3D-printed stereolithographic fluidic devices for automatic nonsupported microelectromembrane extraction and clean-up of wastewater samples.

BACKGROUND: There is a quest of novel functional and reliable platforms for enhancing the efficiency of microextraction approaches in troublesome matrices, such as industrial wastewaters. 3D printing has been proven superb in the analytical field to act as the springboard of microscale extraction approaches. RESULTS: In this work, low-force stereolithography (SL) was exploited for 3D printing and prototyping bespoke fluidic devices for accommodating nonsupported microelectromembrane extraction (µEME). The analytical performance of 3D-printed µEME devices with distinct cross-sections, including square, circle, and obround, and various channel dimensions was explored against that of commonly used circular polytetrafluoroethylene (PTFE) tubing in flow injection systems. A computer-controlled millifluidic system was harnessed for the (i) automatic liquid-handling of minute volumes of donor, acceptor, and organic phases at the low µL level that spanned from 3 to 44 µL in this work, (ii) formation of three-phase µEME, (iii) in-line extraction, (iv) flow-through optical detection of the acceptor phase, and (v) solvent removal and regeneration of the µEME device and fluidic lines. Using methylene blue (MB) as a model analyte, experimental results evinced that the 3D-printed channels with an obround cross-section (2.5 mm × 2.5 mm) were the most efficient in terms of absolute extraction recovery (59%), as compared to PTFE tubing of 2.5 mm inner diameter (27%). This is attributed to the distinctive convex interface of the organic phase (1-octanol), with a more pronounced laminar pattern, in 3D-printed SL methacrylate-based fluidic channels against that of PTFE tubing on account of the enhanced 1-octanol wettability and lower contact angles for the 3D-printed devices. The devices with obround channels were leveraged for the automatic µEME and in-line clean-up of MB in high matrix textile dyeing wastewater samples with relative recoveries ≥81%, RSD% ≤ 17.1% and LOD of 1.3 mg L-1. The 3D-printed nonsupported µEME device was proven superb for the analysis of wastewater samples with an elevated ionic strength (0.7 mol L-1 NaCl, 5000 mg L-1 Na2CO3, and 0.013 mol L-1 NaOH) with recorded electric currents below 12 µA. NOVELTY: The coupling of 3D printing with nonsupported µEME in automatic flow-based systems is herein proposed for the first time and demonstrated for the clean-up of troublesome samples, such as wastewaters.

Screening of organic chemicals associated to virgin low-density polyethylene microplastic pellets exposed to the Mediterranean Sea environment by combining gas chromatography and liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry.

In this work, organic chemicals associated with microplastics (MPs) exposed to a coastal anthropogenized environment for up to eight weeks have been screened for, in order to discern the (de)sorption dynamics of chemicals in the marine ecosystem. Low-density polyethylene (LDPE) pellets were studied since they represent primary MPs used by the plastic industry and a relevant input of MPs into the oceans. To maximize the coverage of chemicals that could be detected, both liquid and gas chromatography coupled to quadrupole-time-of-flight (GC-QTOF and LC-QTOF, respectively) were used. In the case of LC-QTOF, an electrospray ionization source was employed, and the compounds were investigated by combining suspect and non-target screening workflows. The GC-QTOF was equipped with an electron ionization source and compounds were screened in raw and derivatized (silylated) extracts by deconvolution and contrast to high- and low-resolution libraries. A total of 50 compounds of multifarious classes were tentatively identified. Among them, melamine and 2-ethylhexyl salicylate (EHS) were detected in the original MPs but were rapidly desorbed. Melamine was completely released into the marine environment, while EHS was partly released but a portion remained bound to the MPs. On the other hand, many other chemicals of both anthropogenic (e.g. phenanthrene or benzophenone) and natural origin (e.g. betaine and several fatty acids) accumulated onto MPs over time. Quantification of 12 unequivocally identified chemicals resulted into a total concentration of 810 µg/kg after MPs exposure for 8 weeks.

Analytical probing of membranotropic effects of antimicrobial copper nanoparticles on lipid vesicles as membrane models.

