Phenolic Compounds Profiling of Virgin Olive Oils from Different Varieties Cultivated in Mendoza, Argentina, by Using Liquid Chromatography-Mass Spectrometry.

The aim of this work was to achieve a preliminary characterization of the profile of the phenolic fraction of virgin olive oils (VOOs) from Maipú (Mendoza, Argentina). Thus, 25 commercial VOO samples from Arauco, Arbequina, Picual, Frantoio, Changlot, Empeltre, Nevadillo, Manzanilla, and Coratina (both monovarietals and blends) were analyzed using LC-ESI-QTOF MS and LC-ESI-IT MS for identification and quantification purposes, respectively. A rapid LC method (15 min) accomplished quantitative information about a total of 40 phenolic compounds, including secoiridoid derivatives, which have not been evaluated before in samples coming from the subregion so-called Maipú (Mendoza province, Argentina). The results make evident that olive oils coming from Mendoza can be considered as important sources of phenolic bioactive compounds, exhibiting similar phenolic compound levels to those shown by oils from other typical world production regions. Moreover, some distinctive features of the Arauco variety (Argentinean autochthonous variety) were pointed out; indeed, a correlation between flavonoids content and botanical variety was established herewith.

Metabolomic analysis of avocado fruits by GC-APCI-TOF MS: effects of ripening degrees and fruit varieties.

In order to investigate avocado fruit ripening, nontargeted GC-APCI-TOF MS metabolic profiling analyses were carried out. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to explore the metabolic profiles from fruit samples of 13 varieties at two different ripening degrees. Mannoheptulose; pentadecylfuran; aspartic, malic, stearic, citric and pantothenic acids; mannitol; and ß-sitosterol were some of the metabolites found as more influential for the PLS-DA model. The similarities among genetically related samples (putative mutants of "Hass") and their metabolic differences from the rest of the varieties under study have also been evaluated. The achieved results reveal new insights into avocado fruit composition and metabolite changes, demonstrating therefore the value of metabolomics as a functional genomics tool in characterizing the mechanism of fruit ripening development, a key developmental stage in most economically important fruit crops.

Assessment of the stability of proanthocyanidins and other phenolic compounds in cranberry syrup after gamma-irradiation treatment and during storage.

Shelf life of commercial cranberry syrup irradiated with gamma radiation at a rate of 5 kGy and stored for 6 months at 25 °C and 60% relative humidity (RH) and under accelerated stability conditions was investigated. High-performance liquid chromatography coupled to electrospray ionisation quadrupole-time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS) was used to characterise cranberry syrup. Afterwards, these compounds were quantified by HPLC-ESI-QTOF-MS and 4-dimethylaminocinnamaldehyde (DMAC) assay. A significant increase in the content of procyanidin B isomer 1 (from 4.4 to 7.0 µg/ml) and procyanidin A2 (from 83 to 93 µg/ml) was observed after irradiation and compared with the non-irradiated syrup. Procyanidin B isomers and prodelphinidin were stable at 25 °C during the first month of storage, whereas quercetin and some derivatives remained constant for 3 months of storage at this temperature. In short, after gamma-irradiation in dose of 5 kGy, most compounds were highly stable for a month at 25 °C.

A novel tridentate bis(phosphinic acid)phosphine oxide based europium(III)-selective Nafion membrane luminescent sensor.

