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In-vitro oral bioaccessibility tests do remain alluring as viable tools for fast assessment of potential human exposure to environmental contaminants to comply with the ethical constraints posed by the REACH guidelines. A variety of biologically-relevant extraction methods to measure solubility of organic compounds as a proxy for bioaccessibility in the human body using mock digestive fluids have been proposed over the past decade as integral part of holistic exposome studies. However, analytical methods in several instances are not properly validated and do not report proper quality control/quality assurance (QA/QC) issues. This review provides insights into the analytical validation of physiologically relevant gastrointestinal assays for organic contaminants (namely, organophosphate and brominated flame retardants, fluorinated organic species, and (myco)toxins) in risk assessment studies of potentially contaminated environmental solids and (sea)food commodities. Sample preparation and (micro)extraction methods for processing of the gastrointestinal extracts prior to liquid chromatography (LC)/LC-mass spectrometry (MS) and gas chromatography (GC)-MS assays are critically evaluated. We also propose a fully detailed analytical workflow embracing QA/QC tools to serve as a tutorial for proper validation of new physiologically-based extraction tests.

An automatic mesofluidic system combining dynamic oral bioaccessibility with lab-on-valve (LOV)-based sorptive microextraction is herein proposed for the first time for exploring the kinetics of leaching of incurred rather than spiked organic emerging contaminants (viz., methyl paraben, butyl paraben, diclofenac, and triclosan) from exposed mussels on the basis of the Versantvoort's fed-state physiological extraction test. Our method capitalizes on programmable flow analysis, in which gastrointestinal extracts are obtained online by pumping a simulated biorelevant gastrointestinal fluid across a large-bore column (maintained at 37.0 ± 2.0 °C) loaded with 250 mg of freeze-dried and powdered mussel onto a polyvinyldiene difluoride filter membrane. The physiologically relevant extracts are then cleaned up, and the analytes are preconcentrated onto a dedicated reversed-phase solid-phase extraction (Oasis PRIME-HLB) microcolumn that is captured into the channels of an LOV mesofluidic platform. The aim behind this is to obtain analyte-laden eluates with ACN/MeOH (90:10, v/v) in unsupervised mode for direct injection into LC-MS. The LOV minicolumn (≤25 mg) is automatically disposed of and renewed for every individual fraction on account of the strong retention of (phospho)lipids by the copolymeric sorbent. The proposed dynamic bioaccessibility test features a significant shortening of the extraction time against the batch method (28 vs 240 min) while avoiding overestimation of potentially bioavailable fractions. The trueness of the online gastrointestinal extraction method was confirmed using mass-balance validation following ultrasonic-assisted solid-liquid extraction of the original mussel sample and the residual (nonbioaccessible) fraction of emerging contaminants.

Mesofluidic lab-on-a-valve (LOV) platforms have been proven suitable to accommodate automatic micro-solid-phase extraction (μSPE) approaches with on-chip handling of micrometer-bead materials in a fully disposable mode to prevent sample cross-contamination and pressure-drop effects. The efficiency of the extraction process notably depends upon the sorptive capacity of the material because the sorbent mass is usually down to 10 mg in LOV devices. Nanomaterials, capitalizing upon their enhanced surface-to-volume ratio and diversity of potential chemical moieties, are appealing alternatives to microbead sorbents. However, the handling and confinement of nanomaterials in fluidic chip structures have been challenging to date. This is most likely a consequence of the aggregation tendency of a number of nanomaterials, including carbon-based sorbents, that leads to excessive back-pressure in flowing systems along with irreproducible bead loading. This paper addresses these challenges by ad hoc synthesis of hybrid nanomaterials, such as porous carbon-coated titanium dioxide nanotubes (TiO2-NT@pC). Tailoring of the surface polarity of the carbon coating is proven to foster the dispersion of TiO2-NT@pC in LOV settings while affording superior extraction capability of moderately nonpolar species from aqueous matrices. The determination of trace-level concentrations of butylparaben (BPB) and triclosan (TCS) in seawater samples is herein selected as a proof-of-concept of the exploitation of disposable nanomaterials in LOV. The mesofluidic platform accommodating μSPE features online hyphenation to liquid chromatography/tandem mass spectrometry (LC/MS/MS) for reliable determination of the target analytes with excellent limits of detection (0.5 and 0.6 ng/L for BPB and TCS, respectively) and intermediate precision (relative standard deviation

