Bioavailability of Aflatoxins in Cultured Fish and Animal Livers Using an In Vitro Dialyzability Approach.

The objective of the present study was to investigate the bioavailability of aflatoxins (AFs) from fish, and chicken and rabbit livers using an in vitro dialyzability approach. Ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to assess the aflatoxin content in samples, as well as in dialyzate and residue fractions after the in vitro procedure. A vortex-assisted dispersive liquid-liquid microextraction (VALLME) technique was used for preconcentrating AFs before determination. Raw samples showed bioavailability ratios of 41-45% for aflatoxin B1 (AFB1), 28-38% for aflatoxin B2 (AFB2), and 42% for aflatoxin G2 (AFG2). Aflatoxin G1 (AFG1) was not detected. The culinary process (steaming or grilling) was found to change AFs' bioavailability (higher bioavailability ratios were found in cooked samples). AFB2 was found to be transformed into other compounds during the in vitro process, and the presence of AFB2 and AFB2 transformation/degradation products was investigated and confirmed by high-resolution mass spectrometry (HRMS).

Caco-2 in vitro model of human gastrointestinal tract for studying the absorption of titanium dioxide and silver nanoparticles from seafood.

Titanium dioxide nanoparticles (TiO2 NPs) are widely used in industry as a white pigment (paints, paper industry and toothpastes), photocatalysts (environmental decontamination and photovoltaic cells), inorganic UV filter (sunscreens and personal care products) and as a food additive (E171) and antimicrobial food packaging material. Silver nanoparticles (Ag NPs) are used in photonics, microelectronics, catalysis and medicine due to their catalytic activity, magnetic and optical polarizability, electrical and thermal conductivities and enhanced Raman scattering. They also have antibacterial, antifungal and antiviral activities, as well as anti-inflammatory potential. The huge increase in the use of nano-based products, mainly metallic NPs, implies the presence of nanomaterials in the environment, and hence, the unintentional human ingestion through water or foods (gastrointestinal tract is the main pathway of NPs intake in humans). The presence of TiO2 NPs and Ag NPs in seafood samples was firstly established using an ultrasound assisted enzymatic hydrolysis procedure and sp-ICP-MS analysis. Several clams, cockles, mussels, razor clams, oysters and variegated scallops, which contain TiO2 NPs and Ag NPs, were subjected to an in vitro digestion process simulating human gastrointestinal digestion in the stomach and in the small and large intestine to determine the bioaccessibility of these NPs. Caco-2 cells were selected as model of human intestinal epithelium for transport studies because of the development of membrane transporters that are responsible for the uptake of chemicals. Parameters as transepithelial electrical resistance (TEER) and permeability of Lucifer Yellow were studied for establishing cell monolayer integrity. TiO2 NPs and Ag NPs transport as well as total Ti and Ag concentrations passing through the gastrointestinal epithelial barrier model (0-2 h) were assessed by sp-ICP-MS and ICP-MS in several molluscs.

Biopersistence rate of metallic nanoparticles in the gastro-intestinal human tract (stage 0 of the EFSA guidance for nanomaterials risk assessment).

The European Food Safety Authority has published a guidance regarding risk assessment of nanomaterials in food and feed. Following these recommendations, an in vitro gastrointestinal digestion has been applied to study the biopersistence of TiO2 and Ag NPs in standards, molluscs and surimi. TiO2 NPs standards and TiO2 NPs/ TiO2 microparticles from E171 were not found to be degraded. Ag NPs proved to be more degradable than TiO2 NPs, but the biopersistence rates were higher than 12%, which means that Ag NPs are also biopersistent. Findings for seafood are quite similar to those obtained for TiO2 NPs and Ag NPs standards, although the calculation of the biopersistence rate proposed by the EFSA was not found to be straightforward for foodstuff (the use of the NPs concentration in the sample instead of the NPs concentration at initial time (sample mixed with the gastric solution before enzymatic hydrolysis) has been proposed.

In vitro assessment of major and trace element bioaccessibility in tea samples.

