A simple and fast method for metabolomic analysis by gas liquid chromatography-mass spectrometry.

INTRODUCTION: The metabolomic profile is an essential tool for understanding the physiological processes of biological samples and their changes. In addition, it makes it possible to find new substances with industrial applications or use as drugs. As GC-MS is a very common tool for obtaining the metabolomic profile, a simple and fast method for sample preparation is required. OBJECTIVES: The aim of this research was to develop a direct derivatization method for GC-MS to simplify the sample preparation process and apply it to a wide range of samples for non-targeted metabolomic analysis purposes. METHODS: One pot combined esterification of carboxylic acids with methanol and silylation of the hydroxyl groups was achieved using a molar excess of chlorotrimethylsilane with respect to methanol in the presence of pyridine. RESULTS: The metabolome profile obtained from different samples, such as bilberry and cherry cuticles, olive leaves, P. aeruginosa and E. coli bacteria, A. niger fungi and human sebum from the ceruminous gland, shows that the procedure allows the identification of a wide variety of metabolites. Aliphatic fatty acids, hydroxyfatty acids, phenolic and other aromatic compounds, fatty alcohols, fatty aldehydes dimethylacetals, hydrocarbons, terpenoids, sterols and carbohydrates were identified at different MSI levels using their mass spectra. CONCLUSION: The metabolomic profile of different biological samples can be easily obtained by GC-MS using an efficient simultaneous esterification-silylation reaction. The derivatization method can be carried out in a short time in the same injection vial with a small amount of reagents.

Rice-Associated Rhizobacteria as a Source of Secondary Metabolites against Burkholderia glumae.

Various diseases, including bacterial panicle blight (BPB) and sheath rot, threaten rice production. It has been established that Burkholderia glumae (B. glumae) is the causative agent of the above mentioned pathologies. In the present study, antagonistic activity, growth promotion, and the metabolite profiles of two rhizobacteria, isolated in different paddy fields, were assessed against B. glumae. Strains were identified based on 16S rRNA gene sequences, and the phylogenetic analyses showed that both strains belong to the genus Enterobacter, with high similarity to the strain Enterobacter tabaci NR146667.2 (99%). The antagonistic activity was assessed with the disc diffusion method. Active fractions were isolated through a liquid/liquid extraction with ethyl acetate (EtOAc) from the fermentation media, and their antibacterial activities were evaluated following the Clinical and Laboratory Standards Institute (CLSI) guidelines. The Pikovskaya modified medium was used to test the ability of in vitro inorganic phosphorus solubilization, and BSB1 proved to be the best inorganic phosphorus solubilizer, with a solubilization index (SI) of 4.5 ± 0.2. The glass-column fractionation of the EtOAc extracted from BCB11 produced an active fraction (25.9 mg) that inhibited the growth of five B. glumae strains by 85-95%. Further, metabolomic analysis, based on GC-MS, showed 3-phenylpropanoic acid (3-PPA) to be the main compound both in this fraction (46.7%), and in the BSB1 extract (28.6%). This compound showed antibacterial activity against all five strains of B. glumae with a minimum inhibitory concentration (MIC) of 1000 mg/L towards all of them. The results showed that rice rhizosphere microorganisms are a source of compounds that inhibit B. glumae growth and are promising plant growth promoters (PGP).

Combined Analysis of Primary Metabolites and Phenolic Compounds to Authenticate Commercial Monovarietal Peach Purees and Pear Juices.

Here we authenticated single-varietal peach purees and pear juices on the basis of primary metabolite and phenolic compound analysis by Proton Nuclear Magnetic Resonance (1H-NMR) and Ultra Performance Liquid Chromatography coupled to Photodiode Array and Tandem Mass Spectrometry (UPLC-PDA-MS/MS), respectively. After suitable preprocessing, the 1H-NMR and chromatographic data were evaluated by principal component analysis (PCA). The PCA combining data from primary metabolites and phenolic compounds allowed the separation of the clusters in all cases, allowing discrimination of processed and unprocessed peach purees, both separately and pooled. The PCA of primary metabolites allowed the cluster separation of purees of distinct peach varieties but not between processed and non-processed purees. The PCA of phenolic compounds allowed better cluster separation than of primary metabolites. For pear juices, both PCA approaches allowed satisfactory discrimination of Alejandrina, Conference, and Blanquilla cultivars. These approaches may help to better control cultivar authenticity in fruit products. It could therefore contribute to the development of a process to achieve products characterized by a quality characteristic of a given cultivar.

