Alicyclobacillus spp. in fruit-based products: Isolation, identification, quantitative assessment (SPME/GC-MS) of spoilage compounds and spore's resistance to thermal shocks.

Alicyclobacillus spp. is the cause of great concern for the food industry due to their spores' resistance (thermal and chemical) and the spoilage potential of some species. Despite this, not all Alicyclobacillus strains can spoil fruit juices. Thus, this study aimed to identify Alicyclobacillus spp. strains isolated from fruit-based products produced in Argentina, Brazil, and Italy by DNA sequencing. All Alicyclobacillus isolates were tested for guaiacol production by the peroxidase method. Positive strains for guaiacol production were individually inoculated at concentration of 103 CFU/mL in 10 mL of orange (pH 3.90) and apple (pH 3.50) juices adjusted to 11°Brix, following incubation at 45 °C for at least 5 days to induce the production of the following spoilage compounds: Guaiacol, 2,6-dichlorophenol (2,6-DCP) and 2,6-dibromophenol (2,6-DBP). The techniques of micro-solid phase extraction by headspace (HS-SPME) and gas-chromatography with mass spectrometry (GC-MS) were used to identify and quantify the spoilage compounds. All GC-MS data was analyzed by principal component analysis (PCA). The effects of different thermal shock conditions on the recovery of Alicyclobacillus spores inoculated in orange and apple juice (11°Brix) were also tested. A total of 484 strains were isolated from 48 brands, and the species A. acidocaldarius and A. acidoterrestris were the most found among all samples analyzed. In some samples from Argentina, the species A. vulcanalis and A. mali were also identified. The incidence of these two main species of Alicyclobacillus in this study was mainly in products from pear (n = 108; 22.3 %), peach (n = 99; 20.5 %), apple (n = 86; 17.8 %), and tomato (n = 63; 13 %). The results indicated that from the total isolates from Argentina (n = 414), Brazil (n = 54) and Italy (n = 16) were able to produce guaiacol: 107 (25.8 %), 33 (61.1 %) and 13 (81.2 %) isolates from each country, respectively. The PCA score plot indicated that the Argentina and Brazil isolates correlate with higher production of guaiacol and 2,6-DCP/2,6-DBP, respectively. Heatmaps of cell survival after heat shock demonstrated that strains with different levels of guaiacol production present different resistances according to spoilage ability. None of the Alicyclobacillus isolates survived heat shocks at 120 °C for 3 min. This work provides insights into the incidence, spoilage potential, and thermal shock resistance of Alicyclobacillus strains isolated from fruit-based products.

A general, most basic rule for ion dissociation: Ionized molecules.

Herein we revisit a basic rule for the interpretation of ion chemistry of ionized molecules, first proposed by the pioneers of MS spectra interpretation, but somewhat overlooked over the years. This rule states that, when rationalizing or predicting the dissociation chemistry of an ionized molecule (M+.), a model analog to the "mobile proton model," that is, a "mobile electron model" via "e--jumping" should be considered. Ground-state M+. is indeed the first species to be considered, but "e--jumping" may eventually lead to other more energetic electromers-ionized molecules that differ only in the location of the missing electron-and each one of these electromers may dissociate via distinctive routes. In such a scenario, the route involving not necessarily the ground-state M+., but the most labile electromer could become predominant or even exclusive. We argue that this "most labile electromer" rule, as well as an analogous "most labile protomer" rule that we have proposed for protonated molecules in an accompanying article, with the application of our conventional toolbox of a few cleavages and rearrangements, greatly simplifies the interpretation and prediction of ion chemistry.

A general, most basic rule for ion dissociation: Protonated molecules.

