Characterization of monovarietal virgin olive oils by phenols profiling.

The phenolic profiles of seven monovarietal virgin olive oils (VOOs) - viz. Arbequina, Arbosana, Cornicabra, FS-17, Hojiblanca, Picual and Sikitita - were characterized by using a quantitative strategy based on LC-MS/MS and the selected reaction monitoring (SRM) mode. Data dependent methods, based on precursor ion scanning, product ion scanning and neutral loss scanning, were developed for confirmatory analysis of secoiridoid derivatives. The observed phenolic profiles were used to find correlation between pairs of phenols and similarity trends among the monovarietal VOOs. A Pearson analysis revealed several correlations among phenols with p-value<0.01 and correlation coefficient (R)>0.75 in the seven monovarietal VOOs. Cluster analysis showed two main clusters between VOOs, formed by Arbequina/Hojiblanca/Cornicabra/Picual and Sikitita/Arbosana/FS-17. High correlations (R>0.7) were observed for the following pairs of VOOs: Arbequina/Hojiblanca (R=0.77), essentially supported on levels of hydroxytyrosol acetate (3,4-DHPEA-AC) and dialdehydic forms of secoiridoids; Cornicabra/FS-17 (R=0.81) and Picual/FS-17 (R=0.79), by correlation of flavonoids and secoiridoid derivatives in general. The highest correlation was observed for the pair Picual/Cornicabra (R=0.99). This preliminary study allowed setting similarities and dissimilarities between monovarietal VOOs by analysis of the phenolic profile. The observed connections between phenols for different varieties have been tentatively interpreted according to the main pathways for phenols biosynthesis.

Ultrasound-assisted extraction with LC-TOF/MS identification and LC-UV determination of imazamox and its metabolites in leaves of wheat plants.

INTRODUCTION: imazamox is a herbicide used in many legominous and cereal crops. There are few methods in the literature for determination of imazamox and its metabolites in plants because of the lack of commercial standards or owing to expensive and/or complex synthesis. OBJECTIVE: To develop a method based on liquid chromatography and ultraviolet absorption detection for simultaneous determination of imazamox and its metabolites in plants. METHODS: Sample preparation was based on ultrasound-assisted extraction (70 W power and duty cycle of 0.7 s/s for 10 min) with subsequent filtration of the extracts and clean-up and concentration prior to chromatographic separation and detection at 240 nm. The chromatographic analysis was completed in 30 min using a Luna® HILIC column. Identification and confirmatory analysis of the presence of imazamox and its metabolites in extracts from treated plants was performed by LC-TOF/MS in high resolution mode for precursor ions. The metabolites were quantified using a surrogate approach based on an imazamox standard. The method was validated by analysing wheat samples treated with 200 g per hectare of active ingredient imazamox. RESULTS: The linear dynamic range of the calibration curve was within 0.27-600 µg/mL, with a correlation coefficient of 0.998 and precision--studied at 0.1 and 2 µg/mL--of 2.9% and 5.0% for repeatability, and 4.7% and 6.9% for reproducibility, respectively. CONCLUSION: The analytical characteristics of the method make it recommendable for evaluating the metabolism of imazamox in plants.

Liquid chromatography-diode array detection to study the metabolism of glufosinate in Triticum aestivum T-590 and influence of the genetic modification on its resistance.

The resistance to glufosinate of two lines-genetically modified (GM) and unmodified (T-590 and T-549, respectively)-of Triticum aestivum has been studied. In the GM line, the bar gene was introduced to increase the resistance to glufosinate. Experiments in a controlled growth chamber showed that line T-590 presented a high resistance to glufosinate with an ED50 value of 478.59 g active ingredient per hectare (g ai ha(-1)) versus 32.65 g ai ha(-1) for line T-549. The activity of glutamine synthetase (GS) in leaf extracts from both lines was investigated. The I50 for line T-590 was 694.10 µM glufosinate versus 55.46 µM for line T-549, with a resistance factor of 12.51. Metabolism studies showed a higher and faster penetration of glufosinate in line T-549 than in line T-590. LC-TOF/MS analysis of glufosinate metabolism at 48 h after herbicide treatment (300 g ai ha(-1)) revealed an 83.4% conversion of the herbicide (66.5% in N-acetyl-glufosinate metabolite), while in line T-549 conversion of the herbicide was about 40% (0% to N-acetyl-glufosinate). These results suggest that metabolism of glufosinate by the bar gene is a key mechanism of resistance in line T-590 that explains such high levels of herbicide tolerated by the plant, together with other mechanisms due to unmodified pathway, absorption and loss of glufosinate affinity for its target site.

