Mirela B. Coelho

The Multicomponent Hantzsch Reaction: Comprehensive Mass Spectrometry Monitoring Using Charge‐Tagged Reagents

A novel strategy for the ESI-MS monitoring of reaction solutions involving the alternate use of permanently charge-tagged reagents has been used for comprehensive mass spectrometry monitoring of the multicomponent Hantzsch 1,4-dihydropyridine reaction. By placing a charge tag on either, or both, of the two key reactants, ion suppression effects for ESI were eliminated or minimized, and comprehensive detection of charge-tagged intermediates was achieved. The strategy allowed the trapping and characterization of the important intermediates in the mechanism for 1,4-dihydropyridine formation.

Purification of Legumin-Like Proteins from Coffea arabica and Coffea racemosa Seeds and Their Insecticidal Properties toward Cowpea Weevil (Callosobruchus maculatus) (Coleoptera: Bruchidae)

Legumin-like proteins from seeds of Coffea arabica (CaL-1 and CaL-2) and Coffea racemosa (CrL-1 and CrL-2) were characterized and isolated by gel filtration and reverse-phase chromatography. The insecticidal properties of the purified proteins were tested against Callosobruchus maculatus using artificial diets. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses indicated that CaL-1 is composed of two subunits of 33 and 24 kDa, while CaL-2, CrL-1, and CrL-2 were monomeric with a single band of 14 kDa. The LD(50) values were 0.5% (w/w) for CaL-1 and 0.3% (w/w) for CaL-2, CrL-1, and CrL-2. ED(50) at 0.3% was assessed for all protein concentrations. The legumin-like proteins were not digested by midgut homogenates of C. maculatus until 8 h of incubation. CaL-1 and CaL-2 ( C. arabica ) and CrL-1 and CrL-2 ( C. racemosa ) are chitin-binding proteins, and their insecticidal properties toward C. maculatus larvae might be related to their capacity to bind chitin present in the larval gut and their associated low digestibility.

Purification And Characterization Of A Lectin From Annona Coriacea Seeds

A novel lectin, denominated ACLEC, was isolated from Annona coriacea seeds, belonging to the Annonaceae family. The lectin presented one protein band in SDS-PAGE of 14 kDa. Of the sugars tested, Dglucose and D-mannose were the best inhibitors. A search sequence database showed that ACLEC had homology with other plant lectins, belonging to leguminous lectin family.

Blends of soybean biodiesel with petrodiesel: direct quantitation via mass spectrometry

Quantitation and identification of blends of soybean biodiesel with petrodiesel were performed via mass spectrometry using two ionization techniques: electrospray ionization (ESI) and Venturi easy ambient sonic-spray ionization in its liquid mode (VL-EASI). Different soybean biodiesel/petrodiesel blends (from B0 to B100) were diluted and then directly infused and analyzed by both techniques. To investigate adulteration of Bn blends, different soybean oil/biodiesel and soybean oil/petrodiesel blends were analyzed. Analytical curves were obtained in three replicates. The two techniques were shown to provide reasonably accurate quantitation in the B1-B20 range. These techniques were also successfully used to detect contamination or adulteration of Bn blends with vegetable oils. ESI is a widely used and commercially available technique whereas a VL-EASI source can be easily mounted using common laboratory parts requiring no use of high voltages. Both techniques require no pre-separation or derivatization steps and offer, therefore, simple and fast methods for the quantitation of Bn blends. The comprehensive snapshots of the molecular composition also allow quality control and typification of the biodiesel and eventually of the vegetable oils in illegal admixtures.

High precision and selectivity for quantitation of enrofloxacin and ciprofloxacin in five chicken tissues using solid phase extraction and ESI LC-MS/MS for application in monitoring residues

Fluoroquinolones (FQs) are synthetic antimicrobials commonly used in intensive poultry farming to treat chronic respiratory disease, colibacillosis, and fowl cholera. As these drugs are also important in treating infections in humans, their use in poultry has been restricted in some countries to avoid the development of antimicrobial-resistant bacteria. In this work, a novel simple and efficient LC-MS/MS method using solid phase extraction (SPE), ESI ionization and multiple reaction mode (MRM) has been developed and validated to determine the two most common FQ residues: enrofloxacin (ENR) and ciprofloxacin (CIP) in chicken target tissues (muscle, liver, kidney, fat and skin) and in plasma. The method is shown to be sensitive, with limits of quantification (LOQ) between 1 ng g−1 and 5 ng g−1, recoveries 93–115% and correlation coefficients greater than 0.99. This novel methodology allows faster and simpler sample workflow compared to previously reported methodologies, making it ideal for high-throughput screening and confirmation of both ENR and CIP meeting the international regulation levels.

Assessing melatonin and its oxidative metabolites amounts in biological fluid and culture medium by liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS)

The introduction of precise analytical technologies in the biological area is needed to increase the knowledge of the fundamental processes occurring in the animal and human domain. The objective of this study was to develop a highly sensitive and selective method based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) using electrospray ionization for the simultaneous detection and quantification of melatonin (MEL) and its metabolites, 6-hydroxymelatonin (6-HMEL), N2-acetyl-N1-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-metoxykynuramine (AMK). Using this methodology, we have studied the role of melatonin and its metabolites in the mammalian embryo in vitro production system, which is fundamental due to the antioxidant and signaling roles of these compounds. Samples comprised of bovine follicular fluid (FF) and tissue culture medium (TCM 199). The optimized procedure uses liquid–liquid extraction with MS monitoring via selective reaction monitoring (SRM). Low limits of detection/quantification were obtained, 3/10 pg mL−1 for MEL, AFMK and AMK and 30/100 pg mL−1 for 6-HMEL, respectively using deuterated melatonin (MEL-d4) as the internal standard (IS). Validation and correlation coefficients were higher than 0.999 and recoveries were 80–108%. Precision was evaluated as repeatability and intermediate precision with relative standard deviation values <3.55%. The method has been successfully applied to the analysis of pooled FF and TCM 199 samples and results suggest that MEL and its metabolites are involved in oocyte maturation and that their proper quantitation is essential to monitor and study their role in such a process.

In vitro maturation impacts cumulus–oocyte complex metabolism and stress in cattle

The influence of in vitro maturation (IVM) in oocytes is still not totally understood. The aim of this study was to determine the influence of IVM on the metabolism and homeostasis of bovine cumulus-oocyte complexes. In the present study, we demonstrated that IVM leads to accumulation of neutral lipids associated with differential levels of the mono-, di- and triacylglycerols in both cumulus cells and oocytes. We observed that in vitro-matured oocytes exhibited decreased glutathione and reactive oxygen species levels and a lower ATP/ADP ratio when compared to in vivo-matured oocytes, with no significant differences in metabolism and stress-related mRNA or miRNA levels. Moreover, in addition to an increase in lipids in in vitro-matured cumulus cells, fatty acid synthesis and accumulation as well as glycolysis pathway genes were upregulated, whereas those affiliated with the β-oxidation pathway were decreased. Our gene expression data in cumulus cells suggest the disruption of endoplasmic reticulum stress, apoptosis and cellular stress response pathways during IVM. Furthermore, a total of 19 miRNAs were significantly altered by the maturation process in cumulus cells. These results indicate some new negative influences of the in vitro system in cumulus-oocyte complexes, demonstrating the occurrence of functional disruption in lipid metabolism and stress pathways and showing evidences suggesting the occurrence of altered mitochondrial activity and energy metabolism during IVM, with a massive dysregulation of the corresponding transcripts in the surrounding cumulus cells.