A new europium(III) membrane luminescent sensor based on a new tridentate bis(phosphinic acid)phosphine oxide (3) system has been developed. The synthesis of this new ligand is described and its full characterization by NMR, IR and elemental analyses is provided. The luminescent complex formed between europium(III) chloride and ligand 3 was evaluated in solution, observing that its spectroscopic and chemical characteristics are excellent for measuring in polymer inclusion membranes. Included in a Nafion membrane, all the parameters (ligand and ionic additives) that can affect the sensitivity and selectivity of the sensing membrane as well as the instrumental conditions were carefully optimized. The best luminescence signal (λexc = 229.06 nm and λem = 616.02 nm) was exhibited by the sensing film having a Nafion : ligand composition of 262.3 : 0.6 mg mL(-1). The membrane sensor showed a short response time (t95 = 5.0 ± 0.2 min) and an optimum working pH of 5.0 (25 mM acetate buffer solution). The membrane sensor manifested a good selectivity toward europium(III) ions with respect to other trivalent metals (iron, chromium and aluminium) and lanthanide(III) ions (lanthanum, samarium, terbium and ytterbium), although a small positive interference of terbium(III) ions was observed. It provided a linear range from 1.9 × 10(-8) to 5.0 × 10(-6) M with a very low detection limit (5.8 × 10(-9) M) and sensitivity (8.57 × 10(-7) a.u. per M). The applicability of this sensing film has been demonstrated by analyzing different kinds of spiked water samples obtaining recovery percentages of 95-97%.

Reversed-phase ultra-high-performance liquid chromatography coupled to electrospray ionization-quadrupole-time-of-flight mass spectrometry as a powerful tool for metabolic profiling of vegetables: Lactuca sativa as an example of its application.

Lettuce (Lactuca sativa), a leafy vegetal widely consumed worldwide, fresh cut or minimally processed, constitutes a major dietary source of natural antioxidants and bioactive compounds. In this study, reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled to electrospray ionization-quadrupole-time-of-flight mass spectrometry (ESI-QTOF-MS) was applied for the comprehensive profiling of polar and semi-polar metabolites from three lettuce cultivars (baby, romaine, and iceberg). The UHPLC systems allowed the use of a small-particle-size C18 column (1.8 µm), with very fine resolution for the separation of up to seven isomers, and the QTOF mass analyzer enabled sensitive detection with high mass resolution and accuracy in full scan. Thus, a total of 171 compounds were tentatively identified by matching their accurate mass signals and suggested molecular formula with those previously reported in family Asteraceae. Afterwards, their structures were also corroborated by the MS/MS data provided by the QTOF analyzer. Well-known amino acids, organic acids, sesquiterpene lactones, phenolic acids and flavonoids were characterized, e.g. lactucin, lactucopicrin, caftaric acid, chlorogenic acid, caffeoylmalic acid, chicoric acid, isochlorogenic acid A, luteolin, and quercetin glycosides. For this plant species, this is the first available report of several isomeric forms of the latter polyphenols and other types of components such as nucleosides, peptides, and tryptophan-derived alkaloids. Remarkably, 10 novel structures formed by the conjugation of known amino acids and sesquiterpene lactones were also proposed. Thus, the methodology applied is a useful option to develop an exhaustive metabolic profiling of plants that helps to explain their potential biological activities and folk uses.

Phenylpropanoids and their metabolites are the major compounds responsible for blood-cell protection against oxidative stress after administration of Lippia citriodora in rats.

Lippia citriodora (lemon verbena) has been widely used in folk medicine for its pharmacological properties. Verbascoside, the most abundant compound in this plant, has protective effects associated mostly with its strong antioxidant activity. The purpose of this study was to test the effect of L. citriodora extract intake on the antioxidant response of blood cells and to correlate this response with the phenolic metabolites found in plasma. For this purpose, firstly the L. citriodora extract was characterized and its radical scavenging activity was measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Then, catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GRed) activities were determined in lymphocytes, erythrocytes, and neutrophils isolated from rats after acute intake of L. citriodora. Phenolic metabolites were analyzed in the same plasma samples by HPLC-ESI-TOF-MS. Myeloperoxidase (MPO) activity in neutrophils, which has been proposed as a marker for inflammatory vascular damage, was also determined. After L. citriodora administration, the antioxidant enzymes activities significantly accelerated (p<0.05) while MPO activity subsided, indicating that the extract protects blood cells against oxidative damage and shows potential anti-inflammatory and antiatherogenic activities. The main compounds found in plasma were verbascoside and isoverbascoside at a concentration of 80±10 and 57±4 ng/ml, respectively. Five other metabolites derived from verbascoside and isoverbascoside were also found in plasma, namely hydroxytyrosol, caffeic acid, ferulic acid, ferulic acid glucuronide, and homoprotocatechuic acid, together with another eight phenolic compounds. Therefore, the phenylpropanoids verbascoside and isoverbascoside, as well as their metabolites, seem to be the responsible for the above-mentioned effects, although the post-transcriptional activation mechanism of blood-cell antioxidant enzymes by these compounds needs further investigation.

