Galactose-functionalized methacrylate polymers as affinity sorbents for extraction of food allergen lectins.

In this study, glycidyl methacrylate (GMA)-based materials functionalized with different galactose derivatives were prepared to be used as affinity sorbents for solid-phase extraction (SPE) of several food allergen lectins (such as phytohemagglutinin (PHA)). First, GMA-based polymers were synthesized and then galactose derivatives were immobilized onto the GMA surface using two different synthetic routes. In the first approach, the bare polymer was modified with ethylenediamine and glutaraldehyde, and subsequently two galactose derivatives were immobilized. In the second strategy, the starting polymer was modified with cystamine and gold nanoparticles (AuNPs), on which a thiolated galactose derivative was subsequently anchored. The resulting materials were characterized by scanning electron microscopy and used as SPE sorbents for the isolation of PHA (as probe protein) from food matrices. Different SPE parameters (sample pH, eluent solution composition, binding capacity, sample volume, selectivity and reusability) were evaluated. The material that provided the best PHA recovery (98%) was the one obtained in the second approach, being this material successfully applied to the selective extraction of PHA and other similar lectins from different foods (red and lima dried beans, fresh soybeans and biscuits containing soybean protein traces as indicated in their label). After SDS-PAGE of eluates, all samples only exhibited the characteristic PHA band around 30 kDa, suggesting the high potential of the developed material for application in food allergy field.

Novel Nanozeolitic Imidazolate Framework (ZIF-8)-Luciferase Biocomposite for Nanosensing Applications.

The identification of new strategies to improve the stability of proteins is of utmost importance for a number of applications, from biosensing to biocatalysis. Metal-organic frameworks (MOFs) have been shown as a versatile host platform for the immobilization of proteins, with the potential to protect proteins in harsh conditions. In this work, a new thermostable luciferase mutant has been selected as a bioluminescent protein model to investigate the suitability of MOFs to improve its stability and prompt its applications in real-world applications, for example, ATP detection in portable systems. The luciferase has been immobilized onto zeolitic imidazolate framework-8 (ZIF-8) to obtain a bioluminescent biocomposite with enhanced performance. The biocomposite ZIF-8@luc has been characterized in harsh conditions (e.g., high temperature, non-native pH, etc.). Bioluminescence properties confirmed that MOF enhanced the luciferase stability at acidic pH, in the presence of organic solvents, and at -20 °C. To assess the feasibility of this approach, the recyclability, storage stability, precision, and Michaelis-Menten constants (Km) for ATP and d-luciferin have been also evaluated. As a proof of principle, the suitability for ATP detection was investigated and the biocomposite outperformed the free enzyme in the same experimental conditions, achieving a limit of detection for ATP down to 0.2 fmol.

Iron-Doped Bimodal Mesoporous Silica Nanomaterials as Sorbents for Solid-Phase Extraction of Perfluoroalkyl Substances in Environmental Water Samples.

In this work, sorbets based on UVM-7 mesoporous silica doped with Fe were synthesized and applied to solid-phase extraction of perfluoroalkyl substances from environmental water samples. These emerging pollutants were then determined by liquid chromatography coupled with a mass spectrometry detector. Thus, Fe-UVM-7 mesoporous silica materials with different contents of iron, as well as different pore sizes (by using alkyltrimethilamonium bromide surfactants with different organic tail lengths) were synthesized, and their structure was confirmed for the first time by transmission electron microscopy, nitrogen adsorption-desorption, X-ray diffraction, and Raman spectroscopy. After comparison, Fe50-UVM-7-C12 was selected as the best material for analyte retention, and several extraction parameters were optimized regarding the loading and elution step. Once the method was developed and applied to real matrices, extraction efficiencies in the range of 61-110% were obtained for analytes with C8-C14 chain length, both perfluoroalkyl carboxylates, and perfluoroalkyl sulfonates. Likewise, limits of detection in the range of 3.0-8.1 ng L-1 were obtained for all target analytes. In the analysis of real well-water samples, no target compounds were detected. Spiked samples were analyzed in comparison to Oasis WAX cartridges, and statistically comparable results were achieved.

Mesoporous silica sorbent with gold nanoparticles for solid-phase extraction of organochlorine pesticides in water samples.

