Anna Lisa Piccinelli

Application of dispersive liquid–liquid microextraction for the determination of aflatoxins B1, B2, G1 and G2 in cereal products

The application of dispersive liquid-liquid microextraction (DLLME) technique for the rapid analysis of aflatoxins B(1), B(2), G(1) and G(2) in maize, rice and wheat products has been evaluated. After extraction of aflatoxins from cereal matrices with a mixture of methanol/water 8:2 (v/v), the analytes were rapidly transferred from the extract to another small volume of organic solvent, chloroform, by DLLME. Aflatoxins were determined using high performance liquid chromatography with florescence detection and photochemical post-column derivatization. Parameters affecting both extraction and DLLME procedures, such as extraction solvent, type and volume of DLLME extractant, volume of water and salt effect, were systematically investigated and optimized to achieve the best extraction efficiency. Under the optimal experimental conditions, the whole analytical method provides enrichment factors around 2.5 times and detection limits (0.01-0.17 μg kg(-1)) below the maximum levels imposed by current regulation for aflatoxins in cereals and cereal products intended for direct human consumption. Recoveries (67-92%) and repeatability (RSD<10, n=3), tested in three different cereal matrices, meet the performance criteria required by EC Regulation No. 401/2006 for the determination of the levels of mycotoxins in foodstuffs. The proposed method was successfully applied to the analysis of retail cereal products with quantitative results comparable to the immunoaffinity chromatography (IAC). The main advantages of developed method are the simplicity of operation, the rapidity to achieve a very high sample throughput and low cost.

Cuban and Brazilian red propolis: botanical origin and comparative analysis by high-performance liquid chromatography-photodiode array detection/electrospray ionization tandem mass spectrometry.

Chemical composition of propolis depends on the specificity of the local flora at the site of collection and thus on the geographic and climatic characteristics of this place. This paper describes a comparative analysis of Cuban red propolis (CRP), Brazilian red propolis (BRP), and Dalbergia ecastophyllum exudates (DEE) by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry. The aim of this study was to investigate the overall chemical profile and the botanical origin of red propolis and to suggest similarities and differences between samples collected in different tropical regions. Isoliquiritigenin (1), liquiritigenin and naringenin (2 and 17), isoflavones (3-4 and 16), isoflavans (5-7 and 18), and pterocarpans (8-13) were detected in CRP, BRP, and DEE, whereas polyisoprenylated benzophenones (PPBs) guttiferone E/xanthochymol (14a,b) and oblongifolin A (15) were detected only in BRP. Pigments responsible for the red color of DEE and red propolis were also identified as two C30 isoflavans, the new retusapurpurin B (19) and retusapurpurin A (20). PPBs and pigments were isolated and unambiguously characterized by 1D and 2D NMR analysis. These results show that red propolis samples from different tropical zones have a similar chemical composition. DEE is the main red propolis source, but the presence of PPBs in BRP suggests the contribution of different botanical sources for Brazilian samples. This chemical information is important for quality control of red propolis and its commercial products and for biological study.

HPLC-PDA-MS and NMR Characterization of C-Glycosyl Flavones in a Hydroalcoholic Extract of Citrus aurantifolia Leaves with Antiplatelet Activity

A hydroalcoholic extract of lime ( Citrus aurantifolia) leaves has been developed in Cuba to be used as a nutritional supplement and phytomedicine in the form of tincture (TLL). A HPLC-PDA-ESI/MS/MS method has been used for the comprehensive analysis of C-glycosyl flavones in TLL. Six C-glycosyl flavones were characterized and, to confirm the proposed structures and to elucidate the nature of the sugar units, a preparative procedure was applied, and isolated compounds were characterized by NMR. Apigenin-6,8-di-C-beta-D-glucopyranoside (vicenin II) (1), diosmetin-6,8-di- C-beta- d-glucopyranoside (2), apigenin-8-C-beta-D-glucopyranoside (vitexin) (3), apigenin-8-C-[alpha-L-arabinopyranosyl-(1-->6)]-O-beta-D-glucopyranoside (4), apigenin-6-C-[alpha-l-arabinopyranosyl-(1-->6)]-O-beta-D-glucopyranoside (5). and apigenin-6-C-beta-D-glucopyranoside (isovitexin) (6) were identified in TLL and quantified by HPLC-PDA. Compounds 4 and 5 were two new arabinosyl derivatives of vitexin and isovitexin. Inhibitor effect of TLL on platelet aggregation induced by physiological agonists of platelets was evaluated in human plasma. TLL inhibited significantly ADP and epinephrine-induced platelet aggregation in a concentration-dependent manner (IC 50=0.40 and 0.32 mg/mL, respectively).

