Dynamic continuous-flow dialysis method to simulate intestinal digestion for in vitro estimation of mineral bioavailability of food

A system for dynamic continuous-flow dialysis during intestinal digestion for an in vitro simulation of gastrointestinal digestion is presented as an alternative to human and animal in vivo methods for estimation of the bioavailability of minerals. The method is based on the in vitro batch dialysis method described by Miller, which was developed into a continuous-flow system of a simple design to perform dynamic dialysis in the intestinal digestion stage. A flow dialysis system has the advantages of simulation being close to in vivo physiological conditions because pH change during dialysis is gradual and dialyzed components are continuously removed. The proposed new design performed dialysis during a continuous flow of dialyzing solution (NaHCO₃) around a dialysis bag containing peptic digest, which is placed inside a glass dialysis chamber. Gradual change of dialysis pH, similar to that occurring in the gastrointestinal tract, was obtained by optimization of flow rate and concentration of NaHCO₃. The dialysate collected in fractions was analyzed to determine dialyzed minerals and pH change in the course of dialysis. The method was tested by determination of calcium bioavailability of powder milk and calcium carbonate tablets.     

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 10 kDa 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.     

Use of an in vitro digestion method to evaluate the bioaccessibility of arsenic in edible seaweed by inductively coupled plasma-mass spectrometry

Different types of edible seaweeds (Kombu, Wakame, Nori and Sea Lettuce) harvested in the Galician coast (Northwestern Spain) were analyzed for total arsenic quantification before and after being cooked following manufacturer's instructions. Furthermore, the total arsenic in the bioaccessible fraction obtained after simulating a human digestion by an in vitro process was determined in those raw and cooked seaweeds. The detection of the target element was performed by inductively coupled plasma-mass spectrometry (ICP-MS) which was equipped with a collision cell to avoid polyatomic interferences. Piperazine-NN-bis (2-ethane-sulfonic acid) disodium (PIPES) buffer solution at a pH of 7.0 and dialysis membranes of 10 kDa molecular weight cut-off (MWCO) were used for intestinal digestion. Accuracy of the method was assessed by analyzing a BCR-279 certified reference material. The accuracy of the in vitro procedures was established by a mass balance study for Nori and Sea Lettuce which led to good accuracy of the whole in vitro process, after statistical evaluation (95% confidence interval). Results showed that the effect of cooking seaweed causes the removal of the element into the cooking water. Dialyzability percentages found in raw seaweed samples were comparable to those found in cooked seaweed, except in the case of Sea Lettuce sample for which a lower dialyzability percentage was found when it was cooked.     

Continuous-flow extraction system for elemental association study: a case of synthetic metal-doped iron hydroxide

A continuous-flow extraction system originally developed for sequential extraction was applied to study elemental association of a synthetic metal-doped amorphous iron hydroxide phase. The homogeneity and metal association of the precipitates were evaluated by gradual leaching using the system. Leachate was collected in fractions for determination of elemental concentrations. The result obtained as extractograms indicated that the doped metals were adsorbed more on the outermost surface rather than homogeneously distributed in the precipitates. The continuous-flow extraction method was also used for effective removal of surface adsorbed metals to obtain a homogeneous metal-doped synthetic iron hydroxide by a sequential extraction using acetic acid and small volume of hydroxylamine hydrochloride solution. The system not only ensures complete washing, but the extent of metal immobilization in the synthetic iron hydroxide could be determined with high accuracy from the extractograms. The initial metal/iron mole ratio (M/Fe) in solution affected the M/Fe mole ratio in homogeneous doped iron hydroxide phase. The M/Fe mole ratio of metal incorporation was approximately 0.01–0.02 and 0.03–0.06, for initial solution M/Fe mole ratio of 0.025 and 0.100, respectively.     

Literature study of microwave-assisted digestion using electrothermal atomic absorption spectrometry

The literature on the use of microwave-assisted digestion procedures for subsequent sample analysis by means of electrothermal atomic absorption spectrometry (ETAAS) is reviewed. The literature survey reveals that this digestion technique has been applied mainly for biological materials. The elements most extensively determined by this method are cadmium and lead followed by copper, chromium, nickel and iron. The microwave digestion conditions, ETAAS furnace programmes and analytical details of the developed methodologies have been carefully revised.     