In this work, a novel sorbent, based on UVM-7 mesoporous silica doped with Au, has been proposed for organochlorine pesticides extraction. Cartridges containing this material have been applied to the preconcentration of 20 pesticides from water samples, through a solid-phase extraction (SPE) protocol, with their later determination by gas chromatography with an electron capture detector. First, UVM-7 materials were properly characterized by X-ray diffraction, N2 adsorption-desorption, electron microscopy techniques, and UV-Vis spectroscopy, thus confirming their structure and Au incorporation. After optimization of main extraction parameters, recoveries in the range of 80-110% were obtained for most of the analytes, with enrichment factors comprised between 275 and 430. The obtained sensitivity was comparable with other reported methods, with limits of quantification in the range of 0.3-20 ng L-1, thus allowing the determination of these compounds according to European legislation. The developed method has been successfully applied to the analysis of real spiked samples in comparison with a reference method, thus being this sorbent an alternative for organochlorine pesticide enrichment, through a simple, reusable, cheap, and environmentally friendly SPE procedure.

Highly Efficient Removal of Neonicotinoid Insecticides by Thioether-Based (Multivariate) Metal-Organic Frameworks.

Circumventing the impact of agrochemicals on aquatic environments has become a necessity for health and ecological reasons. Herein, we report the use of a family of five eco-friendly water-stable isoreticular metal-organic frameworks (MOFs), prepared from amino acids, as adsorbents for the removal of neonicotinoid insecticides (thiamethoxam, clothianidin, imidacloprid, acetamiprid, and thiacloprid) from water. Among them, the three MOFs containing thioether-based residues show remarkable removal efficiency. In particular, the novel multivariate MOF {SrIICuII6[(S,S)-methox]1.5[(S,S)-Mecysmox]1.50(OH)2(H2O)}·36H2O (5), featuring narrow functional channels decorated with both -CH2SCH3 and -CH2CH2SCH3 thioalkyl chains-from l-methionine and l-methylcysteine amino acid-derived ligands, respectively-stands out and exhibits the higher removal efficiency, being capable to capture 100% of acetamiprid and thiacloprid in a single capture step under dynamic solid-phase extraction conditions-less than 30 s. Such unusual combination of outstanding efficiency, high stability in environmental conditions, and low-cost straightforward synthesis in 5 places this material among the most attractive adsorbents reported for the removal of this type of contaminants.

Dual mixed-mode poly (vinylpyridine-co-methacrylic acid-co-ethylene glycol dimethacrylate)-based sorbent for acidic and basic drug extraction from oral fluid samples.

In this study, a dual mixed-mode polymer sorbent was prepared via one-step thermally initiated polymerization of 4-vinylpyridine (VP), methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) for the solid-phase extraction (SPE) of basic and acidic drugs. The use of VP and MAA as ionizable functional monomers allowed the tailoring of ion-exchange and hydrophobic features of the polymer. The obtained polymer was characterized by Fourier-transform infrared spectroscopy and scanning electron microscopy. Next, the retention behavior of dual mixed-mode polymer towards basic and acidic drugs was investigated. Moreover, the practical capability of this novel material was tested for the extraction of relevant drugs such as cocaine, 3-methylmethcathinone, and diazepam in oral fluid samples. Recovery values (at different spiked levels in blank oral fluid samples), ranging from 69 to 99%, and limits of detection (LODs), between 0.10 and 0.25 µg L-1, were obtained.

Preparation of monolithic polymer-magnetite nanoparticle composites into poly(ethylene-co-tetrafluoroethylene) tubes for uses in micro-bore HPLC separation and extraction of phosphorylated compounds.

This paper describes the fabrication of a novel microbore monolithic column modified with magnetite nanoparticles (MNPs) prepared in a poly(ethylene-co-tetrafluoroethylene) (EFTE) tubing, and its application as stationary phase for the chromatographic separation of phosphorylated compounds. In order to obtain the composite column, a two-step procedure was performed. The formation of a glycidyl methacrylate-based monolith inside the activated ETFE tube was firstly carried out. Then, two incorporation approaches of MNPs in monoliths were investigated. The generic polymer was modified with 3-aminopropyltrimethoxysilane (APTMS) to be subsequently attached to MNP surfaces. Alternatively, APTMS-coated MNPs were firstly prepared and subsequently used for attachment onto the monolith surface through reaction of epoxy groups present in the generic monolith. This last strategy gave a reproducible layer of MNPs coated onto the polymer monolith as well as robust and permeable chromatographic columns. The retention behaviour of this MNP-based composite monolithic column was studied by using small phosphorylated compounds (adenosine phosphates). It was found that the retention of model analytes was ruled by partitioning and adsorption HILIC mechanisms. The columns also exhibited satisfactory performance in the separation of these target compounds, showing good chromatographic behaviour after two months of continued use. These composite monolithic columns were also successfully applied to the extraction of a tryptic digest of ß-casein.