Chemical Characterization and Biotechnological Applicability of Pigments Isolated from Antarctic Bacteria.

Considering the global trend in the search for alternative natural compounds with antioxidant and sun protection factor (SPF) boosting properties, bacterial carotenoids represent an opportunity for exploring pigments of natural origin which possess high antioxidant activity, lower toxicity, no residues, and no environmental risk and are readily decomposable. In this work, three pigmented bacteria from the Antarctic continent, named Arthrobacter agilis 50cyt, Zobellia laminarie 465, and Arthrobacter psychrochitiniphilus 366, were able to withstand UV-B and UV-C radiation. The pigments were extracted and tested for UV absorption, antioxidant capacity, photostability, and phototoxicity profile in murine fibroblasts (3T3 NRU PT-OECD TG 432) to evaluate their further potential use as UV filters. Furthermore, the pigments were identified by ultra-high-performance liquid chromatography-photodiode array detector-mass spectrometry (UPLC-PDA-MS/MS). The results showed that all pigments presented a very high antioxidant activity and good stability under exposure to UV light. However, except for a fraction of the A. agilis 50cyt pigment, they were shown to be phototoxic. A total of 18 different carotenoids were identified from 23 that were separated on a C18 column. The C50 carotenes bacterioruberin and decaprenoxanthin (including its variations) were confirmed for A. agilis 50cyt and A. psychrochitiniphilus 366, respectively. All-trans-bacterioruberin was identified as the pigment that did not express phototoxic activity in the 3T3 NRU PT assay (MPE < 0.1). Zeaxanthin, ß-cryptoxanthin, ß-carotene, and phytoene were detected in Z. laminarie 465. In conclusion, carotenoids identified in this work from Antarctic bacteria open perspectives for their further biotechnological application towards a more sustainable and environmentally friendly way of pigment exploitation.

Pigments in an iridescent bacterium, Cellulophaga fucicola, isolated from Antarctica.

An iridescent yellow pigmented bacterium isolated from the Antarctic continent, named Cellulophaga fucicola strain 416, was found to be able to tolerate UV-B radiation. Its crude pigment extract was tested for antioxidant capacity, UV light stability and phototoxicity profile against murine fibroblast lines. The pigments were further isolated and chemically identified by ultra-high-performance liquid chromatography with photodiode array and mass spectrometry detectors. The results showed that the pigment extract presented weak stability under exposure to UV light, a phototoxic profile in the 3t3 Neutral Red Uptake test and a very high antioxidant activity, suggesting that it could be used as food and feed colourants. Zeaxanthin and two isomers of zeaxanthin, ß-cryptoxanthin and ß-carotene, were identified using a C18 column. These five carotenoids were the major pigments isolated from C. fucicola 416. In conclusion, the identification of pigments produced by the bacterial strain under study may help us understand how bacteria thrive in high UV and cold environments, and opens avenues for further biotechnological application towards a more sustainable and environmentally friendly way of pigment exploitation.

Elongation of the Hydrophobic Chain as a Molecular Switch: Discovery of Capsaicin Derivatives and Endogenous Lipids as Potent Transient Receptor Potential Vanilloid Channel 2 Antagonists.

