups. Ketones and sulfur compounds were the chemical classes with highest contribution for both groups. Results showed that excretion values of 6 VOMs among the total of 79 detected were found to be statistically different (p < 0.05). A significant increase in the peak area of (−)-4-carene, 3-heptanone, 1,2,4-trimethylbenzene, 2-methoxythiophene and phenol, in VOMs of cancer patients relatively to controls was observed. Statiscally significant lower abundances of dimethyl disulfide were found in cancer patients. Bioanalytical data were submitted to multivariate statistics [principal component analysis (PCA)], in order to visualize clusters of cases and to detect the VOMs that are able to differentiate cancer patients from healthy individuals. Very good discrimination within breast cancer and control groups was achieved. Nevertheless, a deep study using a larger number of patients must be carried out to confirm the results.

Supercritical fluid extraction for pesticide multiresidue analysis in honey: determination by gas chromatography with electron-capture and mass spectrometry detection

An analytical procedure using supercritical fluid extraction (SFE) and capillary gas chromatography with electron-capture detection was developed to determine simultaneously residues of different pesticides (organochlorine, organophosphorus, organonitrogen and pyrethroid) in honey samples. Fortification experiments were conducted to test conventional extraction (liquid-liquid) and optimize the extraction procedure in SFE by varying the CO2-modifier, temperature, extraction time and pressure. Best efficiency was achieved at 400 bar using acetonitrile as modifier at 90 degreesC. For the clean-up step, Florisil cartridges were used for both methods LLE and SFE. Recoveries for majority of pesticides from fortified samples of honey at fortification level of 0.01-0.10 mg/kg ranged 75-94% from both methods. Limits of detection found were less than 0.01 mg/kg for ECD and confirmation of pesticide identity was performed by gas chromatography-mass spectrometry in selected-ion monitoring mode. The multiresidue methods in real honey samples were applied and the results of developed methods were compared. (C) 2004 Elsevier B.V. All rights reserved.