Contrary to the common but potentially misleading belief that when a protonated molecule is excited, it is its most stable protomer that will mandatorily dissociate, we demonstrate herein that, when rationalizing or predicting the chemistry of such ions, we should always search for the most labile protomer. This "most labile protomer" rule, based on the mobile proton model, states therefore that when a protonated molecule is heated, during ionization or by collisions for instance, the loosely bonded proton (H+ ) can acquire enough energy to detach itself from the most basic site of the molecule and then freely "walk through" the molecular framework to eventually find, if available, another protonation site, forming other less stable but more labile protomers, that is, protomers that may display lower dissociation thresholds. To demonstrate the validity of the "most labile protomer" rule as well as the misleading nature of the "most stable protomer" rule, we have selected several illustrative molecules and have collected their ESI(+)-MS/MS. To compare energies of precursors and products, we have also performed PM7 calculations and elaborated potential energy surface diagrams for their possible protomers and dissociation thresholds. We have also applied the "most labile protomer" rule to reinterpret-exclusively via classical charge-induced dissociation cleavages-several dissociation processes proposed for protonated molecules. In an accompanying letter, we have also applied a similar "most labile electromer" rule to ionized molecules.

Chemical evaluation of pyrolysis oils from domestic and industrial effluent treatment station sludges with perspective to produce value-added products.

The use of renewable sources for energy has increased due to the high demand of modern society and the environmental impacts caused by the use of fossil fuels. Environmentally friendly renewable energy production may involve thermal processes, including the application of biomass. We provide a comprehensive chemical characterization of sludges from domestic and industrial effluent treatment stations, as well as the bio-oils produced by fast pyrolysis. A comparative study of the sludges and the corresponding pyrolysis oils was performed, with characterization of the raw materials using thermogravimetric analysis, energy-dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy, elemental analysis, and inductively coupled plasma optical emission spectrometry. The bio-oils were characterized using comprehensive two-dimensional gas chromatography/mass spectrometry that identified compounds classified according to their chemical class, mainly related to nitrogenous (62.2%) and ester (18.9%) for domestic sludge bio-oil, and nitrogenous (61.0%) and ester (27.6%) for industrial sludge bio-oil. The Fourier transform ion cyclotron resonance mass spectrometry revealed a broad distribution of classes with oxygen and/or sulfur (N2O2S, O2, and S2 classes). Nitrogenous compounds (N, N2, N3, and NxOxclasses) were also found to be abundant in both bio-oils, due to the origins of the sludges (with the presence of proteins), making these bio-oils unsuitable for use as renewable fuels, since NOxgases could be released during combustion processes. The presence of functionalized alkyl chains indicated the potential of the bio-oils as sources of high added-value compounds that could be obtained by recovery processes and used for the manufacture of fertilizers, surfactants, and nitrogen solvents.

Mass spectrometry molecular fingerprinting of mineral and synthetic lubricant oils.

The molecular composition of lubricating oils has a strong impact on how automotive engines function, but the techniques used to monitor the quality parameters of these oils only inspect their gross physical-chemical properties such as viscosity, color, and bulk spectroscopy profiles; hence, bad-quality, adulterated, or counterfeit oils are hard to detect. Herein, we investigated the ability of direct infusion electrospray ionization mass spectrometry (ESI-MS) to provide simple, rapid but characteristic fingerprint profiles for such oils of the mineral and synthetic types. After a simple aqueous extraction, ESI-MS analyses, particularly in the positive ion mode, did indeed show characteristic molecular markers with unique profiles, which were confirmed and more clearly visualized by partial least squares-discriminant analysis (PLS-DA). Nuclear magnetic resonance and Fourier transform infrared-attenuated total reflection spectroscopy were also tested for the bulk samples but showed nearly identical spectra, thus failing to reveal their distinct molecular composition and to differentiate the oil samples. To simulate adulteration, mixtures of mineral and synthetic oils were also analyzed by ESI(+)-MS, and additions as low as 1% of mineral oil to synthetic oil could be detected. The technique therefore offers a simple and fast but powerful tool to monitor the molecular composition of lubricant oils, particularly vias their more polar constituents.

In Melting Points We Trust: A Review on the Misguiding Characterization of Multicomponent Reactions Adducts and Intermediates.