Selective solid-phase extraction of organophosphorus pesticides and their oxon-derivatives from water samples using molecularly imprinted polymer followed by high-performance liquid chromatography with UV detection.

A molecularly imprinted polymer was synthesized and characterized to be used as solid-phase extraction sorbent for simultaneous chlorpyrifos and diazinon and their oxon derivatives. Several imprinted polymers were prepared and evaluated in a retention study of these analytes compared with a non-printed polymer. Several parameters affecting the extraction of imprinted polymer such as washing solvent, composition and volume of the eluting solvent and sample volume, were also investigated. Under the optimum conditions, the developed method provided satisfactory limits of detection ranging between 0.07 µg L-1 to 0.12 µg L-1 and the material showed an excellent reusability (> 50 reuses). The method was applied to the extraction and preconcentration of these analytes in water samples. The average recoveries ranged from 79 ± 6 to 104 ± 3 %.

3D printed fluidic platform with in-situ covalently immobilized polymer monolithic column for automatic solid-phase extraction.

In this work, 3D stereolithographic printing is proposed for the first time for the fabrication of fluidic devices aimed at in-situ covalent immobilization of polymer monolithic columns. Integration in advanced flow injection systems capitalized upon programmable flow was realized for fully automatic solid-phase extraction (SPE) and clean-up procedures as a 'front-end' to on-line liquid chromatography. The as-fabricated 3D-printed extraction column devices were designed to tolerate the pressure drop of forward-flow fluidic systems when handling large sample volumes as demonstrated by the determination of anti-microbial agents, plastic additives and monomers as models of emerging contaminants (4-hydroxybenzoic acid, methylparaben, phenylparaben, bisphenol A and triclosan). Decoration of the monolithic phase with gold nanoparticles (AuNPs) was proven most appropriate for the enrichment of phenolic-type target compounds. In particular, the absolute recoveries for the tested analytes ranged from 73 to 92% both in water and saliva samples. The 3D printed composite monolith showed remarkable analytical features in terms of loading capacity (2 mg g-1), breakthrough volume (10 mL), satisfactory batch-to-batch reproducibility (<9% RSD), and easy on-line coupling of the SPE device to HPLC systems. The fully automatic 3D-printed SPE-HPLC hyphenated system was also exploited for the on-line extraction, matrix clean-up and determination of triclosan in 200 µL of real saliva samples.

Comparison of silica-based materials for organophosphorus pesticides sampling and occupational risk assessment.

A novel air sampler has been designed containing a sorbent based on UVM-7 mesoporous silica doped with Ti. The sorbent has been applied for the determination of organophosphorus pesticides in occupational air, followed by gas chromatography with mass spectrometry detection. Thus, several silica materials with different structures (mesoporous UVM-7 and microporous xerogels) were synthesized, and modified with the addition of Ti and Fe. The structure of these materials was proved by transmission electronic microscopy, energy-dispersive X-ray, X-ray diffraction, nitrogen adsorption-desorption and UV-Vis and Raman spectroscopy. The potential of these materials for the retention of pesticides was evaluated and Ti25-UVM-7 was selected as the best solid phase for analyte sorption. Then, several sampling parameters were optimized and analytical features such as breakthrough volume were determined. Using the designed samplers, quantitative retentions were achieved with recoveries in the range 93-107% for all analytes except for diazinon (82%). RSD values below 13% were obtained. Likewise, the sensitivity of the method was studied, and limits of quantification below 0.5 µg m-3 were obtained for all pesticides. The reusability of the material was also proved. The developed procedure has been applied to the air sampling and occupational risk assessment, during and after methyl-chlorpyrifos application in orange plantations. High concentrations and exposure rates above the limit value for ensure safe work conditions were obtained. At the same time, the air was sampled with XAD-2 samplers as a reference method, and results obtained with both devices were statistically comparable.

In syringe hybrid monoliths modified with gold nanoparticles for selective extraction of glutathione in biological fluids prior to its determination by HPLC.

