Accelerated solvent extraction by using an ‘in-line’ clean-up approach for multiresidue analysis of pesticides in organic honey

<p>The worldwide loss of honeybee colonies may be due to their exposure to several contaminants (i.e., pesticides); such contamination may also have impacts on consumers’ health. Therefore, it is essential to develop quick and new methods to detect several pesticide residues in honey samples. In this study, the effectiveness of accelerated solvent extraction (ASE) was compared with QuEChERS methods for the analysis of 53 pesticides in organic honey by gas chromatography-triple quadrupole mass spectrometry. Two simple and rapid ASE methods with ‘in-line’ clean-up were optimised and then compared with QuEChERS. Hexane–ethyl acetate (Hex:EtAc) and Florisil were chosen as extraction solvent and retainer for the first ASE method respectively; acetonitrile and a primary–secondary amine phase (ACN-PSA) were selected for the second ASE method. The methods were validated according to the European Union SANTE/11945/2015 guidelines. The validation parameters showed that QuEChERS and ASE with PSA as retainer had better repeatability than ASE with Hex:EtAc and Florisil. In particular, QuEChERS and ASE (ACN-PSA) showed good recovery, according to the SANTE criteria, for the majority of investigated pesticides. Conversely, when ASE with Hex:EtAc and Florisil was used as the retainer, several compounds showed recoveries lower than the acceptable value of 70%. The ASE in-line method was finally applied to evaluate pesticide concentration in organic honey samples.</p

Endogenous level of acetic acid in yellowfin tuna (<i>Thunnus albacares</i>): a pilot study about a possible controversy on its residue nature

<p>A method based on headspace solid-phase microextraction (HS-SPME) followed by GC-MS analysis was developed for the determination of underivatised acetic acid in fresh tuna fish muscle. Parameters such as the fibre selected and the extraction time and temperature were optimised and the linearity, detection limits and precision of the whole analytical procedure were assessed. The method was then applied to determine the acetic acid concentration in fresh yellowfin tuna muscles (<i>Thunnus albacares</i>) in order to evaluate the endogenous level and its variations during the shelf life under different storage conditions. A qualitative comparison was also made with variations in histamine levels to evaluate the possibility of the joint monitoring of acetic acid and histamine to identify fish stored in poor conditions. The caudal area always had a lower content of acetic acid than the ventral area, independent of the storage time and temperature. A difference was found between the 6- and 3-day time points and day 0 at a storage temperature of 8°C and between the 6-day time point and day 0 at a storage temperature of 0°C, independent of the anatomical area of the sampled tissue. The evaluation of acetic acid could represent an important approach in the field of food safety to detect the illicit use of acetic acid as an antibacterial preservative treatment or to eliminate the unpleasant smell of trimethylamine.</p

“Mean” Relative echogenicity and standard errors of examined foal tendons analyzed in their entirety along four regions of interest in relation to age.

<p>Abbreviations: DDFT = deep digital flexor tendon; SDFT = superficial digital flexor tendon; IM = interosseous muscle; ALDDFT = accessory ligament of deep digital flexor tendon; w = week; m = month. Differing letters indicate significant differences (<i>P</i><0.05).</p

“Mean” Relative echogenicity and standard errors of superficial digital flexor tendon (SDFT) (a), deep digital flexor tendon (DDFT) (b), accessory ligament of deep digital flexor tendon (ALDDFT), and interosseous muscle (IM) in each region of interest in relation to age of foals.

<p>Differing letters indicate significant differences (<i>P</i><0.05). Abbreviations: w = week; m = month.</p

Proteome Speciation by Mass Spectrometry: Characterization of Composite Protein Mixtures in Milk Replacers

The ability of tandem mass spectrometry to determine the primary structure of proteolytic peptides can be exploited to trace back the organisms from which the corresponding proteins were extracted. This information can be important when food products, such as protein powders, can be supplemented with lower-quality starting materials. In order to dissect the origin of proteinaceous material composing a given unknown mixture, a two-step database search strategy for bottom-up nanoscale liquid chromatography–tandem mass spectrometry (nanoLC–MS/MS) data was implemented. A single nanoLC–MS/MS analysis was sufficient not only to determine the qualitative composition of the mixtures under examination, but also to assess the relative percent composition of the various proteomes, if dedicated calibration curves were previously generated. The approach of two-step database search for qualitative analysis and proteome total ion current (pTIC) calculation for quantitative analysis was applied to several binary and ternary mixtures which mimic the composition of milk replacers typically used in calf feeding

Gene Ontology annotation of protein.