An automatic programmable-flow system capitalizing upon mesofluidic Lab-On-Valve (LOV) sample processing coupled on-line to inductively coupled plasma atomic emission spectrometry (ICP-AES) is proposed in this work as a quality control tool for expedient assessment of potential contamination episodes of food safety elements (namely, Cd and Pb) in a variety of undigested ripened honeys, taken as a model of an edible matrix. On-chip micro-solid phase extraction (μSPE) is effected in a bead-injection disposable sorbent mode using a cationic exchange restricted access-like material (viz., Bond Elut Plexa PCX). We have proven that the lifetime of the miniaturized solid reactor on-chip (11 ± 0.6 mg) is limited to processing less than 6 mL of unfiltered sample suspension (5% (w/w) honey buffered at pH 4.5) after which the analytical performance is severely deteriorated because of strong adherence of the sample matrix to the bead surface. Several physicochemical parameters including the nature of the sorbent material and its ionic form were investigated in detail so as to maximize absolute recoveries and enrichment factors of Pb and Cd. Variables for the elution process were explored by means of a full factorial design. Using 4.0 mL of sample and 200 μL of 3.0 mol L−1 HNO3 as an eluent, enrichment factors of ca. 15 with extraction/elution efficiencies close to 80% and relative recoveries ranging from 90–111% in honey were obtained for both Pb and Cd. The limits of detection (LODs), based on the 3sintercept criterion, were 26 ng g−1 and 68 ng g−1 for Cd and Pb, respectively, which are far below those endorsed by current regulatory agencies (namely, 100 and 300–500 ng g−1 for Cd and Pb, respectively). Demonstrated by the analysis of a suite of off-the-shelf honey brands, the proposed LOV-μSPE platform hyphenated to ICP-AES is deemed suitable for the reliable quantitation of trace level concentrations of Pb and Cd in honey and the detection of heavy metal contamination episodes.

An automatic flow-through dynamic extraction method is proposed for the first time for in vitro exploration, with high temporal resolution, of the transit of the chyme from the gastric to the duodenal compartment using the Versantvoort's fed-state physiologically relevant extraction test. The flow manifold was coupled on-line to an inductively coupled plasma optical emission spectrometer (ICP OES) for real-time elucidation of the bioaccessible elemental fraction of micronutrients (viz., Cu, Fe and Mn) in food commodities across the gastrointestinal tract. The simulated intestinal and bile biofluid (added to the gastric phase) was successively pumped at 1.0 mL min−1 through a large-bore column (maintained at 37.0 ± 2.0 °C) initially loaded with a weighed amount of linseed (250 mg) using a PVDF filter membrane (5.0 μm pore size) for retaining of the solid sample and in-line filtration of the extracts. The lack of bias (trueness) of the on-line gastrointestinal extraction method coupled to ICP OES was confirmed using mass balance validation following microwave assisted digestion of the residual (non-bioaccessible) elemental fraction. Mass balance validation yielded absolute recoveries spanning from 79 to 121% for the overall analytes and samples. On-line dynamic extraction was critically appraised against batch counterparts for both gastric and gastrointestinal compartments. Due to the lack of consensus in the literature regarding the agitation method for batch oral bioaccessibility testing, several extraction approaches (viz., magnetic stirring, end-over-end rotation and orbital shaking) were evaluated. Improved gastric extractability of Fe along with bioaccessible data comparable to the dynamic counterpart based on the continuous displacement of the extraction equilibrium was obtained with batchwise magnetic stirring, which is deemed most appropriate for ascertaining worst-case/maximum bioaccessibility scenarios.

