T. Rodríguez-Cabo

Dispersive liquid–liquid microextraction using non-chlorinated, lighter than water solvents for gas chromatography–mass spectrometry determination of fungicides in wine

A novel and low solvent consumption method for the sensitive determination of fungicide residues in wine samples is proposed. Analytes were extracted by dispersive liquid-liquid microextraction (DLLME) and further determined by gas chromatography-mass spectrometry (GC-MS). Under optimized conditions, a binary mixture of acetone and 1-undecanol (0.5 and 0.05 mL, respectively) was used to extract target compounds from diluted (1:1) wine samples. After centrifugation, the floating phase of 1-undecanol was solidified and separated from the liquid hydro-alcoholic matrix. Thereafter, it was allowed to melt at room temperature and injected in the GC-MS system. The method showed relative standard deviations (RSDs, %) below 13%, limits of quantification (LOQs) between 0.2 and 3.2 ng mL(-1) and linear responses for concentrations up to 300 ng mL(-1). The efficiency of the liquid-phase microextraction process was scarcely affected by the characteristics of wine samples, consequently pseudo-external standard calibration (using matrix matched standards of red and white wine) sufficed to achieve acceptable accuracy values: relative recoveries from 81 to 120%. The applicability of the method was demonstrated with commercial wine samples.

Determination of hydroxylated stilbenes in wine by dispersive liquid–liquid microextraction followed by gas chromatography mass spectrometry

Dispersive liquid-liquid microextraction (DLLME) has been proposed, for first time, to determine the concentrations of three hydroxylated stilbenes (trans-isomers of pterostilbene, resveratrol and piceatannol) in wine samples. DLLME was preceded by an acetylation step and followed by gas chromatography mass spectrometry (GC-MS) to achieve limits of quantification (LOQs) comprised between 0.6 and 5 ngmL(-1), linear responses up to 5000 ngmL(-1), excellent accuracy (overall recoveries ranged from 90 to 102% for samples spiked at different levels from 50 to 1000 ngmL(-1)) and standard deviations below 12%. Sample intake and organic solvents consumption remained at 1 and 0.6 mL, respectively, with a total sample preparation (derivatization and concentration) time around 15 min. The trans-isomers of piceatannol and resveratrol were measured in wine samples at concentrations up to 0.5 and 2.6 μgmL(-1), respectively. In addition to their cis-forms, the dihydrogenated analogues of both antioxidants were detected in red wines. To the best of our knowledge, this is the first report describing the presence of dihydropiceatannol in wine. On the other hand, pterostilbene remained below the detection limits of the method in all the processed samples. In addition to the relative low levels of this stilbene existing in grapes, its lipophilic character appears to limit the transfer from grapes skins to wine.

Multiclass semi-volatile compounds determination in wine by gas chromatography accurate time-of-flight mass spectrometry.

The performance of gas chromatography (GC) with accurate, high resolution mass spectrometry (HRMS) for the determination of a group of 39 semi-volatile compounds related to wine quality (pesticide residues, phenolic off-flavours, phenolic pollutants and bioactive stilbenes) is investigated. Solid-phase extraction (SPE) was used as extraction technique, previously to acetylation (phenolic compounds) and dispersive liquid-liquid microextraction (DLLME) concentration. Compounds were determined by GC coupled to a quadrupole time-of-flight (QTOF) MS system through an electron ionization (EI) source. The final method attained limits of quantification (LOQs) at the very low ng mL(-1) level, covering the range of expected concentrations for target compounds in red and white wines. For 38 out of 39 compounds, performance of sample preparation and determination steps were hardly affected by the wine matrix; thus, accurate recoveries were achieved by using pseudo-external calibration. Levels of target compounds in a set of 25 wine samples are reported. The capabilities of the described approach for the post-run identification of species not considered during method development, without retention time information, are illustrated and discussed with selected examples of compounds from different classes.

Investigation of liquid chromatography quadrupole time-of-flight mass spectrometry performance for identification and determination of hydroxylated stilbene antioxidants in wine.