The transient receptor potential vanilloid type-2 (TRPV2) protein is a nonselective Ca2+ permeable channel member of the TRPV subfamily, still considered an orphan TRP channel due to the scarcity of available selective and potent pharmacological tools and endogenous modulators. Here we describe the discovery of novel synthetic long-chain capsaicin derivatives as potent TRPV2 antagonists in comparison to the totally inactive capsaicin, the role of their hydrophobic chain, and how the structure-activity relationships of such derivatives led, through a ligand-based approach, to the identification of endogenous long-chain fatty acid ethanolamides or primary amides acting as TRPV2 antagonists. Both synthetic and endogenous antagonists exhibited differential inhibition against known TRPV2 agonists characterized by distinct kinetic profiles. These findings represent the first example of both synthetic and naturally occurring TRPV2 modulators with efficacy in the submicromolar/low-micromolar range, which will be useful for clarifying the physiopathological roles of this receptor, its regulation, and its targeting in pathological conditions.

A fast and reliable ultrahigh-performance liquid chromatography method to assess the fate of chlorophylls in teas and processed vegetable foodstuff.

A total of 48 chlorophylls and derivatives were identified and successfully determined in tea and processed vegetable and fruit foodstuff by UHPLC with photodiode-array and mass spectrometry detection. The method allowed the proper separation of chlorophyll derivatives resulting from demetallation, dephytilation, decarbomethoxylation, epimerisation and copperisation. The method was performed in less than 12 min, using an optimised ternary gradient (MeOH, iPrOH, MeCN and H2O with 10 mM of ammonium acetate) on an ACQUITY HSS T3 column. Mass spectrometry, applying both ESI and APCI ionization sources, was used for identification purposes. The method was applied to evaluate the degree of processing in teas of different origin and quality. It allowed differentiation between supermarket own-brand tea bags and teas sold by specialised shops. Pheophytins, pheophorbides and pyro derivatives were found mainly in processed green vegetable and fruit products thereof. However, chlorophyll-derived food colorants, such as Cu-chlorophyllins, Cu-pheophytins, Cu-pyropheophytins, Cu-pheophorbides and Cu-pyropheophorbides, were also detected in several products.

Bulk industrial fruit fibres. Characterization and prevalence of the original fruit metabolites.

Here we analysed the content of primary and secondary metabolites in nine types of industrially processed fibres derived from the juice industry. Specifically, we examined fibre from: apple, peach, and pear, as non-citrus fruits; the peel and flesh of orange and tangerine, and lemon flesh, as citrus fruits; and carrot, as vegetable. Regarding primary metabolites, the sugar content ranged from 21.6 mg/g in lemon to 290 mg/g in orange peel and lower mass organic acid content ranged from 25.0 mg/g in pear to 250 mg/g in lemon. The content of fatty acids were constant during fibre processing, ranging from 0.5 to 1.46%. Furthermore, the fatty acid profile was not affect for the processing. Concerning secondary metabolites, industrial processing did not decrease the sterols content, which ranged from 0.51 to 1.66 µg/g. Regarding carotenoids, of note was the presence of epoxycarotenoids, which may reflect the quality of the industrial process, thus giving added value to the by-product.

Development of a SBSE-TD method coupled to GC-MS and chemometrics for the differentiation of variety and processing conditions in peach juices.

Peach juices of distinct varieties, namely yellow- and red-fleshed, and commercial and freshly blended were analyzed. The method used was based on Stir Bar Sorptive Extraction (SBSE) involving a polydimethylsiloxane-coated stir bar with thermal desorption (TD), followed by gas chromatography coupled to mass spectrometry (GC-MS) analysis. The resulting analytical data included 41 compounds belonging to several chemical classes, such as aldehydes, alcohols, lactones, terpenoids, fatty aldehydes, fatty acids and hydrocarbons. Furthermore, chemometric data treatment using unsupervised analysis (PCA) proved useful to classify peach juices on the basis of variety. Stepwise Linear Discriminant Analysis (SLDA) showed that a reduced number of variables (14 compounds), including lactones (6-pentyl-α-pyrone, γ-decalactone, γ-dodecalactone, and δ-dodecalactone), fatty acids (hexadecanoic acid), fatty aldehydes (tetracosanal and octacosanal), hydrocarbons (C23, C26, C27, C29, and C33), and alcohols (phytol and α-tocopherol), were necessary to classify the juice samples according to variety and processing conditions.