A metabolite-profiling approach allows the identification of new compounds from Pistacia lentiscus leaves.

Pistacia lentiscus L., commonly known as Mastic tree or lentisk, is a Mediterranean evergreen shrub widely used in traditional medicine to treat such diseases as eczema, diarrhoea, and throat infections. Furthermore, other properties are currently attributed to P. lentiscus, such as antioxidant capacity, hepatoprotective action, and anti-inflammatory effects. High-performance liquid chromatography with diode array coupled to electrospray ionization mass spectrometry (HPLC-ESI-QTOF-MS) was used for the comprehensive characterization of methanol extract from P. lentiscus leaves. After the optimisation of the HPLC-ESI-QTOF-MS method and the use of the negative ionization mode, 46 different compounds were identified, 20 of which were tentatively characterized for the first time in P. Lentiscus leaves. The majority of the compounds were quantified. Flavonoids, phenolic acids and their derivatives were the most abundant compounds, those with the highest concentrations being myricetin glycoside (6216.13 mg/kg of plant), catechin (3354.78 mg/kg of plant), ß-glucogallin (2214.461 mg/kg of plant), and quercitrin gallate (1160 mg/kg of plant). The importance of the knowledge of plants is increasing and our study may help in the future to formulate nutraceutical preparations and will provide the basis for new investigation into activities of the various compounds found in P. lentiscus.

A metabolite-profiling approach to assess the uptake and metabolism of phenolic compounds from olive leaves in SKBR3 cells by HPLC-ESI-QTOF-MS.

Olive leaves, an easily available natural low-cost material, constitute a source of extracts with significant antitumor activity that inhibits cell proliferation in several breast-cancer-cell models. In this work, a metabolite-profiling approach has been used to assess the uptake and metabolism of phenolic compounds from an olive-leaf extract in the breast-cancer-cell line SKBR3 to evaluate the compound or compounds responsible for the cytotoxic activity. For this, the extract was firstly characterized quantitatively by high-performance liquid chromatography coupled to electrospray ionization-quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS). Then, SKBR3 cells were incubated with 200 µg/mL of the olive-leaf extract at different times (15 min, 1, 2, 24, and 48 h). A metabolite-profiling approach based on HPLC-ESI-QTOF-MS was used to determine the intracellular phenolic compounds, enabling the identification of 16 intact phenolic compounds from the extract and four metabolites derived from these compounds in the cell cytoplasm. The major compounds found within the cells were oleuropein, luteolin-7-O-glucoside and its metabolites luteolin aglycone and methyl-luteolin glucoside, as well as apigenin, and verbascoside. Neither hydroxytyrosol nor any of its metabolites were found within the cells at any incubation time. It is proposed that the major compounds responsible for the cytotoxic activity of the olive-leaf extract in SKBR3 cells are oleuropein and the flavones luteolin and apigenin, since these compounds showed high uptake and their antitumor activity has been previously reported.

Evaluation of different extraction approaches for the determination of phenolic compounds and their metabolites in plasma by nanoLC-ESI-TOF-MS.

