Rita Celano

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.

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.

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.

Chemical composition and antioxidant activity of Algerian propolis.

Chemical composition of propolis samples from north Algeria was characterized by chromatographic and spectroscopic analyses. High-performance liquid chromatorgaphy with diode-array detection (HPLC-DAD) fingerprint of the methanol extracts allowed the definition of two main types of Algerian propolis (AP) directly related to their secondary metabolite composition. Investigation of two representative types of AP by preparative chromatographic procedure and mass spectrometric (MS) and NMR techniques led to the identification of their main constituents: caffeate esters and flavonoids from an AP type rich in phenolic compounds (PAP) and labdane and clerodane diterpenes, together with a polymethoxyflavonol, from an AP type containing mainly diterpenes (DAP). Subsequently, two specific HPLC-MS/MS methods for detection of PAP and DAP markers were developed to study the chemical composition of propolis samples of different north Algerian regions. Antioxidant activity of AP samples was evaluated by 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay, and a significant free-radical scavenging effect was observed for propolis of the PAP series rich in polyphenols.

Rapid and automated analysis of aflatoxin M1 in milk and dairy products by online solid phase extraction coupled to ultra-high-pressure-liquid-chromatography tandem mass spectrometry.

This study reports a fast and automated analytical procedure for the analysis of aflatoxin M1 (AFM1) in milk and dairy products. The method is based on the simultaneous protein precipitation and AFM1 extraction, by salt-induced liquid-liquid extraction (SI-LLE), followed by an online solid-phase extraction (online SPE) coupled to ultra-high-pressure-liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis to the automatic pre-concentration, clean up and sensitive and selective determination of AFM1. The main parameters affecting the extraction efficiency and accuracy of the analytical method were studied in detail. In the optimal conditions, acetonitrile and NaCl were used as extraction/denaturant solvent and salting-out agent in SI-LLE, respectively. After centrifugation, the organic phase (acetonitrile) was diluted with water (1:9 v/v) and purified (1mL) by online C18 cartridge coupled with an UHPLC column. Finally, selected reaction monitoring (SRM) acquisition mode was applied to the detection of AFM1. Validation studies were carried out on different dairy products (whole and skimmed cow milk, yogurt, goat milk, and powder infant formula), providing method quantification limits about 25 times lower than AFM1 maximum levels permitted by EU regulation 1881/2006 in milk and dairy products for direct human consumption. Recoveries (86-102%) and repeatability (RSD<3, n=6) meet the performance criteria required by EU regulation N. 401/2006 for the determination of the levels of mycotoxins in foodstuffs. Moreover, no matrix effects were observed in the different milk and dairy products studied. The proposed method improves the performance of AFM1 analysis in milk samples as AFM1 determination is performed with a degree of accuracy higher than the conventional methods. Other advantages are the reduction of sample preparation procedure, time and cost of the analysis, enabling high sample throughput that meet the current concerns of food safety and the public health protection.

Chemical profile and cellular antioxidant activity of artichoke by-products

Artichoke by-products, produced from agricultural procedures and the processing industry, represent a huge amount of discarded material. In this research, the main artichoke by-products, bracts and leaves, were characterized in terms of their bioactive constituents (phenolic compounds and inulin) and cellular antioxidant potential to estimate their nutraceutical potential. The ultrahigh-performance-ultraviolet detection-high resolution mass spectroscopy (UHPLC-UV-HRMS) profiles of both by-products show that 5-caffeoylquinic acid and 1,5-dicaffeoylquinic acid are the most abundant bioactive compounds, and the content of flavone glycosides can be used to discriminate between bracts and leaves. Artichoke by-products contain a remarkable overall phenolic content (0.5-1.7 g per 100 g dry matter), whereas they differ widely in the amounts of inulin with higher levels in bracts (3.8-8.2 g per 100 g dry matter). The cellular antioxidant activities of bract and leaf extracts (half maximal effective concentration (EC50) = 26.6-124.1 mg L-1) are better than or similar to that of a commercial leaf extract, and are related to the dicaffeoylquinic acid levels, particularly to 1,5-dicaffeoylquinic acid. These results reveal that artichoke by-products are a promising and cheap source of bioactive compounds. Bracts could be used as a source of inulin and caffeoylquinic acids for the production of food additives and nutraceuticals and also as an alternative to the traditional application of leaf extracts.