Literature study of microwave-assisted digestion using electrothermal atomic absorption spectrometry

The literature on the use of microwave-assisted digestion procedures for subsequent sample analysis by means of electrothermal atomic absorption spectrometry (ETAAS) is reviewed. The literature survey reveals that this digestion technique has been applied mainly for biological materials. The elements most extensively determined by this method are cadmium and lead followed by copper, chromium, nickel and iron. The microwave digestion conditions, ETAAS furnace programmes and analytical details of the developed methodologies have been carefully revised.     

Determination of trace elements in biological materials using tetramethylammonium hydroxide for sample preparation

A method to prepare milk powder, bovine liver and bovine muscle samples for analysis by electrothermal atomic absorption spectrometry (ETAAS) is proposed. Samples are mixed with a small amount of tetramethylammonium hydroxide (TMAH) and a stable and homogeneous slurry is produced in ca. 2 h with heating at 60–70 °C. After such sample preparation and dilution with water, trace elements are determined in certified reference materials. Pyrolysis and atomisation temperatures are optimised for each element, and several modifiers are investigated. External calibration is used for every analyte. Limits of detection (LODs), precision and accuracy are reported for Cd, Pb, Ni, Cr, Cu and Ag and compared with those obtained after conventional acid digestion. The main advantages of the proposed method are the simplicity of sample preparation and the longer lifetime of the graphite tube.     

Speciation of iron in breast milk and infant formulas whey by size exclusion chromatography-high performance liquid chromatography and electrothermal atomic absorption spectrometry

Speciation of iron in milk was carried out by high performance liquid chromatography (HPLC) and electrothermal atomic absorption spectrometry (ETAAS). Milk whey was obtained and low molecular weight protein separation was performed by size exclusion chromatography (SEC) with a TSK Gel SW glass guard (Waters) pre-column and a TSK-Gel G2000 glass (Toso Haas) column. After studying water as a possible mobile phase, this mobile phase was carefully selected in order to avoid alterations of the sample and to make subsequent iron determination in the protein fractions easier by ETAAS. The proposed method is sensitive (limit of detection [LOD] and LOQ 1.4 and 4.7 μg l⁻¹, respectively) and precise (relative standard deviation [RSD]<10%). Iron is principally found in the proteins of 3 and 76 kDa in breast milk, and it is irregularly distributed in infant formulas.     

Determination of Cd, Co, Cr, Cu, Fe, Ni and Pb in Milk, Cheese and Chocolate

 Combined analytical procedures consisting of wet digestion step followed by instrumental determination – differential pulse cathodic stripping voltammetry (DPCSV) or electrothermal atomic absorption spectrometry (ETAAS) – as well as a direct analysis method – slurry sampling ETAAS – for the determination of Cd, Co, Cr, Cu, Fe, Ni and Pb in milk, cheese and chocolate are described and compared. Wet digestion using a mixture of HNO₃-HClO₄-H₂O₂ is proposed for complete matrix decomposition prior to trace analyte determinati on by DPCSV or ETAAS. A mixture of HNO₃-H₂O₂ is used for slurry preparation. Optimal instrumental parameters for trace analyte measurements are presented. The reliability of the procedures has been verified by analyzing standard reference materials. Results obtained are in good agreement with the certified values and the relative standard deviations (for these results) are in the range 5–10% for wet digestion DPCSV or ETAAS and 3–9% for slurry sampling ETAAS in the range of 2 μgċg⁻¹ (Cd) to 12 μgċg⁻¹ (Fe). Received August 24, 1999. Revision January 20, 2000.     