Dispersive liquid-liquid microextraction and injection-port derivatization for the determination of free lipophilic compounds in fruit juices by gas chromatography-mass spectrometry.

A method consisting of dispersive liquid-liquid microextraction (DLLME) followed by injection-port derivatization and gas chromatography-mass spectrometry (GC-MS) for the analysis of free lipophilic compounds in fruit juices is described. The method allows the analysis of several classes of lipophilic compounds, such as fatty acids, fatty alcohols, phytosterols and triterpenes. The chromatographic separation of the compounds was achieved in a chromatographic run of 25.5min. The best conditions for the dispersive liquid-liquid microextraction were 100µL of CHCl3 in 1mL of acetone. For the injection-port derivatization, the best conditions were at 280°C, 1min purge-off, and a 1:1 sample:derivatization reagent ratio (v/v) using N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA):pyridine (1:1) as reagent. Quality parameters were assessed for the target compounds, giving a limits of detection (LODs) ranging from 1.1 to 5.7ng/mL and limits of quantification (LOQs) from 3.4 to 18.7ng/mL for linoleic and stearic acid, respectively. Repeatability (%RSD, n=5) was below 11.51% in all cases. In addition, the method linearity presented an r2 ≥0.990 for all ranges applied. Finally, the method was used to test the lipophilic fraction of various samples of commercial fruit juice.

A rapid gas chromatographic injection-port derivatization method for the tandem mass spectrometric determination of patulin and 5-hydroxymethylfurfural in fruit juices.

A novel method consisting of injection-port derivatization coupled to gas chromatography-tandem mass spectrometry is described. The method allows the rapid assessment of 5-hydroxymethylfurfural (HMF) and patulin content in apple and pear derivatives. The chromatographic separation of the compounds was achieved in a short chromatographic run (12.2min) suitable for routine controls of these compounds in the fruit juice industry. The optimal conditions for the injection-port derivatization were at 270°C, 0.5min purge-off, and a 1:2 sample:derivatization reagent ratio (v/v). These conditions represent an important saving in terms of derivatization reagent consumption and sample preparation time. Quality parameters were assessed for the target compounds, giving LOD of 0.7 and 1.6µg/kg and LOQ of 2 and 5µg/kg for patulin and HMF, respectively. These values are below the maximum patulin concentration in food products intended for infants and young children. Repeatability (%RSD n=5) was below 12% for both compounds. In addition, the method linearity ranged between 25 and 1000µg/kg and between 5 and 192µg/kg for HMF and patulin, respectively. Finally, the method was applied to study HMF and patulin content in various fruit juice samples.

Injection-port derivatization coupled to GC-MS/MS for the analysis of glycosylated and non-glycosylated polyphenols in fruit samples.

Polyphenols, including glycosylated polyphenols, were analyzed via a procedure based on injection-port derivatization coupled to gas chromatography-tandem mass spectrometry (GC-MS/MS). The polyphenols in lyophilized fruit samples were extracted with an acidified MeOH mixture assisted by ultrasound. Samples were dried under vacuum, and carbonyl groups were protected with methoxylamine. Free hydroxyl groups were subsequently silylated in-port. Mass fragmentations of 17 polyphenol and glycosylated polyphenol standards were examined using Multiple Reaction Monitoring (MRM) as the acquisition mode. Furthermore, in-port derivatization was optimized in terms of optimal injection port temperature, derivatization time and sample: N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) volume ratio. A C18 solid-phase-extraction clean-up method was used to reduce matrix effects and injection liner degradation. Using this clean-up method, recoveries for samples spiked at 1 and 10µg/g ranged from 52% to 98%, depending on the chemical compound. Finally, the method was applied to real fruit samples containing the target compounds. The complete chromatographic runtime was 15min, which is faster than reported for recent HPLC methods able to analyze similar compounds.

Comprehensive Two-dimensional Gas Chromatography Time-of-flight Mass Spectrometry to Assess the Presence of α,α-Trehalose and Other Disaccharides in Apple and Peach.