We discuss herein the problems associated with using melting points to characterize multicomponent reactions' (MCRs) products and intermediates. Although surprising, it is not rare to find articles in which these MCRs final adducts (or their intermediates) are characterized solely by comparing melting points with those available from other reports. A brief survey among specialized articles highlights serious and obvious problems with this practice since, for instance, cases are found in which as many as 25 quite contrasting melting points have been attributed to the very same MCR adduct. Indeed, it seems logical to assume that the inherent non-confirmatory nature of melting points could be vastly misleading as a protocol for structural confirmation, but still many publications (also in the Q1 and Q2 quartiles) insist on using it. This procedure contradicts best practices in organic synthesis, and articles fraught with limitations and misleading conclusions have been published in the MCRs field. The drawbacks inherent to this practice are indeed serious and have misguided MCRs advances. We therefore suggest some precautions aimed at avoiding future confusions.

Urine Metabolites Enable Fast Detection of COVID-19 Using Mass Spectrometry.

The COVID-19 pandemic boosted the development of diagnostic tests to meet patient needs and provide accurate, sensitive, and fast disease detection. Despite rapid advancements, limitations related to turnaround time, varying performance metrics due to different sampling sites, illness duration, co-infections, and the need for particular reagents still exist. As an alternative diagnostic test, we present urine analysis through flow-injection-tandem mass spectrometry (FIA-MS/MS) as a powerful approach for COVID-19 diagnosis, targeting the detection of amino acids and acylcarnitines. We adapted a method that is widely used for newborn screening tests on dried blood for urine samples in order to detect metabolites related to COVID-19 infection. We analyzed samples from 246 volunteers with diagnostic confirmation via PCR. Urine samples were self-collected, diluted, and analyzed with a run time of 4 min. A Lasso statistical classifier was built using 75/25% data for training/validation sets and achieved high diagnostic performances: 97/90% sensitivity, 95/100% specificity, and 95/97.2% accuracy. Additionally, we predicted on two withheld sets composed of suspected hospitalized/symptomatic COVID-19-PCR negative patients and patients out of the optimal time-frame collection for PCR diagnosis, with promising results. Altogether, we show that the benchmarked FIA-MS/MS method is promising for COVID-19 screening and diagnosis, and is also potentially useful after the peak viral load has passed.

Fragmenting nanoPutians: Capturing admiration to the rationality, predictability, and beauty of ion chemistry in mass spectrometry.

The fragmentation chemistry of the protonated and ionized nanoPutian 1 has been studied in the gas phase via electrospray ionization and tandem mass spectrometry. A direct analogy was observed between the fragmentation chemistry of this fascinating "humanoid molecule" and "cleavages" at certain parts of the human body. We argue that such direct analogy and illustrative schemes for the fragmentation of molecular ions of 1 offer a ludic and efficient tool to teach and capture attention to ion chemistry in mass spectrometry. Using the changes in mass for the two heavier nanoPutians with different head styles but the same body design, the analogy has also been used to predict mass spectra. The concepts of isotopic labelling and dissociation thresholds have also been illustrated. For many years, the approach has been successfully used by one of us in classes and lectures, mainly when presenting ion chemistry to students and audiences from fields other than Chemistry, most particularly from Biology, Medicine, and Forensic Chemistry.

Rapid Screening of COVID-19 Directly from Clinical Nasopharyngeal Swabs Using the MasSpec Pen.

The outbreak of COVID-19 has created an unprecedent global crisis. While the polymerase chain reaction (PCR) is the gold standard method for detecting active SARS-CoV-2 infection, alternative high-throughput diagnostic tests are of a significant value to meet universal testing demands. Here, we describe a new design of the MasSpec Pen technology integrated to electrospray ionization (ESI) for direct analysis of clinical swabs and investigate its use for COVID-19 screening. The redesigned MasSpec Pen system incorporates a disposable sampling device refined for uniform and efficient analysis of swab tips via liquid extraction directly coupled to an ESI source. Using this system, we analyzed nasopharyngeal swabs from 244 individuals including symptomatic COVID-19 positive, symptomatic negative, and asymptomatic negative individuals, enabling rapid detection of rich lipid profiles. Two statistical classifiers were generated based on the lipid information acquired. Classifier 1 was built to distinguish symptomatic PCR-positive from asymptomatic PCR-negative individuals, yielding a cross-validation accuracy of 83.5%, sensitivity of 76.6%, and specificity of 86.6%, and validation set accuracy of 89.6%, sensitivity of 100%, and specificity of 85.3%. Classifier 2 was built to distinguish symptomatic PCR-positive patients from negative individuals including symptomatic PCR-negative patients with moderate to severe symptoms and asymptomatic individuals, yielding a cross-validation accuracy of 78.4%, specificity of 77.21%, and sensitivity of 81.8%. Collectively, this study suggests that the lipid profiles detected directly from nasopharyngeal swabs using MasSpec Pen-ESI mass spectrometry (MS) allow fast (under a minute) screening of the COVID-19 disease using minimal operating steps and no specialized reagents, thus representing a promising alternative high-throughput method for screening of COVID-19.