A novel biomimetic extraction procedure that allows for the in-line handing of ≥400 mg solid substrates is herein proposed for automatic ascertainment of trace element (TE) bioaccessibility in soils under worst-case conditions as per recommendations of ISO norms. A unified bioaccessibility/BARGE method (UBM)-like physiological-based extraction test is evaluated for the first time in a dynamic format for accurate assessment of in-vitro bioaccessibility of Cr, Cu, Ni, Pb and Zn in forest and residential-garden soils by on-line coupling of a hybrid flow set-up to inductively coupled plasma atomic emission spectrometry. Three biologically relevant operational extraction modes mimicking: (i) gastric juice extraction alone; (ii) saliva and gastric juice composite in unidirectional flow extraction format and (iii) saliva and gastric juice composite in a recirculation mode were thoroughly investigated. The extraction profiles of the three configurations using digestive fluids were proven to fit a first order reaction kinetic model for estimating the maximum TE bioaccessibility, that is, the actual worst-case scenario in human risk assessment protocols. A full factorial design, in which the sample amount (400-800 mg), the extractant flow rate (0.5-1.5 mL min(-1)) and the extraction temperature (27-37°C) were selected as variables for the multivariate optimization studies in order to obtain the maximum TE extractability. Two soils of varied physicochemical properties were analysed and no significant differences were found at the 0.05 significance level between the summation of leached concentrations of TE in gastric juice plus the residual fraction and the total concentration of the overall assayed metals determined by microwave digestion. These results showed the reliability and lack of bias (trueness) of the automatic biomimetic extraction approach using digestive juices.

In-line microdialysis is in this work hyphenated to electrothermal atomic absorption spectrometry via a dedicated flow-based interface for monitoring the batchwise leaching test endorsed by the Standards, Measurements and Testing Program (SMT) of the European Commission. The bioaccessible pool of lead in soils is measured using 0.43 mol/L AcOH as extractant. The proposed method allows to gain knowledge of leaching kinetics at real-time, simplify the overall procedure by accurate detection of steady-state conditions and overcome sample filtration or centrifugation. Soil leachates were automatically sampled at specified timeframes (e.g, every 20 or 80 min), processed in an external container (where dilution can be applied at will) and further injected into the atomizer. The method was experimentally validated by comparison of in situ microdialysis sampling results with in-line microfiltration in two soils of varying physicochemical properties. A mathematical framework was used for discrimination of different metal fractions (that is, readily mobilizable against slowly mobilizable lead) and also for estimating the total extractable lead under actual steady-state conditions. We have demonstrated that bioaccessibility tests lasting 16 h as endorsed by SMT might not suffice for ascertainment of maximum (steady-state) bioaccessibility of lead in terrestrial environments as demanded in risk assessment programs.

In situ automatic microdialysis sampling under batch-flow conditions is herein proposed for the first time for expedient assessment of the kinetics of lead bioaccessibility/bioavailability in contaminated and agricultural soils exploiting the harmonized physiologically based extraction test (UBM). Capitalized upon a concentric microdialysis probe immersed in synthetic gut fluids, the miniaturized flow system is harnessed for continuous monitoring of lead transfer across the permselective microdialysis membrane to mimic the diffusive transport of metal species through the epithelium of the stomach and of the small intestine. Besides, the addition of the UBM gastrointestinal fluid surrogates at a specified time frame is fully mechanized. Distinct microdialysis probe configurations and membranes types were investigated in detail to ensure passive sampling under steady-state dialytic conditions for lead. Using a 3-cm-long polysulfone membrane with averaged molecular weight cutoff of 30 kDa in a concentric probe and a perfusate flow rate of 2.0 μL min(-1), microdialysis relative recoveries in the gastric phase were close to 100%, thereby omitting the need for probe calibration. The automatic leaching method was validated in terms of bias in the analysis of four soils with different physicochemical properties and containing a wide range of lead content (16 ± 3 to 1216 ± 42 mg kg(-1)) using mass balance assessment as a quality control tool. No significant differences between the mass balance and the total lead concentration in the suite of analyzed soils were encountered (α = 0.05). Our finding that the extraction of soil-borne lead for merely one hour in the GI phase suffices for assessment of the bioavailable fraction as a result of the fast immobilization of lead species at near-neutral conditions would assist in providing risk assessment data from the UBM test on a short notice.