Sample preparation is an important step for the determination of phenolic compounds in biological samples. Different extraction methods have been tested to determine phenolic compounds and their metabolites in plasma by nano-liquid chromatography coupled to electrospray ionisation-time-of-flight mass spectrometry (nanoLC-ESI-TOF-MS). The sample treatment optimisation was performed using commercial foetal bovine serum spiked with representative phenolic standards, namely naringenin, luteolin, verbascoside, apigenin, rutin, syringic acid and catechin. Different protein-precipitation conditions were evaluated as well as enzymatic digestion with trypsin and solid-phase extraction using different phases such as C-18, ABN and ENV+, working at different pH values. The optimum extraction procedure consisted of a previous protein-precipitation step using HCl 200 mmol/L in methanol for 2.5 h at 50 °C followed by a solid-phase extraction using C-18 cartridges at pH 2.5. This procedure was finally applied to the plasma of rats overfed with a phenolic-rich Lippia citriodora extract. These samples were analysed by nanoLC-ESI-TOF-MS, enabling the identification of five compounds previously found in the administered L. citriodora extract and one metabolite.

Development of polymeric sensing films based on a tridentate bis(phosphinic amide)-phosphine oxide for detecting europium(III) in water.

A novel europium(III) membrane luminescence sensor based on a tridentate bis(phosphinic amide)-phosphine oxide, PhPO(C(6)H(4)POPhN(CH(CH(3))(2))(2))(2) (1), is described. The new luminescent complex, [Eu(1)(2)]Cl(3)2, which is formed between europium(III) and ligand 1 and has a 1 : 2 stoichiometry, has been evaluated in solution. It has the excellent spectroscopic and chemical characteristics that make it appropriate for sensing film applications. All the parameters (polymer, plasticizer, ligand and ionic additive) that can affect the sensitivity and selectivity of the membrane sensor and instrumental conditions have been carefully optimized. The best sensing response (λ(exc) = 229.04 nm, λ(em) = 616.02 nm) was observed for 33.4 : 65.1 : 1.5 (%, w/w) PVC : DOS : 1. The sensing film shows a good response time (10 min) and a very good selectivity toward europium(III) with respect to other lanthanides(III) ions, such as La, Sm, Tb and Yb. The newly-developed sensing film has a linear range from 1.6 × 10(-7) to 5.0 × 10(-6) mol L(-1) for Eu ions with a very low detection limit (4.8 × 10(-8) mol L(-1)) and good sensitivity (9.41 × 10(-7) a.u. mol(-1) L(-1)) to europium. Complexes of [Eu(1)(2)]Cl(3) (2) and [Eu(1)]Cl(3) (4) were isolated by mixing ligand 1 with Eu(Cl(3))·6H(2)O in acetonitrile at room temperature in ligand : metal molar ratios of 1 : 2 and 1 : 1, respectively. The 1 : 1 derivative is the product of thermodynamic control when a molar ratio of ligand to europium salt of 1 : 1 is used. The new compounds have been characterized in both the solid form (IR, MS-TOF, elemental analysis, TGA and X-ray diffraction) and in solution (multinuclear magnetic resonance). In both europium complexes, the ligand acts as a tridentate chelate. Thermogravimetric (TG) studies demonstrated that neither complex 2 or 4 possess any water molecules directly bound to the lanthanide metal, which corroborates the X-ray structure. The investigation of the solution behaviour of the Y(III) complexes with pulsed gradient spin-echo (PGSE) NMR diffusion measurements showed that average structures with 1 : 1 and 1 : 2 stoichiometries are retained in acetonitrile solutions.

Application of nanoLC-ESI-TOF-MS for the metabolomic analysis of phenolic compounds from extra-virgin olive oil in treated colon-cancer cells.

Crude phenolic extracts (PE) have been obtained from naturally bearing Spanish extra-virgin olive oil (EVOO) showing different polyphenol families such as secoiridoids, phenolic alcohols, lignans, and flavones. EVOO-derived complex phenols (especially from the Arbequina variety olive) have been shown to suppress cell growth of SW480 and HT29 human colon adenocarcinoma cell lines. Inhibition of proliferation by EVOO-PE Arbequina variety extract was accompanied by apoptosis in both colon-cancer-cell lines and a limited G2M cell-cycle arrest in the case of SW480 cells. The metabolized compounds from EVOO-PE in culture medium and cytoplasm of both cell lines were analyzed using nano-liquid chromatography (nanoLC) coupled with electrospray ionization-time-of-flight-mass spectrometry (ESI-TOF-MS). The results showed many phenolic compounds and their metabolites both in the culture medium as well as in the cytoplasm. The main compounds identified from EVOO-PE were hydroxylated luteolin and decarboxymethyl oleuropein aglycone.