Pool of resistance mechanisms to glyphosate in Digitaria insularis.

Digitaria insularis biotypes resistant to glyphosate have been detected in Brazil. Studies were carried out in controlled conditions to determine the role of absorption, translocation, metabolism, and gene mutation as mechanisms of glyphosate resistance in D. insularis. The susceptible biotype absorbed at least 12% more (14)C-glyphosate up to 48 h after treatment (HAT) than resistant biotypes. High differential (14)C-glyphosate translocation was observed at 12 HAT, so that >70% of the absorbed herbicide remained in the treated leaf in resistant biotypes, whereas 42% remained in the susceptible biotype at 96 HAT. Glyphosate was degraded to aminomethylphosphonic acid (AMPA), glyoxylate, and sarcosine by >90% in resistant biotypes, whereas a small amount of herbicide (up to 11%) was degraded by the susceptible biotype up to 168 HAT. Two amino acid changes were found at positions 182 and 310 in EPSPS, consisting of a proline to threonine and a tyrosine to cysteine substitution, respectively, in resistant biotypes. Therefore, absorption, translocation, metabolism, and gene mutation play an important role in the D. insularis glyphosate resistance.

Targeting metabolomics analysis of the sunscreen agent 2-ethylhexyl 4-(N,N-dimethylamino)benzoate in human urine by automated on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry with liquid chromatography-time-of-flight/mass spectrometry confirmation.

The in vivo metabolism of the xenobiotic agent 2-ethylhexyl 4-(N,N-dimethylamino)benzoate (EDP), a UV filter commonly used in sunscreen cosmetic products, was studied by targeting metabolomics analysis in human urine. The metabolomic study involved the use of urine from male and female volunteers before and after application of an EDP-containing sunscreen cosmetic. The metabolism of EDP in urine was studied by using the triple quadrupole detector in a combination of Precursor Ion Scanning and Neutral Loss Scanning modes, with and without enzymatic hydrolysis. Detected metabolites were subsequently confirmed as glucuronide conjugates of 4-(N,N-dimethylamino)benzoic acid and 4-(N-methylamino)benzoic acid by liquid chromatography-time-of-flight/mass spectrometry (LC-TOF/MS) in the accurate mass mode. In this way, the existence of phase II metabolism in the detoxification of EDP by effects of the lipophilic character of this sunscreen agent was confirmed. Hence, to study the in vivo metabolism of EDP, a fully automated method using a solid-phase extraction (SPE) workstation connected on-line to a liquid chromatograph and a triple quadrupole mass analyzer (LC-MS/MS) was developed. The ensuing hyphenated method is very simple and requires minimal human intervention. Following thorough optimization of the SPE and LC-MS/MS conditions, the analytical procedure was validated and standard addition calibration used for the quantitative correction of matrix effects. The proposed method was applied to determine the phase I metabolites of EDP in urine samples and afforded limits of detection from 0.1 to 1.1 ng and accuracy of 91-107% with relative standard deviations in the range 1.5-8.7% (sample volume: 100 µL). Based on the results of in vivo percutaneous absorption of a single application of the sunscreen, about 0.5% of the amount of the applied EDP is excreted in urine.

Determination of glyphosate and its metabolites in plant material by reversed-polarity CE with indirect absorptiometric detection.

A simple CE method for simultaneous determination of glyphosate and its metabolites (i.e. aminomethylphosphonic acid, glyoxylate, sarcosine and formaldehyde) in plants is reported here. A BGE of pH 7.5, 10% ACN, 7.5 mM phthalate, containing 0.75 mM hexadecyltrimethylammonium bromide as an electro-osmotic flow modifier, an applied voltage of -20 kV and absorptiometric monitoring at 220 nm were the optimal chemical and instrumental parameters. The method, with development time 20 min, shows linear calibrations within the range 5-500 microg/mL (for all target analytes) with correlation coefficients between 0.999 and 0.998. It has been validated by application to samples of Lolium spp. The electroinjection mode hinders most interferents to enter the capillary, thus providing a clean electropherogram and making unnecessary long sample-preparation steps.

Temporal metabolomic analysis of o-glucoside phenolic compounds and their aglycone forms in olive tree and derived materials.