Studies on the constituents of yellow Cuban propolis: GC-MS determination of triterpenoids and flavonoids.

In this study, on the basis of the information supplied by NMR and HPLC-PDA data, we reported a quali-quantitative GC-MS study of 19 yellow Cuban propolis (YCP) samples collected in different regions of Cuba. The profiles of YCP samples allowed us to define two main types of YCP directly related to their secondary metabolite classes: type A, rich in triterpenic alcohols and with the presence of polymethoxylated flavonoids as minor constituents, and type B, containing acetyl triterpenes as the main constituents. For the first time, triterpenoids belonging to oleanane, lupane, ursane, and lanostane skeletons were reported as major compounds in propolis. Also, the presence of polymethoxylated flavones or flavanones was found for the first time in propolis.

The identification of a novel natural activator of p300 histone acetyltranferase provides new insights into the modulation mechanism of this enzyme.

Many severe human pathologies are related to alterations of the fine balance between histone acetylation and deacetylation; because not all such diseases involve hypoacetylation, but also hyperacetylation, compounds able to enhance or repress the activities of histone acetyltransferases (HATs) could be promising therapeutic agents. We evaluated in vitro and in cell the ability of eleven natural polyisoprenylated benzophenone derivatives to modulate the HAT activity of p300/CBP, an enzyme that plays a pivotal role in a variety of cellular processes. Some of the tested compounds bound efficiently to the p300/CBP protein: in particular, guttiferone A, guttiferone E and clusianone inhibit its HAT activity, whereas nemorosone showed a surprising ability to activate the enzyme. The ability of nemorosone to penetrate cell membranes and modulate histone acetylation into the cell together with its high affinity for the p300/CBP enzyme made this compound a suitable lead for the design of optimized anticancer drugs. Besides, the studies performed at a cellular and molecular level on both the inhibitors and the activator provided new insights into the modulation mechanism of p300/CBP by small molecules.

Determination of phenolic compounds in honey using dispersive liquid-liquid microextraction.

Honey is a valuable functional food rich in phenolic compounds with a broad spectrum of biological activities. Analysis of the phenolic compounds in honey is a very promising tool for the quality control, the authentication and characterization of botanical origin, and the nutraceutical research. This work describes a novel approach for the rapid analysis of five phenolic acids and 10 flavonoids in honey. Phenolic compounds were rapidly extracted and concentrated from diluted honey by dispersive liquid-liquid microextraction (DLLME) and then analyzed using high performance liquid chromatography with UV absorbance detection (HPLC-UV). Some important parameters, such as the nature and volume of extraction and dispersive solvents, pH and salt effect were carefully investigated and optimized to achieve the best extraction efficiency. Under the optimal conditions, an exhaustive extraction for twelve of the investigated analytes (recoveries >70%), with a precision (RSD<10%) highly acceptable for complex matrices, and detection and quantification limits at ppb levels (1.4-12 and 4.7-40ngg(-1), respectively) were attained. The proposed method, compared with the most widely used method in the analysis of phenolic compounds in honey, provided similar or higher extraction efficiency, except in the case of the most hydrophilic phenolic acids. The capability of DLLME to the extraction of other honey phytochemicals, such as abscisic acid, was also demonstrated. The main advantages of developed method are the simplicity of operation, the rapidity to achieve a very high sample throughput and low cost.

GC-MS determination of isoflavonoids in seven red Cuban propolis samples.

In the present study, the phenolic composition analysis of seven red varieties of propolis, collected in different regions of Cuba, was evaluated by gas chromatography/mass spectrometry (GC-MS). Seventeen compounds were identified in all samples by the interpretation of their mass spectra. This appears to be the first report on the GC-MS analysis of isoflavonoids in the propolis. The results confirmed the presence of the main isoflavonoids isolated previously and suggested the general structure for the other five isoflavonoids. Vestitol, 7-O-methylvestitol, and medicarpin were present in high amounts in all propolis samples analyzed. This result indicates that propolis samples rich in isoflavonoids are not exclusively found in Pinar del Rio province and proves that GC-MS technique is a useful and alternative tool for the chemical analysis of tropical red propolis.

Ultra-preconcentration and determination of selected pharmaceutical and personal care products in different water matrices by solid-phase extraction combined with dispersive liquid-liquid microextraction prior to ultra high pressure liquid chromatography tandem mass spectrometry analysis.