In this work, a simple device for extraction glutathione (GSH) in biological fluids using a hybrid monolithic material within a polypropylene syringe is developed. For this purpose, glycidyl methacrylate-based monolith was firstly prepared within this housing material, and the polymer was modified with different ligands (ammonia, cysteamine and cystamine). The resulting materials (containing amine or thiol groups, respectively) were then functionalized with gold nanoparticles (AuNPs). The hybrid material that gave the largest AuNPs coverage was selected as solid-phase (SPE) sorbent and several variables affecting the extraction recovery of this compound were investigated. Under optimal conditions, GSH was quantitatively retained at pH 6.0, and then it was desorbed with aqueous dithiothreitol solution and determined, after derivatization with o-phthaldialdehyde, via reversed-phase LC with fluorometric detection. The limit of detection was ca. 1.5 ng mL-1, and the reproducibility between extraction units was below 8% (expressed as relative standard deviation), which demonstrates the robustness of the method. The developed material was also applied for the extraction of GSH in saliva and urine samples yielding recoveries ranging from 86 to 105%.

Hybrid monoliths with metal-organic frameworks in spin columns for extraction of non-steroidal drugs prior to their quantitation by reversed-phase HPLC.

A (glycidyl methacrylate)-co-(ethylene glycol dimethacrylate) polymer (poly(GMA-co-EDMA)) was functionalized with metal-organic frameworks (MOF) and used as a sorbent for solid-phase extraction (SPE). The polymeric sorbent was prepared in-situ by photopolymerization in a previously wall-modified spin column, and then modified with an amino-modified MOF of type NH2-MIL-101(Cr). The sorbents were used for the extraction of nonsteroidal anti-inflammatory drugs (NSAIDs) from human urine samples. The sorbent was compared with the parent monolith and embedded approach, where the MOF particles are admixed in the polymerization mixture before the in-situ polymerization in the modified spin column. SPE is performed by percolating the sample solutions in a centrifuge, which streamlines the SPE steps. The hybrid composites were characterized by scanning electron microscopy and nitrogen intrusion porosimetry. Three NSAIDs (ketoprofen, flurbiprofen, and ibuprofen) were tested. They were eluted from the sorbent with acidified water-acetonitrile mixtures and subsequently analyzed by reversed-phase HPLC with UV detection. The detection limits varied in the range from 0.1 to 7 µg·L-1, and the precisions (relative standard deviation) were <14% in all the cases. The recoveries were between 71.0 and 78.0% in spiked urine samples. Graphical abstractA hybrid monolith modified with amino-modified MOF [named NH2-MIL-101(Cr)] in wall-modified spin columns was prepared. The resulting micro-extraction device was applied to the extraction and preconcentration of non-steroidal anti-inflammatory drugs.

Study of silica-structured materials as sorbents for organophosphorus pesticides determination in environmental water samples.

A novel sorbent based on a UVM-7 mesoporous silica doped with Ti has been synthesized and used for solid-phase extraction of several organophosphorus pesticides in environmental water samples followed by gas chromatography coupled to a nitrogen-phosphorus selective detector. Thus, mesoporous silica materials doped with Ti and Fe as well as immobilized cyclodextrin silica-based supports were prepared and morphologically characterized by several techniques such as transmission electronic microscopy, nitrogen adsorption-desorption and X-ray diffraction. These sorbents were comparatively evaluated, and Ti25-UVM-7 material was selected as the best solid phase. After optimization of extraction parameters such as amount of solid-phase, type and volume of eluent, pH and ionic strength and breakthrough volume, recoveries between 81% and 104.5% were achieved, with RSD values below 7.8% and 12% for intra-day and inter-day repeatability respectively. Moreover, limits of quantification in the range 0.5-4.4 µg L-1 were achieved for all target compounds using mass spectrometry detector. In addition, the developed method was applied for analysis of real water samples and it was validated with commercial C18 cartridges. Matrix effect was demonstrated in complex environmental matrices and the good reusability of synthesized material was also proved.

Zwitterionic codeine-derived methacrylate monoliths for enantioselective capillary electrochromatography of chiral acids and chiral bases.