INTRODUCTION: Maturity is one of the most important factors associated with evaluation of the quality of fruit and vegetables. In olive oil, maturation plays a key role in the kinetics of biosynthetic pathways of the secondary metabolism. One of the most relevant pathways is that catalysed by beta-glucosidases, which are involved in olive oil debittering. Therefore, the knowledge of this influence can be of particular interest for olive oil industry. OBJECTIVE: To monitor the profile of O-glucoside phenols and their aglycone forms in olive oil, alperujo (the semisolid residue resulting in the production of olive oil), stones, leaves and branches in order to interpret its evolution according to the sample and the period of the season (October, when olive drupes are green; December, when these drupes are green-purple; and February, when they are mostly black). METHODOLOGY: Targeted phenols were extracted by previously optimised methods assisted by auxiliary energies (i.e. ultrasound, microwaves or superheated liquids) according to the characteristics of the sample. The analysis was carried out by liquid chromatography-tandem mass spectrometry with a triple quadrupole mass detector. Highly selective identification and sensitive determination of metabolites was performed in multiple-reaction monitoring mode. Statistical analysis to evaluate differences in the profile of the target compounds was based on principal compounds analysis. RESULTS: The evolution of the analytes concentration is strongly related to the role of beta-glucosidases. An explanation for this evolution in olive oil, alperujo and stones is given by relation to the industrial process for olive oil production. For leaves and small branches, the concentration was practically constant over the season, which was foreseeable because of the perennial character of olive trees. Leaves and branches were found to be highly concentrated in O-glucoside derivatives, demonstrating their capacity for phenolic compounds storage. CONCLUSIONS: Targeted metabolomic profiling has proved a useful tool to monitor O-glucoside phenolic compounds and their aglycone forms in olive materials. The profile of target compounds enables interpretion of their evolution according to the olive material and the period of the season.

Liquid chromatography/triple quadrupole tandem mass spectrometry with multiple reaction monitoring for optimal selection of transitions to evaluate nutraceuticals from olive-tree materials.

Optimal transitions have been selected for the identification and quantitation of the most interesting hydrophilic biophenols in extracts from olive-tree materials, which are of interest because of their nutraceutical properties. The tested materials were extra virgin olive oil, waste from oil production (known as alperujo), and olive-tree materials such as leaves, small branches and fruit stones. The identification and determination steps of the target biophenols are based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a triple quadrupole (QQQ) mass detector. The interface between the chromatograph and the QQQ was an electrospray ionization source operated in the negative ion mode. Highly selective identification of the biophenols was confirmed by multiple reaction monitoring (MRM) using the most representative transitions from the precursor ion to the different product ions. Quantitative MS/MS analysis was carried out by optimization and selection of the most sensitive transition for each analyte, which resulted in estimated detection limits of 5.10 to 11.65 ng/mL for the extracts. The biophenols were extracted from the tested samples by different methods: liquid-liquid extraction for virgin olive oil, microwave-assisted leaching for olive leaves, branches and stones, and pressurized liquid leaching for alperujo. This study provides valuable information about the most suitable source for the isolation of each nutraceutical biophenol and enables us to obtain a complete profile of them in Olea Europaea.

Identification and determination of fat-soluble vitamins and metabolites in human serum by liquid chromatography/triple quadrupole mass spectrometry with multiple reaction monitoring.

A method for determination of fat-soluble vitamins K(1), K(3), A, D(2), D(3) and E (as alpha- and delta-tocopherol) and metabolites 25-hydroxyvitamin D(2) and D(3) and 1,25-dihydroxyvitamin D(3) in human serum by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in positive mode is proposed. Highly selective identification of the target compounds in serum was confirmed by the most representative transitions from precursor ion to product ion. Quantitative MS/MS analysis was carried out by multiple reaction monitoring optimizing the most sensitive transition for each analyte in order to achieve low detection limits (from 0.012 to 0.3 ng/mL estimated with serum). The analysis was performed with 1 mL of serum, which was subjected to protein precipitation, liquid-liquid extraction to an organic phase, evaporation to dryness and reconstitution with methanol. The precision of the overall method ranged from 3.17-6.76% as intra-day variability and from 5.07-11.53% as inter-day variability. The method, validated by the standard addition method, provides complete information on the fat-soluble vitamins profile, which is of interest in clinical and metabolomics studies.