Speciation of the bio-available iodine and bromine forms in edible seaweed by high performance liquid chromatography hyphenated with inductively coupled plasma-mass spectrometry

A bioavailability study based on an in vitro dialyzability approach has been applied to assess the bio-available fractions of iodine and bromine species from edible seaweed. Iodide, iodate, 3-iodo-tyrosine (MIT), 3,5-diiodo-tyrosine (DIT), bromide and bromate were separated by anion exchange chromatography under a gradient elution mode (175 mM ammonium nitrate plus 15% (v/v) methanol, pH 3.8, as a mobile phase, and flow rates within the 0.5–1.5 mL min⁻¹ range). Inductively coupled plasma-mass spectrometry (ICP-MS) was used as a selective detector for iodine (¹²⁷I) and bromine (⁷⁹Br). Low dialyzability ratios (within the 2.0–18% range) were found for iodine species; whereas, moderate dialyzability percentages (from 9.0 to 40%) were obtained for bromine species. Iodide and bromide were the major species found in the dialyzates from seaweed, although MIT and bromate were also found in the dialyzates from most of the seaweed samples analysed. However, DIT was only found in dialyzates from Wakame, Kombu, and NIES 09 (Sargasso) certified reference material; whereas, iodate was not found in any dialyzate. Iodine dialyzability was found to be dependent on the protein content (negative correlation), and on the carbohydrate and dietary fibre levels (positive correlation). However, bromine dialyzability was only dependent on the protein amount in seaweed (negative correlation).     

Comparative determination of Ba, Cu, Fe, Pb and Zn in tea leaves by slurry sampling electrothermal atomic absorption and liquid sampling inductively coupled plasma atomic emission spectrometry

The comparative determination of barium, copper, iron, lead and zinc in tea leaf samples by two atomic spectrometric techniques is reported. At first, slurry sampling electrothermal atomization atomic absorption spectrometry (ETAAS) was applied. The results of Ba and Pb determination were calculated using the method of standard additions, and results of Cu, Fe and Zn from the calibration graphs based on aqueous standards. These results were compared with the results obtained after microwave-assisted wet (nitric+hydrochloric+hydrofluoric acids) digestion in closed vessels followed by inductively coupled plasma-atomic emission spectrometric (ICP-AES) determination with the calibration by means of aqueous standards. The exception was lead determined after a wet digestion procedure by ETAAS. The accuracy of the studied methods was checked by the use of the certified reference material Tea GBW-07605. The recoveries of the analytes varied in the range from 91 to 99% for slurry sampling ETAAS, and from 92.5 to 102% for liquid sampling ICP-AES. The advantages of slurry sampling ETAAS method are simplicity of sample preparation and very good sensitivity. Slurry sampling ETAAS method is relatively fast but if several elements must be determined in one sample, the time of the whole microwave-assisted digestion procedure and ICP-AES determination will be shorter. However, worse detection limits of ICP-AES must also be taken into the consideration in a case of some analytes.     

Gravitational field-flow fractionation in combination with flow injection analysis or electrothermal AAS for size based iron speciation of particles

A simple gravitational field-flow fractionation (GrFFF) system was used for size separation of micron sized silica particles coated with hydrous iron oxide (geothite). The amount of iron on the particles was monitored either on-line by reverse-flow injection analysis (r-FIA) with chemiluminescence detection using luminol or off-line by electrothermal atomic absorption spectrophotometry (ETAAS). The combination of GrFFF with reverse FIA or with ETAAS has been demonstrated to be a cost-effective tool for size based iron speciation of particles.     

Comparison of the In Vitro Bioavailability of Selected Minerals from Gluten-Free Breads Enriched with Grains and Synthetic Organic and Non-Organic Compounds

Introduction: Despite the constant efforts of scientists to improve the texture, sensory properties, and nutritional value of gluten-free bread, obtaining high bioavailability of minerals is still a huge challenge. Gluten-free bakery products are characterized by a low bioavailability of minerals. The aim of this study was to design gluten-free bread with high bioavailability of minerals commonly found in deficiencies in people struggling with gluten intolerance. Material and methods: The material consisted of gluten-free breads designed to obtain the highest possible content of minerals in the bread while maintaining a good structure and taste. Results: Higher contents of all the analyzed minerals were obtained in breads with natural and synthetic additives, both in rice and buckwheat bread, compared to basic bread. There was also a higher content of the analyzed minerals in buckwheat bread in comparison to rice bread for each type of additive. Higher bioavailability of iron, copper, calcium, and magnesium was noted in rice bread, while the bioavailability of zinc was higher in buckwheat bread. Conclusion: The additives used increased the bioavailability of the analyzed minerals from the gluten-free breads. The use of various variants of flour (rice, buckwheat) influenced the bioavailability of iron, zinc, copper, calcium, and magnesium. The release of minerals from gluten-free bread depends on the element and added components (seeds or synthetic additives).     