INTRODUCTION: Carbohydrates are important constituents in fruits. Among the carbohydrates, disaccharides have rarely been studied in apple and peach. Indeed, the abiotic stress biomarker and preservation agent α,α-trehalose is a disaccharide. OBJECTIVES: To establish a comprehensive method based on two-dimensional gas chromatography combined with time-of-flight MS detection (GC × GC-ToF/MS) to analyse the disaccharide composition of apple and peach. METHODS: The sample preparation was based on aqueous-methanolic extraction of the analytes, followed by oxime formation and trimethylsilylation of the disaccharides. First, three columns were tested with standards on the one-dimensional system. Next, to perform the sample analysis using GC × GC-MS (which offers significant advantages over conventional GC because it allows higher separation efficiencies), various column configurations were assessed on the two-dimensional system to obtain enhanced separation and low detection limits. The column sets tested included non-polar/semi-polar, semi-polar/polar and polar/non-polar. RESULTS: Using the method that proved to be more efficient, namely the method developed with the semi-polar/non-polar configuration, ten disaccharides were identified, based on analytical standards, retention index and mass spectra. These compounds were quantified in several varieties of apple and peach fruit using the developed GC × GC method and linear curve calibration, resulting in substantial differences among the fruits. However, cultivars within the fruits exhibited no significant differences. CONCLUSION: The proposed method allowed for the identification and quantification of several disaccharides in apple and peach, including the biomarker α,α-trehalose.

Characterisation of phenolic compounds in processed fibres from the juice industry.

The content of phenolic compounds was determined in nine industrially processed fibres derived from the juice industry. Apple, peach, and pear as non-citrus fruit fibres were examined, as well as orange peel and flesh, tangerine peel and flesh, and lemon flesh as citrus fruit fibres, and carrot as vegetable fibre. The extractable phenolic profile of all fibres was obtained by UPLC-PDA-FLR-MS/MS. Forty phenolic compounds were identified and their concentrations determined. In addition, bound phenolic acids and proanthocyanidins were measured in solid residues in order to determine the phenolic compounds remaining. Also, to allow the comparison of the profiles and contents in the fresh fruit and fibres, we analysed extractable and bound phenolic compounds in lyophilized peel and pulp from fresh fruit. The profile and phenolic content of the fibres was similar to that of the fresh fruit, except for flavan-3-ols, which registered lower values.

Ultra performance liquid chromatography analysis to study the changes in the carotenoid profile of commercial monovarietal fruit juices.

We have developed an analytical method that allows the simultaneous determination of epoxycarotenoids, hydroxycarotenoids and carotenes in monovarietal fresh homemade and industrially processed fruit products. Analyses were carried out using ultra performance liquid chromatography (UPLC). The extraction method was optimized using methanol as the first extraction solvent for lyophilized samples followed by a saponification step. Recoveries ranged between 75% and 104% depending on the compound. Repeatability was better than 10% for all compounds (%RSD, n=3). The chromatographic analysis takes less than 17min. In this short period, up to 27 carotenoids were identified in apple, peach and pear products. The developed method allowed us to differentiate juice from six varieties of apple by their carotenoid profile. Moreover, the methodology allows us to differentiate the carotenoid profiles from commercial juices and homemade fresh peach and pear juices, as well as to study the rearrangements of 5,6- to 5,8-epoxycarotenoids.

UHPLC-MS analysis of juvenile hormone II in Mediterranean corn borer (Sesamia nonagrioides) hemolymph using various ionization techniques.

The juvenile hormones (JHs) have been considered the most versatile hormones in the animal kingdom. JH-II is the most abundant JH in Sesamia nonagrioides, important maize pests in the Mediterranean basin. This study compared the sensitivities and matrix effects of four ionization modes on analyzing JH-II in S. nonagrioides hemolymph using ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS) in single ion monitoring (SIM) mode. The ionization techniques tested were electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), and APPI with the lamp turned off, which corresponds to atmospheric pressure thermospray ionization (APTSI). ESI was discarded because of the high matrix effect. APPI was discarded because the correlation responses between solvent and matrix on the instrumental quality parameters were worse than those for APTSI and APCI. In our analytical conditions, APCI has shown the best validation parameter values. APCI ionization is widely available in instrumental laboratories.