Comparison of different extraction procedures for the comprehensive characterization of bioactive phenolic compounds in Rosmarinus officinalis by reversed-phase high-performance liquid chromatography with diode array detection coupled to electrospray time-of-flight mass spectrometry.

In the present work, a comparative study between two environmentally friendly and selective extraction techniques, such as supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE) have been carried out focusing in the bioactive phenolic compounds present in Rosmarinus officinalis. For the analysis of the SFE and PLE extracts, a new methodology for qualitative characterization has been developed, based on the use of reversed-phase high-performance liquid chromatography (RP-HPLC), equipped with two different detection systems coupled in series: diode array detector (DAD) and time of flight mass spectrometry (TOF-MS) detector connected via an electrospray ionization interface (ESI). The use of a small particle size C(18) column (1.8 µm) provided a great resolution and made possible the separation of several isomers. Moreover, UV-visible spectrophotometry is a valuable tool for identifying the class of phenolic compounds, whereas MS data enabled to structurally characterize the compounds present in the extracts. The applied methodology was useful for the determination of many well-known phenolic compounds present in R. officinalis, such as carnosol, carnosic acid, rosmadial, rosmanol, genkwanin, homoplantaginin, scutellarein, cirsimaritin and rosmarinic acid, as well as other phenolic compounds present in other species belonging to Lamiaceae family.

Synthesis and characterization of a molecularly imprinted polymer optosensor for TEXs-screening in drinking water.

A molecularly imprinted polymer (MIP) was synthesized using toluene as template, and was implemented in a fluorescence optosensor (λ(exc)=260 nm, λ(em)=284 nm) for the screening of TEXs (toluene, ethylbenzene and xylenes) in drinking water. All the parameters which can affect the sensitivity and selectivity of the optical sensing phase, were carefully optimized. The screening test runs without the need for any pre-concentration step, thus rendering it suitable for routine use in water-quality-control laboratories. The test recognizes contaminated samples rapidly (81 s) and inexpensively with a cut-off level of 700 µg L(-1) ethylbenzene which corresponds with the maximum contaminant level (MCL) established by US Environmental Protection Agency (EPA) in drinking water. The threshold value of the screening test for the cut-off level was 8.27±0.57 a.u. (95% confidence level, n=10). The reliability of the screening test was 32% false positives and 0% false negatives for 50 samples, and its applicability has been demonstrated by analyzing 15 samples of mineral, tap and river waters obtaining 0% false negatives.

Novel luminescent Ir(III) dyes for developing highly sensitive oxygen sensing films.

New sensing films have been developed for the detection of molecular oxygen. These films are based on luminescent Ir(III) dyes incorporated either into polystyrene (with and without plasticizer) or metal oxide, nanostructured material. The preparation and characterization of each film have been investigated in detail. Due to their high sensitivity for low oxygen concentration, the parameters p(O2) (S=1/2) and DeltaI(1%) have been also evaluated in order to establish the most sensitive membrane for controlling concentrations between 0 and 10% and low oxygen concentrations (lower than 1%), respectively. The results show that the use of nanostructured material increased the sensitivity of the film; the most sensitive membrane for controlling O(2) between 0 and 10% is based on N1001 immobilized in AP200/19 (k(sv)=2848+/-101 bar(-1) and p(O2) (S=1/2)=0.0006), and the complex N969 incorporated into AP200/19 seems to be the most suitable for applications in oxygen trace sensing (DeltaI(1%)=93.13+/-0.13%).

The aqueous extract of Hibiscus sabdariffa calices modulates the production of monocyte chemoattractant protein-1 in humans.