Thiol-ene click reaction of N-acetyl-L-cysteine methyl ester to codeine, followed by reaction with allyl isocyanate and hydrolysis to the corresponding zwitterionic chiral selector and its subsequent bonding to the surface of a methacrylate monolith provided a new chiral capillary column for enantiomer separation of chiral acids and chiral bases. First, the epoxy groups of a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith were converted into amine residues, followed by reaction with allylglycidyl ether. In this way, a spacer arm was bonded to the surface before coating and cross-linking poly(3-mercaptopropyl methylsiloxane) (PMPMS) via radical addition (thiol-ene click reaction) to the surface. In order to improve the performance of the monolithic chiral stationary phase, thio ether and residual thiol groups were oxidized to sulfonyl and sulphonate groups, respectively. This novel chiral stationary phase (CSP) was evaluated by capillary electrochromatography (CEC) using two chiral model compounds, namely N-3,5-dinitrobenzoyl-R,S-leucine (retained by anion-exchange mechanism) and mefloquine (by cation-exchange process). The ion-exchange retention mechanism on the CSP was characterized for these two counterionic model solutes by varying the mobile phase composition, including the nature of solvents, the concentration of counter-ions and co-ions, and the acid-to-base ratio. A series of chiral ß-blockers and amino acid derivatives was used to further check the performance of the modified monolith under the optimal conditions. Several enantiomers were baseline resolved with reasonable peak efficiencies (up to 60,000 theoretical plates per meter for the second eluted enantiomer).

Poly(ethylene glycol) diacrylate based monolithic capillary columns for the analysis of polar small solutes by capillary electrochromatography.

Monolithic stationary phases based on poly(ethylene glycol) diacrylates for capillary electrochromatography were developed. Several poly(ethylene glycol) diacrylates (Mn 250, 575, and 700) were used as single monomers and the resulting columns were carefully compared. Methanol and ethyl ether were selected as porogenic solvents, and in all cases ultraviolet radiation was selected as initiation method to prepare polymeric monoliths. The influence of the monomer chain length and ratio monomer/porogen on the morphological and electrochromatographic properties of the resulting monoliths was investigated. Several families of compounds with different polarity (alkyl benzenes, organophosphorous pesticides, benzoic acid derivatives, and sulfonamides) were selected to evaluate the performance of the fabricated monolithic columns. The best results were obtained for poly(ethylene glycol) diacrylate 700 monoliths affording efficiencies of 144 000 plates/m for retained polar aromatic small molecules and excellent reproducibility in column preparation (RSD values below 2.5%).

Carbon nanotube-modified monolithic polymethacrylate pipette tips for (micro)solid-phase extraction of antidepressants from urine samples.

This work evaluates the potential of methacrylate monoliths with multi-walled carbon nanotubes incorporated into the polymeric network for the extraction of antidepressants from human urine. The method is based on a micropipette solid-phase extraction tip containing a hybrid monolithic material covalently attached to the polypropylene housing. A polymer layer made from poly(ethylene dimethacrylate) was bound to the inner surface of a polypropylene tip via UV grafting. The preparation of the monolith and the microextraction steps were optimized in terms of adsorption capacity. Limits of detection ranged from 9 to 15 µg·L-1. The average precision of the method varied between 3 and 5% (intra-tips), and from 4 to 14% (inter-tips). The accuracy of the method was evaluated through a recovery study by using spiked samples. Graphical abstract Hybrid polymer monoliths containing multi-walled carbon nanotubes (MWCNTs) were prepared in pipette tips by photo-polymerization approach. The extraction devices were used for the extraction of antidepressants in urine samples.

Proteomic fingerprinting of mistletoe (Viscum album L.) via combinatorial peptide ligand libraries and mass spectrometry analysis.

Combinatorial peptide ligand libraries (CPLLs), coupled to mass spectrometry (MS) analysis, have been used to investigate in depth the proteome of Viscum album L. (VA), commonly named European mistletoe, in order to provide a first proteomic fingerprinting. For this purpose, the proteins were captured via CPLLs at two different pH values (acidic and neutral). A total of 648 non-redundant proteins were identified by using two different databases. The two pH values, chosen for bead incubations, have contributed to increment the capture ability: 56% and 31% of CPLLs species were respectively recognized at pH7.2 and at pH2.2. Finally the biological function of identified proteins was evaluated in order to understand their role on human health and the potential benefits of mistletoe extracts in medicine. SIGNIFICANCE: Viscum album L. (VA) extracts are recently used as supporting medicine for cancer therapy, improving patients' survival and increasing their quality of life in medicine. These anticancer effects are investigated and they are probably due to mistletoe's capability to favor tumor cell's death and to modulate the immune system. Although the increasing interest in VA medical benefits, the role of its components in human health remains unclear. In order to exploit this aspect, it is important to comprehensively study proteins present in Viscum album L. (VA) extracts. Nevertheless, since plant proteomics analysis is in most cases handicapped by the presence of high-abundance proteins masking the detection of the low-abundance ones, it is important to overcome this challenge. In this sense, combinatorial peptide ligand libraries (CPLLs) have been used to reduce the dynamic protein concentration range to enable the identification of a higher amount of proteins than employing conventional methods. In this work, a total of 648 non-redundant proteins were identified: 56% and 31% of CPLLs species were respectively recognized at pH7.2 and at pH2.2. This deep proteome identification was useful to investigate the biological functions of proteins in order to evaluate their potential role in human health.