The performance of liquid chromatography (LC) followed by quadrupole time-of-flight (QTOF) mass spectrometry (MS) for the determination of hydroxylated stilbene compounds in red and white wine samples is assessed. When combined with a solid-phase extraction step, LC-QTOF-MS allowed the selective determination of five target compounds (trans- and cis-resveratrol, trans-piceatannol, trans-piceid and trans-pterostilbene) attaining limits of quantification between 3 and 20ngmL(-1) and providing linear responses up to 4000ngmL(-1). Recoveries, established against standards prepared in methanol, varied between 93% and 115%. The distribution of the above species in wine is illustrated with the analysis of 15 samples. Trans-pterostilbene remained undetected in samples, whereas trans-piceid and trans-resveratrol maximum concentrations exceed the 6000ngmL(-1) level. Values for trans-piceatannol and cis-resveratrol ranged from non detected to 600ngmL(-1), and from 11 to more than 3200ngmL(-1), respectively. Accurate MS and MS/MS scan spectra were used to investigate the existence of reduced (dihydro) and oxidized (dehydro) forms of resveratrol and picetannol in the processed samples. Dihydro derivatives appeared, as free compounds, in 100% (dihydro-piceatannol) and 40% (dihydro-resveratrol) of the samples. On the other hand, dehydro derivatives were noticed as conjugated (glycosylated) species, with detection frequencies of 100% and 47% for dehydro-glucosyl-resveratrol and dehydro-glucosyl-piceatannol, respectively. Above findings confirm the suitability of LC-QTOF-MS for the comprehensive study of hydroxylated stilbene antioxidants in wine samples.

Comprehensive evaluation of the photo-transformation routes of trans-resveratrol

Liquid chromatography (LC) combined with accurate mass spectrometry (MS), based on the use of a hybrid quadrupole time-of-flight (TOF) MS system, is employed to systematically investigate the photo-transformation routes of trans-resveratrol. Experiments were performed in quartz tubes, containing ethanolic solutions (12% v/v) of the precursor compound, exposed to different ultraviolet (UV) sources and to solar light. Time-courses of trans-resveratrol and transformation products (TPs) were investigated by direct injection of different reaction times aliquots in the LC-QTOF-MS system. Structural elucidation of detected TPs was derived from interpretation of their accurate product ion scan spectra. Trans-resveratrol labelled with (13)C6 in the mono-hydroxylated ring was also employed to further confirm the exact positions of some substituents in the generated TPs. In addition to the well-known trans-/cis-isomerization process, three different main reactions pathways were noticed under all the investigated conditions: (1) water addition to the exocycle double bond followed by oxidation to a ketone and cleavage of the molecule, (2) intramolecular cyclization to render a trihydroxylated phenanthrene, and (3) oxidation of the phenanthrene-like derivative to generate an orto-diquinone. Both, the trihydroxylated phenanthrene and the orto-diquinone underwent further aromatic hydroxylation reactions. The above transformation routes were also noticed for cis-resveratrol and the two analogue phytoalexins piceid and piceatannol. In addition to above transformation pathways, under solar light exposure, resveratrol underwent a molecular re-arrangement rendering the so-called resveratrone, whose structure consists of two fused aromatic rings bonded to a linear chain containing a carbonyl group conjugated with a double bond.

Selective extraction and determination of neonicotinoid insecticides in wine by liquid chromatography–tandem mass spectrometry

A simplified, high throughput procedure for the determination of five neonicotinoid insecticides in red and white wines, using liquid chromatography (LC)-tandem mass spectrometry (MS/MS), is presented. The effects of different experimental parameters (extraction sorbent, solvent elution and clean-up conditions) in the efficiency and the selectivity of the sample preparation process were assessed through calculation of the extraction yields and the matrix effects (MEs). Wines (10mL) were concentrated using OASIS HLB cartridges, on-line connected to Florisil clean-up cartridges, with acetonitrile serving as the elution solvent. The extract (5mLvol) was concentrated to 1mL and injected in the LC-ESI-MS/MS system. The optimized procedure provided quantitative extraction yields at the same time that the efficiency of ESI ionization remained unchanged between standards and sample extracts. Overall recoveries, calculated against authentic standards in ACN, varied between 77 and 119% and the attained limits of quantification remained below 0.2ngmL(-1). Analysis of commercial wines revealed imidacloprid residues in more than 50% of processed samples, with a maximum level of 14ngmL(-1).

Dispersive liquid-liquid microextraction with non-halogenated extractants for trihalomethanes determination in tap and swimming pool water.