Bioaccessibility of trace elements (Li, Be, Ti, Ga, Cu, Ag, Hg, Cd, Cs, Pt, Tl, Pb, As, Cr, Co, Ni, V, Se, Sn and Sb) and major elements (Rb, Ba, Al, Fe, Zn, Si, Ca, Mg, Mn, Mo, Sr, P and K) in tea infusions has been assessed using an in vitro dialyzability protocol. Gastric simulation (using pepsin solution) and intestinal simulation (using pancreatin and bile salts) were used to perform the in vitro digestion. ICP-MS, ICP-OES and FAES were used for elements determination in digested tea leaves, their infusions and the dialyzate fractions from tea infusions. Microwaves assisted acid digestion was used for the total element determination in tea leaves, while tea infusions were prepared by brewing tea leaves for 5 min in boiling water. The LODs for elements determined in tea leaves were in the range of 0.11-656 ng g-1 and 0.02-145.6 µg g-1 for trace and major elements, respectively. For elements' determination in tea infusions, the LODs were ranged between 0.23 and 399.9 ng L-1 for trace elements and 0.2-1248 µg L-1 for major elements. The LODs for the elements in the dialyzable fraction varied from 0.018 to 142 µg L-1. The accuracy of the total element determination was evaluated using certified reference materials (Tea Leaves INCT-TL-1 and Rye Grass). The analytical recoveries were also assessed for analyzed elements in digested tea leaves (95-114%) and their infusions (92-115%), showing good recoveries. Among the studied elements, K was the most abundant element in tea leaves and tea infusions in almost all samples, followed by Ca, Mg, and P. Zn, Cs, and K showed the highest dialyzability percentages up to 84%, 76%, and 54%, respectively, followed by Si and Ca and K that show moderate to high dialyzability percentages. The accuracy of the dialysis process was evaluated using a mass-balance study.

Miniaturized vortex assisted-dispersive molecularly imprinted polymer micro-solid phase extraction and HPLC-MS/MS for assessing trace aflatoxins in cultured fish.

A dispersive micro-solid phase extraction approach using a molecularly imprinted polymer as an adsorbent has been developed for pre-concentrating aflatoxins from cultured fish. Aflatoxins were first isolated from fish muscle and liver by an ultrasound assisted extraction procedure using a 60 : 40 acetonitrile/0.1 M KH2PO4 aqueous buffer (pH 6.0) mixture. Polymeric adsorbent beads were synthesized using 5,7-dimethoxycoumarin as a dummy template, methacrylic acid as a functional monomer, divinylbenzene as a crosslinker, and 2,2'-azobisisobutyronitrile as an initiator. Parameters affecting the steps of extraction procedure, including the sample (fish extract) pH, adsorption stirring speed and time, desorption stirring speed and time, elution solvent ratio, and polymer capacity, were investigated and optimized. The limit of detection was found to vary from 0.29 to 0.61 µg kg-1 for the several aflatoxins. The proposed method was shown to be accurate and precise. Intraday and inter-day relative standard deviations were lower than 20%, and intraday and inter-day analytical recoveries were within the 80-100% range. The prepared adsorbent in the dispersive micro-solid phase extraction format was re-usable, and the pre-concentration procedure was found to be simple, rapid and highly selective and sensitive to identify/quantify AFs in fish.

Room temperature phosphorescent determination of aflatoxins in fish feed based on molecularly imprinted polymer - Mn-doped ZnS quantum dots.