Rapid and automated on-line solid phase extraction HPLC–MS/MS with peak focusing for the determination of ochratoxin A in wine samples

This study reports a fast and automated analytical procedure based on an on-line SPE-HPLC-MS/MS method for the automatic pre-concentration, clean up and sensitive determination of OTA in wine. The amount of OTA contained in 100μL of sample (pH≅5.5) was retained and concentrated on an Oasis MAX SPE cartridge. After a washing step to remove matrix interferents, the analyte was eluted in back-flush mode and the eluent from the SPE column was diluted through a mixing Tee, using an aqueous solution before the chromatographic separation achieved on a monolithic column. The developed method has been validated according to EU regulation N. 519/2014 and applied for the analysis of 41 red and 17 white wines. The developed method features minimal sample handling, low solvent consumption, high sample throughput, low analysis cost and provides an accurate and highly selective results.

Countercurrent chromatography separation of saponins by skeleton type from Ampelozizyphus amazonicus for off-line ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry analysis and characterisation

Ampelozizyphus amazonicus Ducke (Rhamnaceae), a medicinal plant used to prevent malaria, is a climbing shrub, native to the Amazonian region, with jujubogenin glycoside saponins as main compounds. The crude extract of this plant is too complex for any kind of structural identification, and HPLC separation was not sufficient to resolve this issue. Therefore, the aim of this work was to obtain saponin enriched fractions from the bark ethanol extract by countercurrent chromatography (CCC) for further isolation and identification/characterisation of the major saponins by HPLC and MS. The butanol extract was fractionated by CCC with hexane - ethyl acetate - butanol - ethanol - water (1:6:1:1:6; v/v) solvent system yielding 4 group fractions. The collected fractions were analysed by UHPLC-HRMS (ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry) and MSn. Group 1 presented mainly oleane type saponins, and group 3 showed mainly jujubogenin glycosides, keto-dammarane type triterpene saponins and saponins with C31 skeleton. Thus, CCC separated saponins from the butanol-rich extract by skeleton type. A further purification of group 3 by CCC (ethyl acetate - ethanol - water (1:0.2:1; v/v)) and HPLC-RI was performed in order to obtain these unusual aglycones in pure form.

A new cineol derivative, polyphenols and norterpenoids from Saharan myrtle tea (Myrtus nivellei): Isolation, structure determination, quantitative determination and antioxidant activity

The phytochemical profile of decoction and infusion, obtained from the dried leaves of M. nivellei, consumed as tea in Saharan region, was characterized by UHPLC-PDA-HRMS. Fourteen compounds were characterized and, to confirm the proposed structures a preparative procedure followed by NMR spectroscopy was applied. Compound 3 (2-hydroxy-1,8-cineole disaccharide) was a never reported whereas a bicyclic monoterpenoid glucoside (2), two ionol glucosides (1 and 12), a tri-galloylquinic acid (4), two flavonol glycosides (5 and 9), and a tetra-galloylglucose (7), were reported in Myrtus spp. for the first time. Five flavonol O-glycosides (6, 8, 10-11, and 14) togheter a flavonol (13) were also identified. Quantitative determination of phenolic constituents from decoction and infusion has been performed by HPLC-UV-PDA. The phenolic content was found to be 150.5 and 102.6mg/g in decoction and infusion corresponding to 73.8 and 23.6mg/100mL of a single tea cup, respectively. Myricetin 3-O-β-d-(6″-galloyl)glucopyranoside (5), isomyricitrin (6) and myricitrin (8) were the compounds present in the highest concentration. The free-radical scavenging activities of teas and isolated compounds was measured by the DPPH assay and compared with the values of other commonly used herbal teas (green and black teas). Decoction displayed higher potency in scavenging free-radicals than the infusion and green and black teas.

Oil distillation wastewaters from aromatic herbs as new natural source of antioxidant compounds

Distillation wastewaters (DWWs) are generated during the essential oil steam distillation from aromatic herbs. Despite of growing interest on novel source of natural antioxidant compounds as food additives, studies on DWWs are scarse. Herein, the potential of DWWs produced by the distillation of packaged fresh basil, rosemary and sage wastes was evaluated by chemical and antioxidant characterization. HPLC-DAD-HRMS profiling revealed that DWWs contain water-soluble phenolic compounds, mainly caffeic acid derivatives and flavonoid glycosides, with rosmarinic acid (RA) as predominant components (29-135mg/100mL). DWWs demonstrated high levels of total phenolic compounds (TPC, 152-443mg GAE/100mL) and strong antioxidant capacities, in ORAC, DPPH and ABTS assays (1101-4720, 635-4244 and 571-3145μmol TE/100mL, respectively). Highly significant correlations of TEAC values with TPC and RA contents revealed that phenolic compounds and high RA content were responsible of DWWs antioxidant properties.Thus, DWWs are proposed as a new promising source of natural food additives and/or functional ingredients for cosmetic, nutraceutical and food applications.