Diet supplementation and/or modulation is an important strategy to significantly improve human health. The search of plants as additional sources of bioactive phenolic compounds is relevant in this context. The aqueous extract of Hibiscus sabdariffa is rich in anthocyanins and other phenolic compounds including hydroxycitric and chlorogenic acids. Using this extract we have shown an effective protection of cultured peripheral blood mononuclear cells from the cellular death induced by H(2)O(2) and a significant role in the production of inflammatory cytokines. In vitro, the extract promotes the production of IL-6 and IL-8 and decreases the concentration of MCP-1 in supernatants in a dose-dependent manner. In humans, the ingestion of an acute dose of the extract (10g) was well tolerated and decreased plasma MCP-1 concentrations significantly without further effects on other cytokines. This effect was not due to a concomitant increase in the antioxidant capacity of plasma. Instead, its mechanisms probably involve a direct inhibition of inflammatory and/or metabolic pathways responsible for MCP-1 production, and may be relevant in inflammatory and chronic conditions in which the role of MCP-1 is well established. If beneficial effects are confirmed in patients, Hibiscus sabdariffa could be considered a valuable traditional herbal medicine for the treatment of chronic inflammatory diseases with the advantage of being devoid of caloric value or potential alcohol toxicity.

High-performance liquid chromatography with diode array detection coupled to electrospray time-of-flight and ion-trap tandem mass spectrometry to identify phenolic compounds from a Cistus ladanifer aqueous extract.

INTRODUCTION: Cistus ladanifer is an aromatic shrub that is widespread in the Mediterranean region. The labdanum exudate is used in the fragrance industry and has been characterised. However, there is not enough information about the phenolic content of the raw plant, the aerial part of it being a very rich source of bioactive compounds. OBJECTIVE: Characterisation of the bioactive compounds of the raw plant and its aerial parts. METHODOLOGY: High-performance liquid chromatography with diode array and electrospray ionisation mass spectrometric detection was used to carry out the comprehensive characterisation of a Cistus ladanifer shrub aqueous extract. Two different MS techniques were coupled to HPLC: time-of-flight mass spectrometry and tandem mass spectrometry. RESULTS: Many well-known compounds present in Cistus ladanifer were characterised, such as flavonoids, phenolic acids, ellagitanins, hexahydroxydiphenoyl and derivatives, and other compounds. CONCLUSION: The method described simultaneously separated a wide range of phenolic compounds and the proposed characterisation of the major compounds of this extract was carried out. It is important to highlight that, to our knowledge, this is the first time that a Cistus ladanifer aqueous extract from the raw plant has been characterised.

High-performance liquid chromatography with diode array detection coupled to electrospray time-of-flight and ion-trap tandem mass spectrometry to identify phenolic compounds from a lemon verbena extract.

High-performance liquid chromatography with diode array and electrospray ionization mass spectrometric detection was used to carry out the comprehensive characterization of a lemon verbena extract with demonstrated antioxidant and antiinflammatory activity. Two different MS techniques have been coupled to HPLC: on one hand, time-of-flight mass spectrometry, and on the other hand, tandem mass spectrometry on an ion-trap. The use of a small particle size C18 column (1.8 microm) provided a great resolution and made possible the separation of several isomers. The UV-visible spectrophotometry was used to delimit the class of phenolic compound and the accurate mass measurements on time-of-flight spectrometer enabled to identify the compounds present in the extract. Finally, the fragmentation pattern obtained in MS-MS experiments confirmed the proposed structures. This procedure was able to determine many well-known phenolic compounds present in lemon verbena such as verbascoside and its derivatives, diglucuronide derivatives of apigenin and luteolin, and eukovoside. Also gardoside, verbasoside, cistanoside F, theveside, campneoside I, chrysoeriol-7-diglucuronide, forsythoside A and acacetin-7-diglucuronide were found for the first time in lemon verbena.

Use of high-performance liquid chromatography with diode array detection coupled to electrospray-Qq-time-of-flight mass spectrometry for the direct characterization of the phenolic fraction in organic commercial juices.