Copper nanoparticles (CuNPs) are antimicrobial agents that are increasingly being used in several real-life goods. However, concerns are arising about their potential toxicity and thus, appropriate legislation is being issued in various countries. In vitro exploration of the permeability and the distribution of nanoparticles in cell membranes should be explored as the first step towards the investigation of the toxicity mechanisms of metal nanoantimicrobials. In this work, phosphatidylcholine-based large unilamellar vesicles have been explored as mimics of cellular membranes to investigate the effect of ultra-small CuNPs on the physicochemical features of phospholipid membranes. 4 nm-sized CuNPs were synthesized by a wet-chemical route that involves glutathione as a stabilizer, with further characterization by UV-vis absorption spectroscopy, fluorescence spectroscopy, transmission electron microscopy, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FTIR) spectroscopy. Two fluorescent membrane probes bearing naphthalene moieties (laurdan and prodan) were used to monitor the bilayer structure and dynamics, as well as to demonstrate the strong membranotropic effects of CuNPs. The fluorescence spectroscopic studies were supported by dynamic light scattering (DLS) measurements and the calcein leakage assay. Additionally, the degree of perturbation of the phospholipid bilayer by CuNPs was compared against that of Cu2+ ions, the latter resulting in negligible effects. The findings suggested that CuNPs are able to damage the phospholipid membranes, leading to their agglomeration or disruption.

Biomimetic Dispersive Solid-Phase Microextraction: A Novel Concept for High-Throughput Estimation of Human Oral Absorption of Organic Compounds.

There is a quest for a novel in vitro analytical methodology that is properly validated for the prediction of human oral absorption and bioaccumulation of organic compounds with no need of animal models. The traditional log P parameter might not serve to predict bioparameters accurately inasmuch as it merely accounts for the hydrophobicity of the compound, but the actual interaction with the components of eukaryotic cells is neglected. This contribution proposes for the first time a novel biomimetic microextraction approach capitalized on immobilized phosphatidylcholine as a plasma membrane surrogate onto organic polymeric sorptive phases for the estimation of human intestinal effective permeability of a number of pharmaceuticals that are also deemed contaminants of emerging concern in environmental settings. A comprehensive exploration of the conformation of the lipid structure onto the surfaces is undertaken so as to discriminate the generation of either lipid monolayers or bilayers or the attachment of lipid nanovesicles. The experimentally obtained biomimetic extraction data is proven to be a superb parameter against other molecular descriptors for the development of reliable prediction models of human jejunum permeability with R2 = 0.76, but the incorporation of log D and the number of aromatic rings in multiple linear regression equations enabled improved correlations up to R2 = 0.88. This work is expected to open new avenues for expeditious in vitro screening methods for oral absorption of organic contaminants of emerging concern in human exposomics.

Total analysis system for the determination of uremic toxins in human plasma based on bead injection solid phase extraction hyphenated to mass spectrometry.

Indoxyl sulfate (INDS) and p-cresol sulfate (pCS) are two of the most relevant uremic toxins that are recognized to have an essential role in chronic kidney disease (CKD) progression and associated cardiovascular risk. Thus, it is crucial to accurately assess their circulating levels in the body. Aiming at establishing an analytical strategy for quantification of INDS and pCS in human plasma, an automatic on-line micro-solid-phase extraction (µSPE) procedure hyphenated to tandem mass spectrometry (MS/MS) detection without previous chromatographic separation was herein developed. The bead injection (BI) concept was used to implement the µSPE procedure in the lab-on-valve (LOV) format. After studying the extraction conditions, the anion-exchange OASIS WAX sorbent beads (10 mg) and 99% ACN-H2O (15:85, v/v)-1% (v/v) NH4OH were chosen as sorbent and eluent, respectively, as they provided the highest analyte recoveries. Subsequently, the µSPE-BI-LOV system was hyphenated on-line to a MS/MS detector and the full analytical cycle, comprising sample preparation and analytes detection, was completed in <20 min. The developed µSPE-BI-LOV-MS methodology presented good linearity (r2 > 0.999) for quantification of the target analytes at concentrations ranging from 18 to 360 µg mL-1 in plasma. LOQ values were 2 µg mL-1 for INDS and 7 µg mL-1 for pCS in plasma. Human plasma samples from healthy subjects and individuals with CKD were successfully analyzed using the developed approach. The proposed automatic methodology can be described as an eco-friendly strategy, with a favorable score of 0.64 after greenness evaluation using the AGREE metric.