Unveiling the mechanism of N-methylation of indole with dimethylcarbonate using either DABCO or DBU as catalyst.

Depending on the catalyst used, N-methylation of indole with dimethylcarbonate (DMC)-an environmentally friendly alkylation agent-yields different products. With 1,4-diazabicyclo[2.2.2]octane (DABCO), the reaction forms only N-methylated indole, but with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), both N-methylated and N-methoxycarbonylated indole are formed. Using direct ESI(+)-MS monitoring to collect actual snapshots of the changing ionic composition of the reaction solution, we report on the interception and characterization of key intermediates for such reactions. Although a mechanism has been proposed with methoxycarbonylated base as the key intermediate for both DBU and DABCO, the ESI(+)-MS data and B3LYP-D3/6-311+G** calculations suggest that the reaction of DMC with indole under either DABCO or DBU catalysis follows contrasting mechanisms.

How and Why to Investigate Multicomponent Reactions Mechanisms? A Critical Review.

We review the most innovative efforts and greatest challenges faced when elucidating multicomponent reactions (MCRs) mechanisms. When compared to traditional reactions, the often two or more concurrent reactions pathways and the greater number of possible intermediates in MCRs turn their mechanistic investigation both a harder and trickier task. The common approaches used to investigate reaction mechanisms are often unable to clarify MCRs mechanisms; hence few but clever approaches are currently used to determine these mechanisms and to depict their key transformations. Their complexity has required most innovative approaches and the use of a number of unique techniques that have shed light over the favored pathway selected from the myriad of alternatives theoretically available for MCRs. This review focuses on the most successful efforts applied by a few leading groups to perform these puzzlingly investigations.

Comparison of generational effect on proteins and metabolites in non-transgenic and transgenic soybean seeds through the insertion of the cp4-EPSPS gene assessed by omics-based platforms.

This work evaluates different generations of transgenic (cp4-EPSPS gene) and non-transgenic soybean plants through proteomics and metabolomics. For proteomics purpose, 24 differentially abundant protein spots were found through 2-D DIGE, being 4 belonging to transgenic plants. From this total, 19 were successfully identified, storage proteins as predominant class. Some identified proteins are involved in growing and cell division, and stress response, such as LEA and dehydrin. For metabolomics, 17 compounds were putatively annotated, mainly belonging to the secondary metabolism, such as flavonoids. From these analyzes, all generations and varieties of the soybean are prone to be differentiate by PLS-DA. According to our results, transgenic plants appear to be more stable than non-transgenic ones. In addition, the omics-based approaches allowed access some relations between those differential spot proteins and metabolites, mainly those storage proteins and flavonoid.

Distribution of major toxins in Rhinella marina parotoid macroglands using Desorption-Electrospray-Ionization mass spectrometry imaging (DESI-MSI).

Amphibian cutaneous glands secrete toxins used in different vital functions including passive defense. Through Desorption Electrospray Ionization-Imaging we analyzed the distribution of the major toxins of the toad Rhinella marina parotoid macroglands. Alkaloids and steroids showed characteristic distribution and intensity within the glands and were also present at lower levels on the skin surface. A comprehensive overview of toxins distribution in toads' skin might help to understand their full biological role within the amphibians.

Multiplatform Investigation of Plasma and Tissue Lipid Signatures of Breast Cancer Using Mass Spectrometry Tools.