Slurry Sampling Electrothermal Atomic Absorption Spectrometric Determination of Sodium and Iron Impurities in Optical Crystals of Rubidium Titanyl Phosphate

A simple and rapid slurry sampling electrothermal atomic absorption spectrometric (ETAAS) method was developed for the determination of traces of sodium and iron in single crystals of rubidium titanyl phosphate (RTP). The finely ground crystal material was dispersed in 5mL of 2% (v/v) HNO₃, containing 0.005% (v/v) Triton-X-100. The fast furnace ETAAS analysis was performed using a pyrolytic platform coated with titanium carbide. No matrix interference was registered for either of the analytes in slurries containing 4 to 10mgmL^–1 of RTP, which permitted simple calibration against aqueous solutions. The precision was about 2% RSD. The results for sodium and iron content in RTP were in good agreement with those obtained by flame AAS after sample digestion.     

In vitro mineral availability from digested tea: a rich dietary source of managanese

Tea is potentially a rich source of some dietary metals and approximately 70 l are drunk per capita per year in the UK. In particular, tea may be an important source of Mn, since leaf tea contains 350-900 micrograms g-1 of this essential element. However, the leaching and bioavailability of Mn from tea have been little studied, so a recently developed in vitro assay was applied to compare the bioavailability of Mn from tea infusions with that of other major and trace essential elements. Analysis of tea infusions before digestion showed that 1.0 l contained 115% of the average daily dietary intake of Mn but < 6% of all other minerals. Samples of these infusions were incubated with human gastric juice (37 degrees C, 1 h) and some were then adjusted to pH 6.5 to simulate intestinal pH. All were centrifuged through ultrafilters with molecular mass cut-offs of 3, 10 and 30 kDa. The percentages of ultrafilterable (< 3 kDa) elements following simulated gastrointestinal digestion were (n = 3; mean +/- s) Ca 47.7 +/- 10.7, Cu 45.3 (n = 1), Fe < 5, Mg 66.4 +/- 1.6, Mn 39.8 +/- 11.4, K 40.3 +/- 2.2, Na 100.0 +/- 5.3 and Zn 33.7 +/- 1.1. Hence the ultrafilterability of elements showed the general trend M+ > M2+ > M3+, which is probably the inverse of the order of their strengths of binding to tea polyphenols. However, Mn was the only element found in significant dietary amounts in tea, and under simulated intestinal conditions was still 40% bioavailable.     

ETAAS determination of Cd and Pb in plants

Simple and rapid analytical procedures for the ETAAS determination of Cd and Pb in plant (poplar, clover, plantain) leaves are described. Optimal conditions are presented for the wet digestion of plant matrices with a mixture of nitric acid and hydrogen peroxide along with optimal temperature programmes for subsequent modifier-free electrothermal determination of Cd and Pb. Slurry preparation procedures using tetramethylammonium hydroxide or a mixture of 1 mol/L HNO₃ + 1.5 mol/L H₂O₂ are proposed as another approach for rapid and accurate routine analysis of Cd and Pb in plant leaves. It has been found that single standard addition can be used for quantitative determination in the case of wet digestion ETAAS and standard addition to each analysed matrix is recommended in the case of slurry ETAAS. For all plant leaves investigated good agreement was achieved between the concentrations determined by wet digestion ETAAS and slurry ETAAS. The wet digestion method provides relative standard deviations for Cd ranging from 3% to 17% and for Pb from 2% to 16%. For the slurry method the RSD values are in the range of 4–31% for Cd and 4–30% for Pb, depending for both cases on the magnitude of the measured concentrations. Validity and versatility of the methods are verified by the analyses of standard reference materials. Received: 17 March 1997 / Revised: 2 May 1997 / Accepted: 8 May 1997     

Cloud point extraction of aluminum (III) in water samples and determination by electrothermal atomic absorption spectrometry,flame atomic absorption spectrometry and UV-visible spectrophotometry