Possibilities of room temperature spectrometry based on Mn-doped ZnS quantum dots coated with a molecularly imprinted polymer based nanosensor have been explored for the sensitive and selective determination of aflatoxins. Synthesized polymeric nanoparticles exhibit intense room temperature phosphorescence (total decay time of 0.004 s) and aflatoxins quench the room temperature phosphorescence when interact with the recognition cavities of the molecularly imprinted polymer attached to the phosphorescent quantum dots. Room temperature phosphorescence was recorded by scanning from 520 nm to 720 nm (maximum peak intensity at 594 nm) after excitation at 290 nm. The prepared imprinted material was found to have higher adsorption capacity than those based non-imprinted quantum dots, demonstrating high adsorption uptake for aflatoxins. In addition, selectivity studies have demonstrated that the material offers a specific recognition for aflatoxins. Room temperature phosphorescence quenching by aflatoxins was found to be linear within the 2-20 µg L-1 range, and a limit of detection of 3.56 µg kg-1 was obtained. This value was lower than the maximum acceptable/residual level (aflatoxins in feeds) published by the European Commission. The results indicate a simple room temperature phosphorescence nanosensor for aflatoxins detection in fish feed as a versatile tool having excellent sensitivity and selectivity.

Combining ultrasound-assisted extraction and vortex-assisted liquid-liquid microextraction for the sensitive assessment of aflatoxins in aquaculture fish species.

Although aflatoxins contamination in feedstuff is a well-known problem, and hence these residues are controlled in poultry products, there is scarce information regarding the presence of these toxic substances in aquaculture fish, facilities that use several feedstuff for fish breeding. A simple, rapid, and sensitive method has been therefore developed for aflatoxins (B1, B2, G1, and G2) assessment in aquaculture products by combining ultrasound probe-assisted extraction and vortex-assisted liquid-liquid microextraction as a sample pretreatment, and high-performance liquid chromatography-tandem mass spectrometry as a separation/detection system. Aflatoxins were extracted from fish flesh/liver with a 60:40 acetonitrile/aqueous phosphate buffer (pH 7.0) mixture before preconcentration and clean-up by vortex-assisted liquid-liquid microextraction under the following optimized conditions: 5.0 mL of fish extract at pH 7.0 and NaCl at 0.5% (w/v), 400 µL of chloroform as extracting solvent, and vortex shaking at 2000 rpm for 1 min. The proposed method is shown to be precise and accurate, and the limit of quantitations (from 0.20 to 1.10 µg kg-1 ) were lower than the value established by the European Commission Regulation for aflatoxins in foodstuff. Results have shown that fish flesh is free of aflatoxins, but aflatoxins B2 and G1 were quantified in fish liver.

Ultrasound assisted combined molecularly imprinted polymer for the selective micro-solid phase extraction and determination of aflatoxins in fish feed using liquid chromatography-tandem mass spectrometry.

A combined procedure based on using ultrasounds for target isolation followed by porous membrane-protected micro solid phase extraction using a molecularly imprinted polymer as an adsorbent has been developed as a highly selective extraction and clean-up procedure for isolating aflatoxins B1, B2, G1, and G2 from fish feed before ultra-high-performance liquid chromatography tandem mass spectrometry determination. Polymeric adsorbent beads have been synthesized by the precipitation polymerization method which guarantees a homogeneous particles size distribution and the integrity of the generated imprinted cavities. In addition, polymerization was performed using a higher proportion of organic solvent (toluene) in the porogen mixture, which generates MIP particles adequate for interacting with targets dissolved in organic (hydro-organic) mixtures (extracts from fish feed). These approaches led to a selective and high efficient pre-concentration method for AFs. Ultrasound-assisted extraction (10 mL of 60:40 acetonitrile/0.1 M KH2PO4 pH 6.0, 40% amplitude, continuous sonication for 7.0 min) allowed an efficient aflatoxins isolation from fish feed. In addition, the resulting pH of the extract (pH 7.0) has been found to be the optimum for performing clean-up/pre-concentration (enrichment factor of 33.3) by molecularly imprinted polymer based micro-solid phase extraction (orbital horizontal shaking speed at 150 rpm for 10 min for loading, and 5 mL of 95:5 acetonitrile/formic acid as eluting solution using ultrasounds 35 kHz for 15 min). The current proposal was shown to be an accurate and precise method through relative standard deviation of intraday and inter-day tests below 20% and analytical recoveries in the range of 80-100%. The limits of detection were within the 0.42-1.15 µg kg-1 range, quite lower than those established by European Commission guidelines for aflatoxins in animal feeds.

