A complementary LC-ESI-MS and MALDI-TOF approach for screening antibacterial proteomic signature of farmed European sea bass mucus.

Antibacterial protection in the mucus is provided by antimicrobial compounds and till now few numbers of AMP and proteins were identified. Herein, mass spectral profiling of fresh mucus from farmed sea bass (Dicentrarchus labrax) using Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometer (MALDI-TOF) and liquid chromatography mass spectrometry is investigated in order to survey the infective/healthy status of the mucus. We identify AMP peptides of 2891.7, 2919.45 and 2286.6 Da molecular weight respectively and characterize Chrysophsins in the mucus of Dicentrarchus labrax. These peptides display broad-spectrum bactericidal activity against Gram-negative (Minimum Inhibitory Concentrations namely MICs < 0.5 µM) and Gram-positive bacteria (MICs < 0.5 µM) including Escherichia coli and Bacillus subtilis. Furthermore, sensitivity to yeast Candida albicans is reported for the first time and shows interesting MICs of less than 2 µM. We also demonstrate that the fish pathogen Aeromonas salmonoicida is sensitive to Chrysophsins (MICs ranging between 5 and 14 µM). Our mucus molecular mass mapping developed approach allows for fast exploration of immune status. Our data provides evidence that Chrysophsins are secreted by immune cells and are released in mucus of non-challenged farmed European sea bass. These results suggest that Chrysophsins, secreted by gills of red sea bream, are an important widespread component of Teleostei defense against disease.

Multi-residue analysis and ultra-trace quantification of 36 priority substances from the European Water Framework Directive by GC-MS and LC-FLD-MS/MS in surface waters.

A multi residue analysis was developed for screening, quantification and confirmation of 36 priority organic compounds included in the 2000/60/EC European Water Framework Directive. The compounds analyzed included 19 pesticides, 8 PAH, 5 endocrine-disruptors and 4 organochlorine compounds. The method was developed in three steps. First, automated off-line solid-phase extraction using Strata X cartridges was optimized to trap simultaneously the 36 studied compounds. Second, the more volatile compounds were analysed by gas chromatography coupled to mass spectrometry with electron impact ionisation in selected ion monitoring mode (SIM). Third, the last 20 compounds were detected and quantified, in one run, by liquid chromatography coupled to fluorescence detector and tandem mass spectrometry. The excellent selectivity and sensitivity allowed us satisfactory quantification and confirmation at levels as low as 0.2-67 ng L(-1) with recoveries between 59 and 105%. Such methodology was then applied to French surface waters: all the waters present organic contaminants, and their concentration varied according to the origin and nature of substances.

Analysis of neem oils by LC-MS and degradation kinetics of azadirachtin-A in a controlled environment. Characterization of degradation products by HPLC-MS-MS.

Since it was first isolated, the oil extracted from seeds of neem (Azadirachtin indica A juss) has been extensively studied in terms of its efficacy as an insecticide. Several industrial formulations are produced as emulsifiable solutions containing a stated titer of the active ingredient azadirachtin-A (AZ-A). The work reported here is the characterization of a formulation of this insecticide marketed under the name of Neem-azal T/S and kinetic studies of the major active ingredient of this formulation. We initially performed liquid-liquid extraction to isolate the neem oil from other ingredients in the commercial mixture. This was followed by a purification using flash chromatography and semi-preparative chromatography, leading to (13)C NMR identification of structures such as azadirachtin-A, azadirachtin-B, and azadirachtin-H. The neem extract was also characterized by HPLC-MS using two ionization sources, APCI (atmospheric pressure chemical ionization) and ESI (electrospray ionization) in positive and negative ion modes of detection. This led to the identification of other compounds present in the extract-azadirachtin-D, azadirachtin-I, deacetylnimbin, deacetylsalannin, nimbin, and salannin. The comparative study of data gathered by use of the two ionization sources is discussed and shows that the ESI source enables the largest number of structures to be identified. In a second part, kinetic changes in the main product (AZ-A) were studied under precise conditions of pH (2, 4, 6, and 8), temperature (40 to 70 degrees C), and light (UV, dark room and in daylight). This enabled us to determine the degradation kinetics of the product (AZ-A) over time. The activation energy of the molecule (75+/-9 kJ mol(-1)) was determined by examining thermal stability in the range 40 to 70 degrees C. The degradation products of this compound were identified by use of HPLC-MS and HPLC-MS-MS. The results enabled proposal of a chemical degradation reaction route for AZ-A under different conditions of pH and temperature. The data show that at room temperature and pH between 4 and 5 the product degrades into two preferential forms that are hydrolyzed to a single product over time and as a function of pH change.

Analysis of pesticide residues in essential oils of citrus fruit by GC-MS and HPLC-MS after solid-phase extraction.

A robust reliable method for the analysis of residues of pesticides in citrus groves was developed. Residues of twelve pesticides were extracted from citrus essential oils by SPE, separated by liquid chromatography and analyzed by GC-MS. In addition, ten pesticides were extracted by SPE, separated and analyzed by electrospray HPLC-MS. In the case of lemon essential oils, all twenty residues were separated by liquid/solid extraction on a mixed Florisil-C(18) cartridge. The method enabled the analysis of the twenty pesticide residues at levels of 2 to 30 ppm with limits of detection ranging between 0.02 to 0.50 mg L(-1).

Determination of residual pesticides in olive oil by GC-MS and HPLC-MS after extraction by size-exclusion chromatography.

This work describes the development of a method for analyzing pesticide residues in olive oil by GC-MS and HPLC-MS. Pesticides were separated from the oily matrix by size-exclusion chromatography. After extraction, 20 pesticides were separated and analyzed by GC-MS and 11 others HPLC-MS in electrospray mode. The development of this method enabled us to identify and quantify the pesticides of interest.