<p>To have a general picture of the molecular functions (a), biological processes (b), and protein class (c), of identified proteins, the Protein ANalysis THrough Evolutionary Relationships (PANTHER) Database was used.</p

1d-SDS-PAGE.

<p>Proteins were resolved by Any kD Mini-PROTEAN TGX precast polyacrylamide gels. Gel line was sliced up in 7 pieces; gel bands were destained, reduced, alkylated, and digested with trypsin; peptides were resuspended and analyzed by LC-MS/MS.</p

IPA analysis.

<p>List of signaling networks associated with identified proteins.</p

IPA analysis.

<p>In the figure are shown the top 6 signaling networks: 1) Cellular Movement, Hematological System Development and Function and Immune Cell Trafficking; 2) Cell Morphology, Cellular Movement, Digestive System Development and Function; 3) Free Radical Scavenging, Connective Tissue Disorders and Inflammatory Disease; 4) Cell-To-Cell Signaling and Interaction, Cellular Function and Maintenance, Hematological System Development and Function; 5) Organ Morphology, Skeletal and Muscular System Development and Function, Tissue Morphology; 6) Organ Morphology, Skeletal and Muscular System Development and Function, Cancer.</p

MOESM1 of Ex-vivo characterization of circulating colon cancer cells distinguished in stem and differentiated subset provides useful biomarker for personalized metastatic risk assessment

Additional file 1: Figure S1. Sensibility, specificity and purity of CTCs detection methodology. The sensitivity of the methodology was calculated through the formula employing mean values (expressed in percentage) for each CTCs subsets identified by the combined expression of CK20 and CD45, found in the total cellular suspension collected from the working density phase. The sensitivity or the capability to detect the real subset of CTCs CK20pos corresponded to 91 %. The specificity, corresponding to the probability of a negative test, was calculated at about 87 %. Finally, the purity was at 75 %. Figure S2. Resolution of CTCs detection methodology. To verify that the collected fraction was enriched in cancer cells, HCT 116 cells were infected with pAdenoVator-CMV-IRES-GFP reporter. Human cancer colon cell lines HCT 116 were cultured in RPMI1640 medium containing 10 % fetal bovine serum (FBS), 2 mmol/l L-glutamine, and 30 mg penicillin G/0.05 g streptomycin. Cells were plated at 8 x 106 per well onto a six-well plate 24 hours before infection, and were infected with adenoviral vector. In order to perform infections, HCT 116 cells were incubated with pAdenoVator-CMV5(CuO)-IRES-GFP (Qbiogene, Carlsbad, CA) in serum free medium for 1 hour at 37 °C. Both vectors were used at multiplicity of infection (m.o.i.) of 3000 physical particles/cell, experimentally determined as the lowest m.o.i. at which the majority of the cell population is infected (as assessed by EGFP expression). Twenty-four hours later, both adherent and floating cells were harvested, washed in PBS and counted. Different concentration of HCT 116-GFP (HCT 116*) were put in entire blood sample (5 ml) and were evaluated through cytometric analysis. The resolution for the minimal concentration of HCT 116* (8 x 103 cell/5 ml) put in a volume of peripheral blood sample of 5ml, useful to detect them in the working density phase, was calculated at 5,8 cells/5 ml (B). Figure S3. DTCs in livers of mice treated with localized and advanced cancer eCTCs. Dot Plots report the expression of CK20 antigen on human colon cancer cells disseminated within liver tissue of mouse submitted to xenograft procedure. In particular, dot plot in (A) shows human colon cancer cell CK20 positive founded in liver tissue of mouse injected with eCTCs-CXCR4negCKneg referred as control. Dot plots in (B) and (C) show human cancer colon cells expressing CK20 marker in liver tissues of mouse injected with eCTCs-CXCR4posCKpos derived from localized (B) and advanced (C) colon cancer cases respectively. Figure S4. xenograft developed with circulating stem cells. Xenograft procedure developed injecting eCTCs-CD45negCD133pos organized in spheres (A). In (B) immunofluorescence positive for CD133 (green staining). In (C) Tumour formations produced within 2 weeks and after 80 days. Immunohistochemical analysis shows the distribution of the cancer colon cells expressing CD133 (brown staining) in the tumour sections of 8 μm (D)