Enzymatic hydrolysis as a sample pre-treatment for titanium dioxide nanoparticles assessment in surimi (crab sticks) by single particle ICP-MS.

A reliable sample pre-treatment based on enzymatic hydrolysis has been fully optimized and validated for TiO2 NPs isolation and determination/characterization in surimi (crab sticks). Efficient extractions have been found when using a pancreatin/lipase mixture at pH 7.4 and 37 °C for 12 h under continuous stirring. The proposed sample pre-treatment procedure has been found not to change TiO2 NPs size distribution, therefore guaranteeing TiO2 NPs integrity. TiO2 NPs determination (TiO2 NPs concentration) and TiO2 NPs characterization (size distribution) were assessed by single-particle inductively coupled plasma mass spectrometry (sp-ICP-MS) working with dwell times in the microsecond range (high frequency of data acquisition). Method validation was performed for TiO2 NPs concentrations and TiO2 NPs sizes. Good repeatability (25% and 8% for TiO2 NPs concentration and TiO2 NPs most frequent size), and sensitivity (limit of detection of 5.28 × 105 NPs g-1for TiO2 NPs concentrations, and 31.3-37.1 nm for TiO2 NPs size) were obtained. Accuracy, calculated through analytical recovery was adequate. Recoveries for TiO2 NPs standards of 50 and 100 nm were 108 ±â€¯5 and 105 ±â€¯4%, respectively. The proposed methodology was applied to several surimi samples for assessing TiO2 NPs concentrations and size distribution. Some surimi samples were found to contain TiO2 NPs (concentrations from 1.40 × 107 to 1.19 × 109 NPs g-1). TiO2 NPs size distributions were very different among the samples, and some of them showed wide size ranges (the most frequent size varied from 53.8 to 62.1 nm; whereas, the mean size values were within the 73.4-217.5 nm range).

In vitro human bioavailability of major, trace and ultra-trace elements in Chilean 'natural' wines from Itata Valley.

In vitro human bioavailability of elements in 'natural' wines from Chile's Itata Valley has been assessed using an in vitro dialyzability approach. The red wines (fifteen samples) were of the Cinsault, Cabernet sauvignon, Carmènére, Malbec, and Pinot noir varieties. All white wines (three samples) were of the Muscat of Alexandria variety. Inductively coupled plasma-mass spectrometry was used for determination. Elements such as Ag, As, Be, Cd, Cr, Hg, Mo, Ni, Pb, Sb, Se, Sn, Ti, Tl, and V were not found to be bioavailable (concentrations lower than the limit of detection in the dialysates). Elements such as Al, Co, and Fe showed low bioavailability ratios (lower than 20%), whereas B, K, Li, Mg, and Mn were found to be of moderate bioavailability (bioavailability ratios within the 20-79% range for most wine samples). Ca, Cu, and Sr bioavailability was moderate (higher than 20%) in some wines, but most of the samples showed Ca, Cu, and Sr bioavailabilty ratios lower than 20%. No differences were found regarding bioavailability ratios among red and white wines, or among the grape varieties.

Polyphenol bioavailability in nuts and seeds by an in vitro dialyzability approach.

An in vitro dialyzability approach has been undertaken to elucidate the bioavailable fraction of the total polyphenols (TPs) of edible nuts and seeds. The TP contents in samples and in dialyzates were assessed by the Folin-Ciocalteu spectrophotometric method. Antioxidant activity was determined in selected samples, using a modified method against Trolox®. TPs and antioxidant activity in nuts/seeds were determined after applying a pressurized liquid extraction sample pre-treatment. High dialyzability ratios were assessed in most nuts/seeds (TP dialyzability percentages within the 25-91% range). The highest TP dialyzability ratios were found in raw Brazil nuts (81 ±â€¯5%), toasted pistachios (88 ±â€¯9%), and fried cashews (89 ±â€¯9%), whereas TPs in pumpkin seeds were found to be very low (TPs were not detected in the dialyzable fraction). TP dialyzability was correlated with the copper content in nuts and seeds.