Release of bioactive compounds from food matrices is regarded as the first step towards their human bioavailability. The objective of this work was the implementation of an affordable and robust flow-through device for expedient dynamic leaching experiments aiming at the assessment of readily bioaccessible antioxidant compounds in solid food commodities. A simple configuration is proposed using commercially available devices containing regenerated cellulose filters placed in polypropylene holders to entrap the solid sample, featuring a disposable, single use extraction chamber. The kinetic extraction profile of fast leachable antioxidants from different food matrices was evaluated using the ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) assay, fitting a first-order reaction model for readily bioaccessible compounds (R>0.9). The leaching rate constant values associated to the fast leachable antioxidant compounds were 0.060–0.446 min −1 and 0.105–0.210 min −1 for water and ethanol/water (1:1, v/v) applied as extractants, respectively. Furthermore, no statistically significant differences were found between the estimated values of bioaccessible antioxidant compounds by the kinetic model and the values attained using conventional batch-wise extraction methodology, ranging from 3.37 to 60.3 µmol of Trolox ((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) per g of sample. Extension of the method using U. S. Pharmacopeia surrogate biological media (stomach (pH 1.2) and intestinal (pH 7.5) fluids without enzymes) to NIST-1570a spinach leaves provided gastrointestinal compartment-dependent kinetic leaching rates (0.120 and 0.198 min −1 , respectively) and total antioxidant content (45.5 and 52.5 µmol of Trolox per g of sample, respectively).

This paper reports the evaluation of in-vitro physiologically relevant extraction tests for ascertainment of the bioaccessible fractions of emerging flame retardants from indoor dust in the gastric and gastrointestinal compartments. Standardized bioaccessibility tests under both fasting (UBM-like test) and fed (FOREhST test) conditions simulating the macronutrient composition of an average child diet were harnessed for investigation of the oral bioaccessibility of chlorinated organophosphate esters, namely, tris(2-chloroethyl) phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCPP) and tris(1,3-dichloro-2-propyl) phosphate (TDCP), in household and automobile cabin dust samples with varying concentration levels of contaminants. Minimal processing of the biomimetic extracts (only protein precipitation using acetonitrile) was proven feasible by analysis with liquid chromatography-mass spectrometric detection (LC-MS/MS). An inversely proportional relationship was identified between log Kow and oral bioaccessibility concentrations for TCEP, TCPP and TDCP in both dust samples with maximum bioaccessibility fractions for TCEP within the range of 50–103%. Non-bioaccessible fractions were determined by matrix-solid phase dispersion. Limits of quantification of LC-MS/MS in surrogate digestive fluids ranging from 0.4–0.8 ng/mL suffice for determination of freely dissolved fractions of the two less hydrophobic species. Our results indicate that lipophilic food commodities used under fed-state gastrointestinal extraction conditions do not increase availability of TCEP, TCPP and TDCP in body fluids, and therefore conservative conditions in human health risk explorations for the target moderately polar flame retardants might be obtained with simplified tests under fasting conditions. This also holds true for the UBM/FOREhST bioaccessibility data for SRM 2585 (organic contaminants in house dust). Estimated average daily intake doses for toddlers incorporating oral bioaccessibility data afforded body burdens for the three chlorinated alkyl phosphates of ca. 3000 to 700 times below reference dose values, which indicate that long-term exposure to chlorinated organophosphate esters via accidental ingestion of indoor dust does not pose health risks to toddlers.