We have developed a direct method for the qualitative analysis of polyphenols in commercial organic fruit juices. The juices were diluted with water (50/50), filtered and directly injected. The analysis of phenolic compounds was carried out by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to photodiode array detection (DAD) and electrospray ionisation-Qq-time-of-flight mass spectrometry (ESI-Qq-TOF-MS). A unique gradient program has been optimized for the separation of several phenolic classes and the analysis time was only 5 min. The fruit juice samples were successfully analysed in positive and negative ionisation modes. In positive mode the anthocyanins were identified whereas the vast majority of polyphenols were identified using the negative ionisation mode. The sensitivity, together with mass accuracy and true isotopic pattern of the Qq-TOF-MS, allowed the identification of the phenolic compounds. Moreover, the advantage of the proposed method is the combined search of MS and MS/MS spectra, which improves the identification of compounds considerably, reducing ambiguities and false positive hits. Therefore the total fragmentation of the compound ion leading to the aglycone ion or other fragments was corroborated by MS-MS. The method was successfully employed to characterize diverse phenolic families in commercially available organic juices from four different fruits and consequently could be used in the future for the quantification purposes to compare different content of polyphenols in juices.

Reversed-phase high-performance liquid chromatography coupled to ultraviolet and electrospray time-of-flight mass spectrometry on-line detection for the separation of eight tetracyclines in honey samples.

Eight antibiotics, chlortetracycline, demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, tetracycline and rolitetracycline, were separated and quantified in Spanish honey extracts of different floral origin using a commercial RP-C18 HPLC column and two different on-line detectors (diode array and electrospray time-of-flight mass spectrometry (ESI-TOF-MS) systems). Operating a linear gradient at a flow of 2 ml min(-1) the HPLC separation of the eight antibiotics was obtained within 10 min with good peak symmetry and an acceptable resolution (2.1) for the critical band pair rolitetracycline and oxytetracycline. Values of the numbers of theoretical plates (N) were comprised between 2328 and 19448 while the limits of detection in honey were within 0.02-1.03 microg kg(-1) in the case of UV detection and 0.05-0.76 microg kg(-1) for ESI-TOF-MS detection (operating in negative mode). A recovery study was carried out by preparing some quality control samples at four levels of concentration (10, 25, 50 and 100 microg kg(-1)) and percentages between 72% and 98% were attained.

Giacomo Castelvetro's salads. Anti-HER2 oncogene nutraceuticals since the 17th century?

We are accumulating evidence to suggest that 17(th) century Renaissance foodways -largely based on the old "Mediterranean dietary traditions"- may provide new nutraceutical management strategies against HER2-positive breast cancer disease in the 21st century. Epidemiological and experimental studies begin to support the notion that "The Sacred Law of Salads" (i.e., "raw vegetables... plenty of generous (olive) oil") -originally proposed in 1614 by Giacomo Castelvetro in its book The Fruit, Herbs & Vegetables of Italy- might be considered the first (unintended) example of customised diets for breast cancer prevention based on individual genetic make-up (i.e., nutraceuticals against human breast carcinomas bearing HER2 oncogene amplification/overexpression). First, the so-called salad vegetables dietary pattern (i.e., a high consumption of raw vegetables and olive oil) appears to exert a protective effect mostly confined to the HER2-positive breast cancer subtype, with no significant influence on the occurrence of HER2-negative breast cancers. Second, all the main olive oil constituents (i.e., the omega-9 monounsaturated fatty acid oleic acid and polyphenolic compounds such as the secoiridoid oleuropein or the lignan 1-[+]-acetoxypinoresinol) dramatically reduce HER2 expression and specifically induce apoptotic cell death in cultured HER2- positive breast cancer cells, with marginal effects against HER2-negative cells. Third, an olive oil-rich diet negatively influences experimental mammary tumorigenesis in rats likewise decreasing HER2 expression levels. If early 1600s Castelvetro's salads can be used as dietary protocols capable to protecting women against biologically aggressive HER2-positive breast cancer subtypes is an intriguing prospect that warrants to be evaluated in human pilot studies in the future. Here, at least, we would like to recognise Giacomo Castelvetro as the father of modern nutritional genomics in oncology.