Bioavailability assessment of essential and toxic metals in edible nuts and seeds.

Bioavailability of essential and toxic metals in edible nuts and seeds has been assessed by using an in vitro dialyzability approach. The samples studied included walnuts, Brazil nuts, Macadamia nuts, pecans, hazelnuts, chestnuts, cashews, peanuts, pistachios and seeds (almond, pine, pumpkin and sunflower). Metals were measured by inductively coupled plasma-mass spectrometry in dialyzates and also in samples after a microwave assisted acid digestion pre-treatment. Low dialyzability percentages were found for Al, Fe and Hg; moderate percentages were found for Ba, Ca, Cd, Co, Cu, K, Li, Mg, Mn, Mo, P, Pb, Se, Sr, Tl and Zn; and high dialyzability ratios were found for As, Cr and Ni. The highest dialyzability percentages were found in raw chestnuts and raw hazelnuts. Metal dialyzability was found to be negatively affected by fat content. Positive correlation was found between carbohydrate content and metal dialyzability ratios. Protein and dietary fibre content did not influence metal bioavailability. Predicted dialyzability for some metals based on fat and protein content could also be established.

Mercury speciation in seawater by liquid chromatography-inductively coupled plasma-mass spectrometry following solid phase extraction pre-concentration by using an ionic imprinted polymer based on methyl-mercury-phenobarbital interaction.

Trace levels of inorganic mercury, methyl-mercury and ethyl-mercury have been assessed in seawater by high performance liquid chromatography (HPLC) hyphenated with inductively coupled plasma-mass spectrometry (ICP-MS) after solid phase extraction (SPE) pre-concentration with a novel synthesized ionic imprinted polymer. The adsorbent material was prepared by trapping a non-vinylated chelating ligand (phenobarbital) via imprinting of a ternary mixed ligand complex of the non-vinylated chelating agent, the template (methyl-mercury), and the vinyl ligand (metacrylic acid, MAA). Ethylene dimetacrylate (EDMA) and 2,2'-azobisisobutyronitrile (AIBN) were used as cross-linker and initiator reagents, respectively; and the precipitation polymerization technique was used in a porogen of acetonitrile/water (4:1). The best retention properties for methyl-mercury, inorganic mercury and ethyl-mercury species from seawater were obtained when loading 200 mL of sample adjusted to pH 8.0 and at a flow rate of 2.0 mL min(-1) on a column-packed with 200mg of the material. Quantitative mercury species recoveries were obtained using 4 mL of an eluting solution consisting of 0.8% (v/v) 2-mercaptoethanol and 20% (v/v) methanol (pH adjusted to 4.5) pumped at a flow rate of 2.0 mL min(-1). Mercury species separation was achieved on a Kinetex C18 column working under isocratic conditions (0.4% (v/v) 2-mercaptoethanol, 10% (v/v) methanol, pH 2.5, flow rate 0.7 mL min(-1)). ICP-MS detection was performed by monitoring the mercury mass to charge ratio of 202. The limits of quantification of the method were 11, 6.7, and 12 ng L(-1), for inorganic mercury, methyl-mercury and ethyl-mercury, respectively (pre-concentration factor of 50); whereas, analytical recoveries ranged from 96 to 106%. The developed method was successfully applied to several seawater samples from unpolluted areas.

In vitro bioavailability of total selenium and selenium species from seafood.