Fast determination of oleic acid in pork by flow injection analysis/mass spectrometry.

In some Mediterranean products such as olive oil or ham, oleic acid is the most abundant component of the total fat. Due to the large volume of trade in these products, it may be necessary to analyze oleic fatty acids in high numbers of samples in short periods of time. However, using classic lipid analysis techniques, it is not always possible to cope with these high demands. To solve this problem, a high-throughput analytical method for oleic fatty acid quantification in pork is presented. The purpose of the method is to avoid liquid chromatography processes using a flow injection analysis (FIA) system based on electrospray ionization mass spectrometry. The use of pentadecanoic fatty acid as an internal standard overcame matrix effects. The oleic FIA technique could be used as a suitable method for discriminating carcass samples for selection and labeling by oleic acid content when large numbers of pork samples must be processed in a short period of time.

From symmetric glycerol derivatives to dissymmetric chlorohydrins.

The anticipated worldwide increase in biodiesel production will result in an accumulation of glycerol for which there are insufficient conventional uses. The surplus of this by-product has increased rapidly during the last decade, prompting a search for new glycerol applications. We describe here the synthesis of dissymmetric chlorohydrin esters from symmetric 1,3-dichloro-2-propyl esters obtained from glycerol. We studied the influence of two solvents: 1,4-dioxane and 1-butanol and two bases: sodium carbonate and 1-butylimidazole, on the synthesis of dissymmetric chlorohydrin esters. In addition, we studied the influence of other bases (potassium and lithium carbonates) in the reaction using 1,4-dioxane as the solvent. The highest yield was obtained using 1,4-dioxane and sodium carbonate.

Preparation of (S)-1-Halo-2-octanols Using Ionic Liquids and Biocatalysts.

Preparation of (S)-1-chloro-2-octanol and (S)-1-bromo-2-octanol was carried out by the enzymatic hydrolysis of halohydrin palmitates using biocatalysts. Halohydrin palmitates were prepared by various methods from palmitic acid and 1,2-octanediol. A tandem hydrolysis was carried out using lipases from Candida antarctica (Novozym 435), Rhizomucor miehei (Lipozyme IM), and "resting cells" from a Rhizopus oryzae strain that was not mycotoxigenic. The influence of the enzyme and the reaction medium on the selective hydrolysis of isomeric mixtures of halohydrin esters is described. Novozym 435 allowed preparation of (S)-1-chloro-2-octanol and (S)-1-bromo-2-octanol after 1-3 h of reaction at 40 degrees C in [BMIM][PF(6)].

Synthesis of allyl esters of fatty acids and their ovicidal effect on Cydia pomonella (L.).

Eight allyl esters of fatty acids were synthesized in moderate to high yields with a novel two-step procedure using glycerol as a starting material. The two-step methodology avoids the use of allyl alcohol. The first step consisted of heating at 80 degrees C for 48 h a 2:1:5 mmol mixture of glycerol, a fatty acid, and chlorotrimethylsilane in a solvent-free medium. The crude compound was then dissolved in butanone and heated at 115 degrees C in the presence of NaI. A tandem Finkelstein rearrangement-elimination reaction occurs, producing the corresponding allyl ester. The activity of these esters against Cydia pomonella (L.) (Lepidoptera: Tortricidae) eggs was tested in the laboratory by topical application of one 0.1 microL drop. All of the compounds showed a concentration-mortality response and caused 100% mortality at the highest concentration tested (10 mg/mL). There was an inverse relationship between the alkyl chain length and the ovicidal activity of the allyl ester; the LC(50) and the LC(90) of the two compounds that have the longer alkyl chains were significantly higher than those of the rest of the compounds. The ovicidal and IGR activities of this kind of compound appear to be unprecedented.