3D-printed chemiluminescence flow cells with customized cross-section geometry for enhanced analytical performance.

Low force stereolithography is exploited for the first time for one-step facile fabrication of chemiluminescence (CL) flow-through cells that bear unrivalled features as compared to those available through milling or blowing procedures or alternative 3D printing technologies. A variety of bespoke cross-section geometries with polyhedral features (namely, triangular, square, and five-side polygon) as well as semicircular cross-section are herein critically evaluated in terms of analytical performance against the standardcircular cross-section in a flat spirally-shape format. The idea behind is to maximize capture of elicited light by the new designs while leveraging 3D printing further for fabrication of (i) customized gaskets that enable reliable attaching of the active mixing zone of the CL cell to the detection window, (ii) in-line 3D-printed serpentine reactors, and (iii) flow confluences with tailorable shapes for enhancing mixing of samples with CL reagents. Up to twenty transparent functional cells were simultaneously fabricated without inner supports following post-curing and surface treatment protocols lasting less than 5 h. In fact, previous attempts to print spirally-shaped cells in one-step by resorting to less cost effective photopolymer inkjet printing technologies were unsuccessful because of the requirement of lengthy procedures (>15 days) for quantitative removal of the support material. By exploiting the phthalazinedione-hydrogen peroxide chemistry as a model reaction, the five-side irregular pentagon cell exhibited superior analytical figures of merit in terms of LOD, dynamic range and intermediate precision as compared to alternative designs. Computational fluid dynamic simulations for mapping velocities at the entry region of the spiral cell corroborated the fact that the 5-side polygon cross-section flow-cell with Y-type confluence permitted the most efficient mixing of reagents and sample while enabling larger flow velocities near the inlet that contribute to a more efficient capture of the photons from the flash-type reaction. The applicability of the 3D-printed 5-side polygon CL cell for automatic determination of hydrogen peroxide using a computerized hybrid flow system was demonstrated for the analysis of high matrix samples, viz., seawater and saliva, with relative recoveries ranging from 83 to 103%.

Sample preparation and chromatographic methods for the determination of protein-bound uremic retention solutes in human biological samples: An overview.

Protein-bound uremic retention solutes, such as indole-3-acetic acid, indoxyl sulfate, p-cresol and p-cresol sulfate, are associated with the development of several pathologies, namely renal, cardiovascular, and bone toxicities, due to their potential accumulation in the human body, thus requiring analytical methods for monitoring and evaluation. The present review addresses conventional and advanced sample treatment procedures for sample handling and the chromatographic analytical methods developed for quantification of these compounds in different biological fluids, with particular focus on plasma, serum, and urine. The sample preparation and chromatographic methods coupled to different detection systems are critically discussed, focusing on the different steps involved for sample treatment, namely elimination of interfering compounds present in the sample matrix, and the evaluation of their environmental impact through the AGREEprep tool. There is a clear trend for the application of liquid-chromatography coupled to tandem mass spectrometry, which requires protein precipitation, solid-phase extraction and/or dilution prior to analysis of biological samples. Furthermore, from a sustainability point of view, miniaturized methods resorting to microplate devices are highly recommended.

Programmable Millifluidic Platform Integrating Automatic Electromembrane Extraction Cleanup and In-Line Electrochemical Detection: A Proof of Concept.