In vitro bioavailability of total selenium and selenium species from different raw seafood has been assessed by using a simulated gastric and intestinal digestion/dialysis method. Inductively coupled plasma-mass spectrometry (ICP-MS) was used to assess total selenium contents after a microwave assisted acid digestion, and also to quantify total selenium in the dialyzable and non-dialyzable fractions. Selenium speciation in the dialyzates was assessed by high performance liquid chromatography (HPLC) coupled with ICP-MS detection. Major Se species (selenium methionine and oxidized selenium methionine) from dialyzate were identified and characterized by HPLC coupled to mass spectrometry (HPLC-MS). Selenocystine was detected at low concentrations while Se-(Methyl)selenocysteine and inorganic selenium species (selenite and selenate) were not detected in the dialyzate. Low bioavailability percentages for total selenium (6.69±3.39 and 5.45±2.44% for fish and mollusk samples, respectively) were obtained. Similar bioavailability percentages was achieved for total selenium as a sum of selenium species (selenocystine plus oxidized selenium methionine and selenium methionine, mainly). HPLC-MS data confirmed SeMet oxidation during the in vitro procedure.

Evaluation of an in vitro method to estimate trace elements bioavailability in edible seaweeds.

Raw edible seaweed harvested in the Galician coast (Northwestern Spain), including two red seaweed types (Dulse and Nori), three brown seaweed (Kombu, Wakame and Sea Spaghetti), one green seaweed (Sea Lettuce) and one microalgae (Spirulina platensis) were studied to assess trace elements bioavailability using an in vitro method (simulated gastric and intestinal digestion/dialysis). Similarly, a cooked seaweed sample (canned in brine) consisting of a mixture of two brown seaweed (Sea Spaghetti and Furbelows) and a derived product (Agar-Agar) from the red seaweed Gelidiumm sesquipedale, were also included in the study. The total trace element content as well as the non-dialyzable fractions was carried out after a microwave acid digestion of the seaweed samples by inductively coupled plasma-mass spectrometry (ICP-MS). The dialyzable fraction was determined without any pre-treatment by ICP-MS. PIPES buffer solution at a pH of 7.0 and dialysis membranes of 10kDa molecular weight cut off (MWCO) were used for intestinal digestion. Accuracy of the method was assessed by analyzing a NIES-09 certified reference material (Sargasso seaweed). The accuracy of the in vitro procedure was established by a mass balance study which led to good accuracy of the whole in vitro process, after statistical evaluation (95% confidence interval). The highest dialyzability percentages (100±0.2%) were obtained for Dulse in Mn and V.

Selenium speciation in cow milk obtained after supplementation with different selenium forms to the cow feed using liquid chromatography coupled with hydride generation-atomic fluorescence spectrometry.

The purpose of this paper is to develop an easy and quick on-line selenium speciation method (LC-UV-HG-AFS) in cow milk obtained after different supplementation to cow feed. This study focuses on selenium speciation in cow milk after the use of different selenium species (organic selenium as selenised yeast and inorganic selenium as sodium selenite) in the supplementation of forages. Separation was carried out on a muBondapack C(18) column with the positively charged ion-pairing agent tetraethylammonium chloride in the mobile phase. The optimization of pre-reduction conditions was carried out; this step was done with UV irradiation and a heating block to improve the reduction of the different Se-compounds. Variables such as exposure time, hydrochloric acid concentration and temperature were studied. The detection limits for SeCyst(2), Se(IV), SeMet and Se(VI) were 0.4, 0.5, 0.9 and 1.0mugl(-1), respectively. The proposed method was applied to cow milk samples. The milk samples obtained after an organic supplementation of feeding as selenised yeast present three species of selenium, SeCyst2, Se(IV) and SeMet, while only SeCyst2 and Se(IV) are present in milk samples obtained after an inorganic supplementation of feeding.

Study of the bioavailability of selenium in cows' milk after a supplementation of cow feed with different forms of selenium.

The purpose of the work described in this paper was to develop an easy and quick in-vitro method for comparing the bioavailability of selenium in cows' milk after different cow feed. The study focuses on bioavailability differences resulting from the use of different selenium species (organic selenium as selenised yeast and sodium selenite) for supplementation of forage. A procedure for determination of selenium in cows' milk and dialysates, by hydride-generation atomic-fluorescence spectrometry (HG-AFS) after microwave-assisted acid digestion, was optimised. The results show it is possible to obtain cows' milk enriched with selenium at different concentration without altering the original composition of the milk. The bioavailability was statistically greater for cows' milk obtained after supplementation of forage with organic selenium at levels of 0.4 and 0.5 microg Se g(-1) than for that obtained after supplementation with inorganic and organic selenium at levels of 0.2 and 0.3 microg Se g(-1).

