Using matrix-assisted ionization method for mass spectral identification of fullerene-dye conjugates

Traditional soft ionization methods are not always suitable for mass spectral analysis of complex compounds. Factors such as laser radiation and heating resulting in fragmentations of sample molecules in the case of matrix-assisted laser desorption/ionization and difficulties in preparing suitable sample solutions in the case of electrospray ionization make it impossible to use these methods in some cases. Matrix-assisted ionization was used to analyze products of chemical synthesis involving pyropheophorbide and fullerene. Mass spectra were acquired using a simple effective modification of the Exactive Orbitrap mass spectrometer electrospray interface. Reliable identification of pyropheophorbide-fullerene dyad ions and its derivatives was carried out. An experimental comparison of a matrix-assisted ionization and an electrospray ionization technique demonstrated the significant advantage in sensitivity to the ions under study (approximately 20 times higher) of the matrix-assisted ionization method in this particular study.

Spray-Drying Microencapsulation of Bioactive Compounds from Lemon Verbena Green Extract

Lippia citriodora has been demonstrated to have a wide variety of phytochemicals which provide benefits to human health acting as antioxidants or anti-obesogenics. In this study, these phytochemicals were recovered using a microwave-assisted technology and applying optimal conditions and microencapsulated using spray drying. In this study, two different carbohydrates, maltodextrin (MD) and inulin (IN), were compared as carriers in the encapsulation procedure. The spray drying process was optimized by using a response surface methodology (RSM) based on a central composite design 22, where air inlet temperature and the sample:encapsulating agent ratio (S:EA) were selected as independent variables. Both designs were analyzed equally to evaluate differences between each carrying agent on polar compounds’ encapsulation (process yield (Y%), encapsulation efficiency (EE%) and recovery of compounds (R%)) during the spray drying. The EE% and R% of each polar compound was monitored by High Performance Liquid Chromatography coupled to Time-of-Flight mass spectrometer by electrospray interface (HPLC-ESI-TOF-MS). The results showed that the use of IN as a carrier increased the powder recovered and the recovery of polar compounds after the spray dry process, whereas MD achieved a higher encapsulation efficiency.

Identification of Suvorexant in Blood Using LC–MS-MS: Important Considerations for Matrix Effects and Quantitative Interferences in Targeted Assays

Suvorexant (Belsomra®) is a novel dual orexin receptor antagonist used for the treatment of insomnia. The prevalence of suvorexant in forensic samples is relatively unknown, which demonstrates the need for robust analytical assays for the detection of this sedative hypnotic in forensic toxicology laboratories. In this study, suvorexant was isolated from whole blood using a simple acidic/neutral liquid–liquid extraction followed by analysis by liquid chromatography tandem mass spectrometry (LC–MS/MS). Matrix effects were evaluated qualitatively and quantitatively using various extraction solvents, proprietary lipid clean-up devices and source conditions. The method was validated in terms of limit of detection, limit of quantitation, precision, bias, calibration model, carryover, matrix effects and drug interferences. Electrospray is a competitive ionization process whereby compounds in the droplet compete for a limited number of charged sites at the surface. As such, it is capacity-limited, and LC–MS-based techniques must be carefully evaluated to ensure that matrix effects or coeluting drugs do not impact quantitative assay performance. In this report, we describe efforts to ameliorate such effects in the absence of an isotopically labeled internal standard. Matrix effects are highly variable and heavily dependent on the physico-chemical properties of the substance. Although there is no universal solution to their resolution, conditions at the electrospray interface can mitigate these issues. Using this approach, the LC–MS/MS assay was fully validated and limits of detection and quantitation of 0.1 and 0.5 ng/mL suvorexant were achieved in blood.

Photooxidation of cyclohexene in the presence of SO₂: SOA yield and chemical composition

Secondary organic aerosol (SOA) formation from a cyclohexene ∕ NOx system with various SO2 concentrations under UV light was investigated to study the effects of cyclic alkenes on the atmospheric environment in polluted urban areas. A clear decrease at first and then an increase in the SOA yield was found with increasing SO2 concentrations. The lowest SOA yield was obtained when the initial SO2 concentration was in the range of 30–40 ppb, while higher SOA yield compared to that without SO2 could not be obtained until the initial SO2 concentration was higher than 85 ppb. The decreasing SOA yield might be due to the fact that the promoting effect of acid-catalysed reactions on SOA formation was less important than the inhibiting effect of decreasing OH concentration at low initial SO2 concentrations, caused by the competition reactions of OH with SO2 and cyclohexene. The competitive reaction was an important factor for SOA yield and it should not be neglected in photooxidation reactions. The composition of organic compounds in SOA was measured using several complementary techniques including Fourier transform infrared (FTIR) spectroscopy, ion chromatography (IC), and Exactive Plus Orbitrap mass spectrometer equipped with electrospray interface (ESI). We present new evidence that organosulfates were produced from the photooxidation of cyclohexene in the presence of SO2.

Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis

Sulfide was used as an electron donor early in the evolution of photosynthesis, with many extant photosynthetic bacteria still capable of using sulfur compounds such as hydrogen sulfide (H2S) as a photosynthetic electron donor. Although enzymes involved in H2S oxidation have been characterized, mechanisms of regulation of sulfide-dependent photosynthesis have not been elucidated. In this study, we have identified a sulfide-responsive transcriptional repressor, SqrR, that functions as a master regulator of sulfide-dependent gene expression in the purple photosynthetic bacterium Rhodobacter capsulatus. SqrR has three cysteine residues, two of which, C41 and C107, are conserved in SqrR homologs from other bacteria. Analysis with liquid chromatography coupled with an electrospray-interface tandem-mass spectrometer reveals that SqrR forms an intramolecular tetrasulfide bond between C41 and C107 when incubated with the sulfur donor glutathione persulfide. SqrR is oxidized in sulfide-stressed cells, and tetrasulfide–cross-linked SqrR binds more weakly to a target promoter relative to unmodified SqrR. C41S and C107S R. capsulatus SqrRs lack the ability to respond to sulfide, and constitutively repress target gene expression in cells. These results establish that SqrR is a sensor of H2S-derived reactive sulfur species that maintain sulfide homeostasis in this photosynthetic bacterium and reveal the mechanism of sulfide-dependent transcriptional derepression of genes involved in sulfide metabolism.

74 THE BOVINE EMBRYO INFLUENCES THE PROTEOME OF THE OVIDUCTAL FLUID

It has been shown that the equine embryo is able to modulate the proteome of the oviduct, increasing the presence of certain proteins involved in the embryo-maternal communication (Smits et al. 2016 Reprod. Fertil. Dev. doi: 10.1071/RD15481). In cattle, the presence of a single embryo did not affect the transcriptome of the oviduct, whereas multiple embryos induced changes (Maillo et al. 2015 Biol. Reprod. 92, 144). The aim of the present study was to examine the effect of the presence of an embryo on the oviduct fluid proteome. Cross-bred beef heifers were synchronized, and those in standing oestrus were randomly allocated to cyclic, not bred (n = 7), or pregnant, artificially inseminated (n = 11), groups. All heifers were slaughtered on Day 3 after oestrus. The oviducts from each animal were isolated, straightened, and cut, separating ampulla and isthmus. Each portion was flushed with 500 μL of PBS free of protein to confirm the presence of an oocyte/embryo. Recovered unfertilized oocytes (cyclic group) and embryos (pregnant group) were located in the isthmus of the oviduct ipsilateral to the corpus luteum. Flushings from 4 cyclic and 4 pregnant heifers (8-cell embryos) were used for proteomic analysis, including blank controls of PBS. Comparison of ipsilateral ampulla and isthmus in pregnant and cyclic heifers did not reveal any differences. Therefore, samples from ipsilateral oviducts were compared between cyclic and pregnant heifers. Total protein (150 μg) from cyclic and pregnant samples was labelled with different cyanine fluorescent probes and separated according to the isoelectric point using immobilized pH gradient strips (pH 3–10, 17 cm, Protean® IEF cell system, Bio Rad, Hercules, CA, USA). Second dimension was performed in a polyacrylamide gel (12%) in the presence of SDS using a Protean II XL system (Bio Rad). The images were obtained with the Typhoon 9410 scanner and analysed with the Progenesis SameSpots software v. 4.0. The results of image analysis showed 42 different spots between the 2 groups (ANOVA P < 0.01 and fold difference >1.5), the majority (38) of which were more abundant in pregnant heifers. Gels were stained with Coomassie blue to detect the spots. The differential spots were digested with trypsin and analysed with Agilent Ion-Trap XCT Plus mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray interface. Twenty spots were highly abundant in pregnant heifers. From these, 3 different proteins were identified with score >5.0 and % SPI >76.0 by mass spectrometry, corresponding to SERPINA1, serum albumin, and serum transferrin. These proteins are abundant in plasma, suggesting that the embryo may be able to activate pathways to increase the permeability of the vascular endothelium in the oviduct. The same proteins were also identified in the equine oviducal proteome in the presence of an embryo, suggesting a possible conserved mechanism between species. In conclusion, presence of an embryo in the bovine oviduct increases the abundance of some proteins that may be related with its early development. This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL-2012–37510 and AGL-2015–70140-R) and Science Foundation Ireland (13/IA/1983).