Plasma and tissue from breast cancer patients are valuable for diagnostic/prognostic purposes and are accessible by multiple mass spectrometry (MS) tools. Liquid chromatography-mass spectrometry (LC-MS) and ambient mass spectrometry imaging (MSI) were shown to be robust and reproducible technologies for breast cancer diagnosis. Here, we investigated whether there is a correspondence between lipid cancer features observed by desorption electrospray ionization (DESI)-MSI in tissue and those detected by LC-MS in plasma samples. The study included 28 tissues and 20 plasma samples from 24 women with ductal breast carcinomas of both special and no special type (NST) along with 22 plasma samples from healthy women. The comparison of plasma and tissue lipid signatures revealed that each one of the studied matrices (i.e., blood or tumor) has its own specific molecular signature and the full interposition of their discriminant ions is not possible. This comparison also revealed that the molecular indicators of tissue injury, characteristic of the breast cancer tissue profile obtained by DESI-MSI, do not persist as cancer discriminators in peripheral blood even though some of them could be found in plasma samples.

Assessing the Metabolic Impact of Ground Chia Seed in Overweight and Obese Prepubescent Children: Results of a Double-Blind Randomized Clinical Trial.

Childhood obesity is a medical condition of major public health concern. Chia seeds are used to treat certain noncommunicable diseases, and they are rich in omega-3 fatty acids, which contribute to the absorption of vitamins. A randomized double-blind clinical trial of 30 obese children was performed. The sample was composed of prepubertal 5- to 10-year-old children of both sexes with body mass indexes equal to or above the 95th percentile who were recruited through the Pediatric Department of the Faculdade de Medicina do ABC. Blood samples were drawn, the children were weighed and measured, and a 24-h dietary recall was obtained before and after the treatment. Not only were significant differences observed for fibrinogen (P = .011) but a correlation between the changes in markers and the presence of fibers was also observed for two inflammatory parameters: tumor necrosis factor-α (P = .027) and nuclear factor-κß (P = .059). These results indicate that chia seeds may have anti-inflammatory effects related to their fiber content in the context of childhood obesity.

In Situ DESI-MSI Lipidomic Profiles of Breast Cancer Molecular Subtypes and Precursor Lesions.

Clinically meaningful molecular subtypes for classification of breast cancers have been established, however, initiation and progression of these subtypes remain poorly understood. The recent development of desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) facilitates the convergence of analytical chemistry and traditional pathology, allowing chemical profiling with minimal tissue pretreatment in frozen samples. Here, we characterized the chemical composition of molecular subtypes of breast cancer with DESI-MSI. Regions of interest were identified, including invasive breast cancer (IBC), ductal carcinoma in situ (DCIS), and adjacent benign tissue (ABT), and metabolomic profiles at 200 µm elaborated using Biomap software and the Lasso method. Top ions identified in IBC regions included polyunsaturated fatty acids, deprotonated glycerophospholipids, and sphingolipids. Highly saturated lipids, as well as antioxidant molecules [taurine (m/z 124.0068), uric acid (m/z 167.0210), ascorbic acid (m/z 175.0241), and glutathione (m/z 306.0765)], were able to distinguish IBC from ABT. Moreover, luminal B and triple-negative subtypes showed more complex lipid profiles compared with luminal A and HER2 subtypes. DCIS and IBC were distinguished on the basis of cell signaling and apoptosis-related ions [fatty acids (341.2100 and 382.3736 m/z) and glycerophospholipids (PE (P-16:0/22:6, m/z 746.5099, and PS (38:3), m/z 812.5440)]. In summary, DESI-MSI identified distinct lipid composition between DCIS and IBC and across molecular subtypes of breast cancer, with potential implications for breast cancer pathogenesis. SIGNIFICANCE: These findings present the first in situ metabolomic findings of the four molecular subtypes of breast cancer, DCIS, and normal tissue, and add to the understanding of their pathogenesis.

Pharmacokinetics, Pharmacodynamic Efficacy Prediction Indexes and Monte Carlo Simulations of Enrofloxacin Hydrochloride Against Bacterial Strains That Induce Common Clinical Diseases in Broiler Chickens.