Two novel dynamic extraction approaches, the so-called sequential injection microcolumn extraction and sequential injection stirred-flow chamber extraction, based on the implementation of a sample-containing container as an external extraction reactor in a sequential injection network, are for the first time, optimized and critically appraised for fractionation assays. The three steps of the original Community Bureau of Reference (BCR) sequential extraction scheme have been performed in both automated dynamic fractionation systems to evaluate the extractability of Cr, Cu, Ni, Pb, and Zn in a standard reference material of coal fly ash (NIST 1633b). In order to find the experimental conditions with the greatest influence on metal leachability in dynamic BCR fractionation, a full-factorial design was applied, in which the solid sample weight (100-500 mg) and the extraction flow rate (3.0-6.0 mL min(-1)) were selected as experimental factors. Identical cumulative extractabilities were found in both sequential injection (SI)-based methods for most of assayed trace elements regardless of the extraction conditions selected, revealing that both dynamic fractionation systems, as opposed to conventional steady-state BCR extraction, are not operationally defined within the selected range of experimental conditions. Besides, the proposed automated SI assemblies offer a significant saving of operational time with respect to classical BCR test, that is, 3.3 h versus 48 h, for complete fractionation with minimum analyst involvement.

A simple and expedient flow-based assembly capitalizing on programmable flow is herein proposed for reliable determination of trace level concentrations of aluminum as a potential contaminant in dialysis concentrate solutions without any prior sample clean-up/preconcentration procedure. Using salicylaldehyde picolinoylhydrazone in weakly acidic media as a turn-on fluorescent probe, the manifold is devised to handle three samples concurrently in stopped-flow reaction mode for simultaneous improvement of the analytical sensitivity and sample throughput. Analytical parameters influencing the sensitivity and repeatability of the assays, namely, probe concentration, reaction temperature and reaction time were investigated using a multivariate optimization protocol composed of a full factorial screening design followed by a Doehlert matrix model. The analysis of the Pareto chart and surface response revealed that the reaction time and amount of fluorescent probe were critical parameters for reliable assays of aluminum at the low ng mL−1 level. Under the optimized chemical and physical variables, a detection limit of 1.1 ng mL−1 Al(III) at the 3sblank level, relative standard deviations better than 1.0%, a dynamic linear range of 5.0–80 ng mL−1 and a sample throughput up to 25 h−1 were obtained with no need for either sample preconcentration or the use of organized supramolecular systems. Demonstrated with the analysis of hemodialysis and peritoneal concentrate solutions, and dialysis waters, the flow-through method copes with the requirements of regulatory bodies (e.g., European Pharmacopeia) for quality control of aluminum in high salinity containing dialysis concentrates.

Dynamic fractionation has been recognized as an appealing alternative to conventional equilibrium-based sequential extraction procedures (SEPs) for partitioning of trace elements (TE) in environmental solid samples. This paper reports the first attempt for harmonization of flow-through dynamic fractionation using two novel methods, the so-called sequential injection microcolumn (SIMC) extraction and rotating coiled column (RCC) extraction. In SIMC extraction, a column packed with the solid sample is clustered in a sequential injection system, while in RCC, the particulate matter is retained under the action of centrifugal forces. In both methods, the leachants are continuously pumped through the solid substrates by the use of either peristaltic or syringe pumps. A five-step SEP was selected for partitioning of Cu, Pb and Zn in water soluble/exchangeable, acid-soluble, easily reducible, easily oxidizable and moderately reducible fractions from 0.2 to 0.5 g samples at an extractant flow rate of 1.0 mL min −1 prior to leachate analysis by inductively coupled plasma-atomic emission spectrometry. Similarities and discrepancies between both dynamic approaches were ascertained by fractionation of TE in certified reference materials, namely, SRM 2711 Montana Soil and GBW 07311 sediment, and two real soil samples as well. Notwithstanding the different extraction conditions set by both methods, similar trends of metal distribution were in generally found. The most critical parameters for reliable assessment of mobilisable pools of TE in worse-case scenarios are the size-distribution of sample particles, the density of particles, the content of organic matter and the concentration of major elements. For reference materials and a soil rich in organic matter, the extraction in RCC results in slightly higher recoveries of environmentally relevant fractions of TE, whereas SIMC leaching is more effective for calcareous soils.