An effective, low solvent consumption, reduced cost and high throughput sample preparation method for the determination of four trihalomethanes (THMs) in tap and swimming pool water samples is presented. THMs extraction and concentration were performed by dispersive liquid-liquid microextraction (DLLME), based on the use of non-halogenated and low volatile solvents as extractants. Analytes were further determined by gas chromatography with micro-electron capture detection (GC-microECD). Under optimized conditions, the proposed method uses 18 mL volume samples, 0.7 mL of acetone (dispersant) and 0.05 mL of 1-undecanol (extractant). Achieved enrichment factors (EFs) varied from 67 to 104 times, the limits of quantification (LOQs) stayed between 0.05 and 1.3 ng mL(-1), and an excellent linearity was noticed up to 100 ng mL(-1). Relative recoveries, measured for spiked aliquots of tap and swimming pool water samples, remained between 79% and 113%, with associated standard deviations below 12%. The applicability of the developed methodology was demonstrated with chlorinated water samples analysis. As regards tap water samples, the sum of THMs concentrations remained under the limit fixed by the European Union (100 ng mL(-1)); however, some samples contained levels close to 80 ng mL(-1), the maximum allowable concentration established by the United States Environmental Protection Agency (EPA).

Liquid chromatography quadrupole time-of-flight mass spectrometry selective determination of ochratoxin A in wine

The performance of liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) for ochratoxin A (OTA) determination in wine is evaluated for the first time. Sample preparation was optimized to obtain quantitative recoveries at the same time that the efficiency of electrospray ionization (ESI) remained unaltered between sample extracts and calibration standards. Under final conditions, samples (20 mL) were concentrated using a reversed-phase solid-phase extraction (SPE) cartridge, followed by OTA elution with 1 mL of ethyl acetate. The absolute recoveries of the method, established against calibration standards, were 91-121% and 90-113% (without and with internal standard correction, respectively), for wines fortified at 3 concentration levels. The attained LOQ (0.05 ng mL(-1)) remained below the maximum permitted OTA concentration (2 ng mL(-1)) in dry wines. The method was applied to different samples, with OTA being found in some dessert wines at concentrations below 1 ng mL(-1). The ethyl ester of OTA (OTC) could be identified in the same wine samples from its accurate full product ion spectra.

Assessment of dispersive liquid–liquid microextraction conditions for gas chromatography time-of-flight mass spectrometry identification of organic compounds in honey

The suitability of the dispersive liquid-liquid microextraction (DLLME) technique for gas chromatography (GC) characterization of minor organic compounds in honey samples is evaluated. Under optimized conditions, samples were pre-treated by liquid-liquid extraction with acetonitrile followed by DLLME using carbon tetrachloride (CCl4, 0.075 mL) as extractant. The yielded settled phase was analyzed by GC using high resolution time-of-flight (TOF) mass spectrometry (MS). The whole sample preparation process is completed in approximately 10 min, with a total consumption of organic solvents below 4 mL, relative standard deviations lower than 12% and with more than 70 organic compounds, displaying linear retention index in the range from 990 to 2900, identified in the obtained extracts. In comparison with HS SPME extraction, higher peak intensities were attained for most volatile and semi-volatile compounds amenable to both extraction techniques. Furthermore, other species such as highly polar and water soluble benzene acids, long chain fatty acids, esters and flavonoids, which are difficult to concentrate by HS SPME, could be identified in DLLME extracts. Some of the compounds identified in DLLME extracts have been proposed as useful for samples classification and/or they are recognized as markers of honeys from certain geographic areas.

Liquid chromatography time‐of‐flight mass spectrometry evaluation of fungicides reactivity in free chlorine containing water samples

Liquid chromatography (LC) combined with tandem mass spectrometry (MS/MS), based on the use of a hybrid quadrupole-time-of-flight mass analyzer, was used to investigate the reactivity of nine fungicides in free chlorine-containing water samples. Three of the selected compounds (fenhexamid, FEN; pyrimethanil, PYR; and cyprodinil, CYP) displayed a poor stability in presence of moderate chlorine levels; thus, the effects of different parameters on their half-lives (t(1/2)) were evaluated. Sample pH, bromide traces, and the water matrix affected their relative stabilities. Despite such variations, the three fungicides are degraded at significant rates not only in ultrapure, but also in surface water spiked with chlorine levels up to 2 µg ml(-1), and when mixed with chlorinated tap water, generating several transformation products (TPs). The time-course of precursor species and their TPs was followed in the LC-MS mode, using the information contained in accurate, full scan mass spectra (MS) to propose the empirical formulae of TPs. Thereafter, their ion product scan (MS/MS) spectra were considered to set their chemical structures; allowing, in some cases, to distinguish between isomeric TPs. The reaction pathway of FEN, the less stable fungicide, involved just an electrophilic substitution of hydrogen per chlorine, or bromine, and cleavage of the molecule to render an amide. PYR and CYP shared common reaction routes consisting of halogenation, hydroxylation, and condensation processes leading to complex mixtures of TPs, which were relatively stable to further transformations.