Pharmacokinetic parameters and efficacy prediction indexes (Cmax/MIC90 and AUC0-24/MIC90) of an enrofloxacin hydrochloride (ENR-HCl) veterinary product soluble in water were determined in healthy broiler chickens of both sexes after a single oral dose of ENR-HCl (equivalent to 10 mg ENR base/kg bw). Monte Carlo simulations targeting Cmax/MIC90 = 10 and AUC0-24/MIC90 =125 were also performed based on a set of MIC (minimum inhibitory concentration) values of bacterial strains that induce common clinical diseases in broiler chickens and that showed to be susceptible to ENR-HCl. Plasma concentrations of ENR and its main metabolite ciprofloxacin (CIP) were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma concentration-time curves were found to fit a non-compartmental open model. The ratio of the area under the plasma concentration-time curve (AUC) of CIP/ENR was 4.91%. Maximum plasma concentrations of 1.35 ± 0.15 µg/mL for ENR-HCl and 0.09 ± 0.01 µg/mL for CIP were reached at 4.00 ± 0.00 h and 3.44 ± 1.01 h, respectively. Areas under the plasma vs. time concentration curve in 24 h (AUC0-24) were 18.91 ± 1.91 h × µg/mL and 1.19 ± 0.12 h × µg/mL for ENR-HCl and CIP, respectively. Using a microbroth dilution method, the minimum inhibitory concentration (MIC90) values were determined for ENR-HCl for 10 bacterial strains (Mycoplasma gallisepticum, Mycoplasma synoviae, Avibacterium paragallinarum, Clostridium perfringens, Escherichia coli, Pseudomonas aeruginosa, Salmonella ser. Enteritidis, Salmonella ser. Gallinarum, Salmonella ser. Pullorum, and Salmonella ser. Typhimurium), which are the most common causes of infectious clinical diseases in broiler chickens. In summary, the PK/PD ratios and Monte Carlo simulation were carried out for ENR-HCl in poultry, which due to its solubility was administered in drinking water. The PK/PD efficacy prediction indexes and Monte Carlo simulations indicated that the ENR-HCl oral dose used in this study is useful for bacterial infections in treating C. perfringens (Gram-positive), E. coli and S. ser. Enteritidis (Gram-negative) and M. gallisepticum bacteria responsible for systemic infections in poultry, predicting a success rate of 100% when MIC ≤ 0.06 µg/mL for E. coli and S. ser. Enteritidis and MIC ≤ 0.1 µg/mL for M. gallisepticum. For C. perfringens, the success rate was 98.26% for MIC ≤ 0.12. However, clinical trials are needed to confirm this recommendation.

The Intermediates in Lewis Acid Catalysis with Lanthanide Triflates.

Lanthanide triflates are effective Lewis acid catalysts in reactions involving carbonyl compounds due to their high oxophilicity and water stability. Despite the growing interest, the identity of the catalytic species formed in lanthanide catalysed reactions is still unknown. We have therefore used mass spectrometry and ion spectroscopy to intercept and characterize the intermediates in a reaction catalysed by ytterbium and dysprosium triflates. We were able to identify a number of lanthanide intermediates formed in a simple condensation reaction between a C-acid and an aldehyde. Results show correlation between the reactivity of lanthanide complexes and their charge state and suggest that the triply charged complexes play a key role in lanthanide catalysed reactions. Spectroscopic data of the gaseous ions accompanied by theoretical calculations reveal that the difference between catalytic efficiencies of ytterbium and dysprosium ions can be explained by their different electrophilicity.

Applicability of MALDI-TOF MS for determination of quinolone residues in fish.

MALDI-TOF MS approach for determination of six quinolones residues in fillets of pangasius (Pangasionodon hypophthalmus) was studied, considering that is a very sensitive analytical technique with simple and high-throughput operation, contributing to knowledge regarding application of this technique to the determination of small-molecular-weight organic compound residues in foods. LIFT-MS/MS showed to be a successful approach to identify the presence of all quinolone residues in the fish fillet, at their respective MRL level. This study opens an important field of research for the development of simple and high-throughput bioanalytical screening methods for the determination of veterinary drug residues in foods.