Cloud point extraction was applied as a preconcentration step for the determination of trace level of Al(III) in water samples with electrothermal atomic absorption spectrometry (ETAAS), flame atomic absorption spectrometry (FAAS) and UV-visible spectrophotometry. The aluminum was extracted as aluminum-Eriochrome Cyanine R (ECR) complex, at pH 6 by micelles of the non-ionic surfactant octylphenoxypolyethoxyethanol (Triton X-114). The investigations showed that the same CPE procedure can be used for different detection techniques. The results obtained from these techniques were evaluated. Under the optimal conditions, limit of detection obtained with ETAAS, FAAS and UV-visible spectrophotometry were 0.03 ng mL^?1, 0.06 µg mL^?1 and 0.01 µg mL^?1, respectively. The accuracy of the procedure was tested by analysing certified reference material. The method was successfully applied to determination of aluminum in water samples and dialysis fluid.     

Microwave sample-digestion procedure for determination of arsenic in moss samples using electrothermal atomic absorption spectrometry and inductively coupled plasma mass spectrometry

Arsenic in moss samples was determined by electrothermal atomic absorption spectrometry (ETAAS) after microwave-assisted sample digestion. Two different sample masses (500 mg and 1000 mg) and three different microwave ovens were used in the digestion. There was a slight difference in the digestion efficiency, as determined by the residual carbon concentrations of 500 mg digested samples, between the microwave ovens. The arsenic results obtained for moss reference samples were, in most cases, satisfactory. However, phosphorus was found to have a reducing influence on the arsenic peak area in the ETAAS determination. According to the results, it was not possible to reduce the phosphorus interference by increasing the amount of Mg(NO(3))(2) in the Pd-Mg chemical modifier. The arsenic results obtained by ETAAS were compared to those obtained by inductively coupled plasma mass spectrometry (ICP-MS).     

Comparative study of pretreatment methods for the determination of metals in atmospheric aerosol by electrothermal atomic absorption spectrometry

A comparative study of pretreatment methods for the determination of 10 elements (As, Cd, Pb, V, Ni, Mn, Cr, Cu, Fe, Al) in atmospheric aerosols by electrothermal atomic absorption spectrometry (ETAAS) was conducted. For the digestion of the particulates collected in filters, six methods were compared using a mixture of HNO₃ and HF with or without the addition of various oxidative agents (HClO₄ or H₂O₂) or acids (HCl). The comparative study was performed using loaded cellulose filter samples, which were digested in Parr bombs and heated in a conventional oven at 170 °C for 5 h. The extraction efficiency and blanks were compared and it was proved that the digestion method using only HNO₃–HF extracted most of the metals and gave the lowest blanks. The HNO₃–HF mixture was selected for the development of an improved microwave digestion method specific for aerosol-loaded filters. The operating parameters were optimized, so that quantitative recovery of the reference materials NIST 1649a urban dust and NIST 1648 urban particulate matter was achieved. The blank of cellulose and teflon filters were also determined and compared. Teflon filters present the lowest blanks for all the elements. The obtained limits of detection for each type of filters were adequate for environmental monitoring purposes. ETAAS instrumental operation was also optimized for the compensation and the elimination of interferences. The temperature optimization was performed for each metal in every type of filter and optimized parameters are proposed for 10 elements.     

Size-based speciation of iron in clay mineral particles by gravitational field-flow fractionation with electrothermal atomic absorption spectrometry

Gravitational field-flow fractionation (FFF) coupled to UV and ETAAS detectors has been tested for micron-size particles in the range of 5–20 μm using three Fe-rich clay samples. The iron content estimated after aqua regia extraction was about 20–40 mg kg⁻¹. The ETAAS analysis was performed both off-line from collected fractions and in an online continuous sampling mode using a specially designed flow through vial placed in the autosampler of the ETAAS. Comparison of the direct injection method with total analysis after aqua regia digestion shows that slurry injection of the dilute samples in the gravitational field-flow fractionation (GrFFF) effluent is quite efficient in these samples. In the majority of cases, more than 90% recovery was obtained for the slurry injection method. Fe mass-based particle size distributions and Fe concentration versus particle diameter plots can be generated using certain assumptions. This provides detailed information on size-based speciation of particulate samples. Generally, the Fe concentrations in the particles decreased slightly with an increase in particle size as is often found for soil and sediment samples.