Selenium content and distribution in cow's milk supplemented with two dietary selenium sources.

The effect of two sources of Se, selenized yeast (Se-Y) and sodium selenite, added to total mixed rations (TMR) fed to cows on Se milk content and distribution in milk components was studied on three farms for 6 weeks. The maximal increase in milk Se was attained with Se-Y supplemented at 0.3 microg g(-1). The effect was immediate, with an increase of 9 microg L(-1) being observed after only 5 days, and remained steady until the last sample at day 40 of Se supplementation. Se distribution in milk components was constant, 53.6, 42.6, and 9.3% in whey, casein, and fat, respectively, and was unaffected by the form of supplementation. The effect of the level of Se-Y supplementation on milk Se was studied on two farms. Increasing dietary Se-Y from 0 to 0.5 microg g(-1) elevated milk Se content from 20 to 39 microg L(-1). Se-enriched cow's milk at different levels can be produced by varying dietary Se supplementation in the form of selenized yeast.

Determination of total selenium and selenium distribution in the milk phases in commercial cow's milk by HG-AAS.

A procedure has been developed for determining the selenium in cow's milk using hydride generation-atomic absorption spectrometry (HG-AAS) following microwave-assisted acid digestion. The selenium distributions in milk whey, fat and micellar casein phases were studied after separating the different phases by ultracentrifugation and determining the selenium in all of them. The detection limits obtained by HG-AAS for the whole milk, milk whey and micellar casein were 0.074, 0.065 and 0.075 microg l(-1), respectively. The accuracy for the whole milk was checked by using a Certified Reference Material CRM 8435 whole milk powder from NIST, and the analytical recoveries for the milk whey and casein micelles were 100.9 and 96.9%, respectively. A mass balance study of the determination of selenium in the different milk phases was carried out, obtaining values of 95.5-100.8%. The total content of selenium was determined in 37 milk samples from 15 different manufacturers, 19 whole milk samples and 18 skimmed milk samples. The selenium levels found were within the 8.5-21 microg l(-1) range. The selenium distributions in the different milk phases were studied in 14 whole milk samples, and the highest selenium levels were found in milk whey (47.2-73.6%), while the lowest level was found for the fat phase (4.8-16.2%). A strong correlation was found between the selenium levels in whole milk and the selenium levels in the milk components.

Application of ultrasound-assisted acid leaching procedures for major and trace elements determination in edible seaweed by inductively coupled plasma-optical emission spectrometry.

A new method using diluted reagents (nitric and hydrochloric acids and oxygen peroxide) and ultrasound energy to assist metals acid leaching with from edible seaweed was optimized. The method uses a first sonication at high temperature with hydrochloric acid as a previous stage to an ultrasound-assisted acid leaching with 7ml of an acid solution containing nitric acid, hydrochloric acid and hydrogen peroxide at concentrations of 3.7, 3.0 and 3.0M, respectively. Optimum conditions for the first sonication step were ultrasound energy at 17kHz, sonication temperature at 65 degrees C, an acid volume of 2ml, an hydrochloric acid concentration of 6.0M and a sonication time of 10min. It has been found that the first sonication stage at high temperature with hydrochloric acid is necessary to obtain quantitative recoveries for As, Ba, Fe and V. Otherwise quantitative recoveries were reached for the other elements investigated (Ca, K, Na, Mg, Cd, Cr, Cu, Mn, Ni, Pb and Zn). The repeatability of the ultrasound-assisted acid leaching method was around 10% for all elements. Adequate limit of detection and limit of quantification were reached by using inductively coupled plasma-optical emission spectrometry (ICP-OES) for measurements. The method resulted accurate after analysing several seaweed certified reference materials (IAEA-140/TM, NIES-03 and NIES-09). The method was finally applied to the multi-element determination in edible seaweed samples.