Pharmaceutical and personal care products (PPCPs) are one of the most important classes of emerging contaminants. The potential of ecological and environmental impacts associated with PPCPs are of particular concern because they continually penetrate the aquatic environment. This work describes a novel ultra-preconcentration technique for the rapid and highly sensitive analysis of selected PPCPs in environmental water matrices at ppt levels. Selected PPCPs were rapidly extracted and concentrated from large volumes of aqueous solutions (500 and 250mL) by solid-phase extraction combined with dispersive liquid-liquid microextraction (SPE-DLLME) and then analyzed using UHPLC-MS/MS. Experimental parameters were carefully investigated and optimized to achieve the best SPE-DLLME efficiency and higher enrichment factors. The best results were obtained using the ternary mixture acetonitrile/methanol/dichloromethane 3:3:4, v/v/v, both as SPE eluent and DLLME extractant/dispersive mixture. DLLME aqueous solution (5% NaCl, 10mgL(-1) TBAB) was also modified to improve the extraction efficiency of more hydrophilic PPCPs. Under the optimal conditions, an exhaustive extraction for most of the investigated analytes (recoveries >70%), with a precision (RSD <10%) and very high enrichment factors were attained for different aqueous matrices (drinking, sea, river and wastewater). Method detection and quantification limits were at very low ppt levels and below 1 and 3ngL(-1), respectively, for 15 of selected PPCPs. The proposed analytical procedure offers numerous advantages such as the simplicity of operation, rapidity, a high enrichment factor and sensitivity. So it is suitable for monitoring and studies of occurrence of PPCPs in different environmental compartments.

Application of pressurized liquid extraction in the analysis of aflatoxins B1, B2, G1 and G2 in nuts.

Aflatoxins (AFs) B(1), B(2), G(1) and G(2) were extracted from nuts by using pressurized liquid extraction (PLE) and the PLE extracts were analyzed using HPLC with fluorescence detection using photochemical post-column derivatization without further cleanup procedures. Several extraction parameters such as temperature (25, 40, 60 and 80 degrees C), pressure (500, 1000, 1500 and 2000 psi), solvent extraction mixture (acetone, acetonitrile, ethyl acetate and methanol), number of cycles (1 and 2), use of dispersing agents and cell size (5 and 11 mL) were investigated for their effects on the extraction performance. The results showed 60 degrees C, 1500 psi, acetonitrile, one cycle and a cell size of 5 mL as most favorable PLE operating conditions. The proposed analytical method provides LODs below the maximum levels established by European Union regulations and the recoveries of the four AFs were between 77 and 93% at spiking levels of 4, 2 and 0.5 microg/kg for AFB(1) and AFG(1) and 1, 0.5 and 0.13 microg/kg for AFB(2) and AFG(2). Validation was carried out using certified reference materials. PLE has been applied for the first time to the analysis of AFs in nuts and offers the possibility for fast simple and accurate quantitative determination of studied mycotoxins.

pH-controlled dispersive liquid-liquid microextraction for the analysis of ionisable compounds in complex matrices: Case study of ochratoxin A in cereals.

A new sample preparation procedure, termed pH-controlled dispersive liquid-liquid microextraction (pH-DLLME), has been developed for the analysis of ionisable compounds in highly complex matrices. This DLLME mode, intended to improve the selectivity and to expand the application range of DLLME, is based on two successive DLLMEs conducted at opposite pH values. pH-DLLME was applied to determination of ochratoxin A (OTA) in cereals. The hydrophobic matrix interferences in the raw methanol extract (disperser, 1mL) were removed by a first DLLME (I DLLME) performed at pH 8 to reduce the solubility of OTA in the extractant (CCl(4), 400μL). The pH of the aqueous phase was then adjusted to 2, and the analyte was extracted and concentrated by a second DLLME (extractant, 150μL C(2)H(4)Br(2)). The main factors influencing the efficiency of pH-DLLME including type and volume of I DLLME extractant, as well as the parameters affecting the OTA extraction by II DLLME, were studied in detail. Under optimum conditions, the method has detection and quantification limits of 0.019 and 0.062μg kg(-1), respectively, with OTA recoveries in the range of 81.2-90.1% (n=3). The accuracy of the analytical procedure, evaluated with a reference material (cereal naturally contaminated with OTA), is acceptable (accuracy of 85.6%±1.7, n=5). The applicability of pH-DLLME to the selective extraction of other ionisable compounds, such as acidic and basic pharmaceutical products was also demonstrated. The additional advantages of pH-DLLME are a higher selectivity and the extension of this microextraction technique to highly complex matrices.