Simultaneous determination of main phenolic acids and flavonoids in tomato by micellar electrokinetic capillary electrophoresis.

A methodology for the determination of tomato phenolic acids and flavonoids has been developed combining MEKC and DAD detection. The influence on polyphenol separation of pH and background electrolyte, BGE (borax, acetonitrile, methanol and SDS concentrations), was studied and optimized using response surface methodology and weighted desirability function. Separation of polyphenols was achieved within 20min at 15°C using 11.3mM borax and 11.2mM SDS adjusted to pH 8.5 as BGE. Validation was performed using standards and tomato extracts. Recoveries ranged from 77 to 106%. Acceptable repeatabilities were obtained for peak area (%RSD <3.1% and <3.7%) and migration times (%RSD <0.2% and <1.4%) for intra- and inter-day respectively. Detection limits ranged between 0.8 and 3.8mgkg-1. Five and seven of these polyphenols were determined in samples of tomato and related species. This methodology will be valuable tool in breeding programs, analyzing a large number of samples.

Evaluation of 2,3-epoxypropyl groups and functionalization yield in glycidyl methacrylate monoliths using gas chromatography.

Poly(glycidyl methacrylate-co-ethylene dimethacrylate) (poly(GMA-co-EDMA)) is most frequently used as parent monolith to obtain stationary phases with a variety of surface chemistries for liquid chromatography and capillary electrochromatography. Functionalization is performed by opening the accessible 2,3-epoxypropyl groups of the monolith with a suitable reagent. The number of 2,3-epoxypropyl groups which are accessible before and after the functionalization reaction, and the grafting yield, are important parameters, required both to optimize functionalization and to interpret the chromatographic performance of functionalized monoliths. In this work, a method capable of providing this information for parent and functionalized poly(GMA-co-EDMA) monoliths prepared both in silica capillaries and in other supports is proposed. First, sulfuric acid and lithium aluminium hydride (LiAlH4) are sequentially used to release the 2,3-epoxypropyl groups as glycerol, which is subsequently determined by GC. About 6.0mmol of 2,3-epoxypropyl groups per gram of monolith was found in this work for the parent monoliths prepared in silica capillaries using UV-initiation. The monoliths were also functionalized using ammonia (NH3), diethylamine (DEA) and epinephrine, and the amount of residual 2,3-epoxypropyl groups, and hence the functionalization yield, were established by also measuring the GC peak of glycerol. The amounts of 2,3-epoxypropyl groups and the derivatization yields were established with RSDs of 1.7 and 3.4%, respectively. The proposed method was also applied to the characterization of poly(GMA-co-EDMA) monoliths prepared in glass vials. Significant differences with respect to those prepared in 100µm I.D. silica capillaries were evidenced.

Fast simultaneous determination of prominent polyphenols in vegetables and fruits by reversed phase liquid chromatography using a fused-core column.

A reversed-phase high-performance liquid chromatography method with photodiode array detection has been developed enabling the joint determination of 17 prominent flavonoids and phenolic acids in vegetables and fruits. A multi-segmented gradient program using a fused-core column for the separation of several phenolic classes (phenolic acids and flavonoids) has been optimised. The influence of extraction conditions (sample freeze-drying, ultrasound extraction, solvent composition and extraction time) has been also optimised using response surface methodology with tomato samples as a model. Complete recoveries (76-108%) were obtained for the phenolic compounds present in tomato. The developed method provided satisfactory repeatability in terms of peak area (RSD<2.9%) and retention time (RSD<0.2%) both for standards and real samples. Detection limits ranged between 3 and 44µgkg(-1) for the detected polyphenols. This method is recommended for routine analysis of large number of samples typical of production quality systems or plant breeding programs.