A fully automatic millifluidic sensing platform coupling in-line nonsupported microelectromembrane extraction (µ-EME) with electrochemical detection (ECD) is herein proposed for the first time. Exploiting the features of the second generation of flow analysis, termed sequential injection (SI), the smart integration of SI and µ-EME-ECD enables (i) the repeatable formation of microvolumes of phases for the extraction step in a membrane-less (nonsupported) arrangement, (ii) diverting the acceptor plug to the ECD sensing device, (iii) in-line pH adjustment before the detection step, and (iv) washing of the platform for efficient removal of remnants of wetting film solvent, all entirely unsupervised. The real-life applicability of the miniaturized sensing system is studied for in-line sample cleanup and ECD of diclofenac as a model analyte after µ-EME of urine as a complex biological sample. A comprehensive study of the merits and the limitations of µ-EME solvents on ECD is presented. Under the optimal experimental conditions using 14 µL of unprocessed urine as the donor, 14 µL of 1-nonanol as the organic phase, and 14 µL of 25 mM NaOH as the acceptor in a 2.4 mm ID PTFE tubing, an extraction voltage of 250 V, and an extraction time of 10 min, an absolute (mass) extraction recovery of 48% of diclofenac in urine is obtained. The proposed flow-through system is proven to efficiently remove the interfering effect of predominantly occurring organic species in human urine on ECD with RSD% less than 8.6%.

Real-time monitoring of Metridia luciferase release from cells upon interaction with model toxic substances by a fully automatic flow setup - A proof of concept.

This manuscript reports on a fully automatic sequential injection system incorporating a 3D printed module for real-time monitoring of the release of Metridia luciferase from a modified liver epithelial cell line. To this end, a simple and effective approach for the automation of flash-type chemiluminescence assays was developed. The 3D printed module comprised an apical and a basal compartment that enabled monitoring membrane processes on both sides of the cell monolayer aimed at elucidating the direction of luciferase release. A natural release was observed after transfection with the luciferase plasmid by online measurement of the elicited light from the reaction of the synthesized luciferase with the coelenterazine substrate. Model substances for acute toxicity from the group of cholic acids - chenodeoxycholic and deoxycholic acids - were applied at the 1.0 and 0.5 mmol L-1 levels. The tested cholic acids caused changes in cell membrane permeability that was accompanied by an increased luciferase release. The obtained kinetic profiles were evaluated based on the delay between the addition of the toxic substance and the increase of the chemiluminescence signal. All experiments were carried out in a fully automatic system in ca. 5 min per sample in 30 min intervals and no manual interventions were needed for a sampling period of at least 6 h.

3D printed spinning cup-shaped device for immunoaffinity solid-phase extraction of diclofenac in wastewaters.

This article reports current research efforts towards designing bespoke microscale extraction approaches exploiting the versatility of 3D printing for fast prototyping of novel geometries of sorptive devices. This is demonstrated via the so-called 3D printed spinning cup-based platform for immunoextraction of emerging contaminants using diclofenac as a model analyte. A new format of rotating cylindrical scaffold (containing a semispherical upper cavity) with enhanced coverage of biorecognition elements, and providing elevated enhancement factors with no need of eluate processing as compared with other microextraction stirring units is proposed. Two distinct synthetic routes capitalized upon modification of the acrylate surface of stereolithographic 3D printed parts with hexamethylenediamine or branched polyethyleneimine chemistries were assayed for covalent binding of monoclonal diclofenac antibody.Under the optimized experimental conditions, a LOD of 108 ng L-1 diclofenac, dynamic linear range of 0.4-1,500 µg L-1, and enrichment factors > 83 (for near-exhaustive extraction) were obtained using liquid chromatography coupled with UV-Vis detection. The feasibility of the antibody-laden device for handling of complex samples was demonstrated with the analysis of raw influent wastewaters with relative recoveries ranging from 102 to 109%. By exploiting stereolithographic 3D printing, up to 36 midget devices were fabricated in a single run with an estimated cost of mere 0.68 euros per 3D print and up to 16 €/device after the incorporation of the monoclonal antibody.

Automated Sequential Injection-Capillary Electrophoresis for Dried Blood Spot Analysis: A Proof-of-Concept Study.

A hyphenated analytical platform that enables fully automated analyses of dried blood spots (DBSs) is proposed by the at-line coupling of sequential injection (SI) to capillary electrophoresis (CE). The SI system, exploited herein for the first time for unattended DBS handling, serves as the "front end" mesofluidic platform for facilitating exhaustive elution of the entire DBS by flow programming. The DBS eluates are thus free from hematocrit and nonhomogeneity biases. The SI pump transfers the resulting DBS eluates into CE sample vials through an internal port of the CE instrument and homogenizes the eluates, whereupon the eluted blood compounds are automatically injected, separated, and quantified by the CE instrument. The SI and CE are commercially available off-the-shelf instruments and are interconnected through standard nuts, ferrules, and tubing without additional instrumental adjustments. They are controlled by dedicated software and are synchronized for a fully autonomous operation. The direct determination of endogenous (potassium and sodium) and exogenous (lithium as a model drug) inorganic cations in DBS samples has been used for the proof-of-concept demonstration. The hyphenated SI-CE platform provides excellent precision of the analytical method with relative standard deviation (RSD) values of peak areas below 1.5 and 3.5% for intraday and interday analyses, respectively, of the endogenous concentrations of the two inorganic cations. For the determination of lithium, calibration is linear in a typical clinical range of the drug (R2 better than 0.9993 for 2-20 mg/L), RSD values of peak areas are below 4.5% (in the entire calibration range), the limit of detection (0.4 mg/L) and the limit of quantification (1.3 mg/L) are well below the drug's minimum therapeutic concentration (4 mg/L), and total analysis time is shorter than 5 min. The SI-CE platform reflects the actual trends in the automation of analytical methods, offers rapid and highly flexible DBS elution/analysis processes, and might thus provide a general solution to modern clinical analysis as it can be applied to a broad range of analytes and dried biological materials.

Mimicking human ingestion of microplastics: Oral bioaccessibility tests of bisphenol A and phthalate esters under fed and fasted states.

Notwithstanding the fact that microplastic fragments were encountered in the human stool, little effort has been geared towards elucidating the impact of chemical additives upon the human health. In this work, standardized bioaccessibility tests under both fasting and fed conditions are herein applied to the investigation of human oral bioaccessibility of plastic additives and monomers (i.e. eight phthalate esters (PAEs) and bisphenol A (BPA)) in low-density polyethylene (LDPE) and polyvinyl chloride (PVC) microplastics. The generation of phthalate monoesters is evaluated in the time course of the bioaccessibility tests. Maximum gastric and gastrointestinal bioaccessibility fractions are obtained for dimethyl phthalate, diethyl phthalate and BPA, within the range of 55-83%, 40-68% and 37-67%, respectively, increasing to 56-92% and 41-70% for dimethyl phthalate and diethyl phthalate, respectively, whenever their hydrolysis products are considered. Bioaccessibility fractions of polar PAEs are dependent upon the physicochemical characteristics of the microplastics, with greater bioaccessibility for the rubbery polymer (LDPE). With the method herein proposed, oral bioaccessible pools of moderately to non-polar PAEs can be also accurately assessed for risk-assessment explorations, with values ranging from 1.8% to 32.2%, with again significantly larger desorption percentages for LDPE. Our results suggested that the highest gastric/gastrointestinal bioaccessibility of the eight PAEs and BPA is reached under fed-state gastrointestinal extraction conditions because of the larger amounts of surface-active biomolecules. Even including the bioaccessibility factor within human risk assessment/exposure studies to microplastics, concentrations of dimethyl phthalate, di-n-butyl phthalate and BPA exceeding 0.3% (w/w) may pose severe risks after oral uptake in contrast to the more hydrophobic congeners for which concentrations above 3% (w/w), except for diethylhexyl phthalate, would be tolerated.

Automatic and renewable micro-solid-phase extraction based on bead injection lab-on-valve system for determination of tranexamic acid in urine by UHPLC coupled with tandem mass spectrometry.

An automatic micro-solid-phase extraction (µSPE) method using on-line renewable sorbent beads followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established for the determination of tranexamic acid (TXA) in urine. The µSPE method was based on the bead injection (BI) concept combined with the mesofluidic lab-on-valve (LOV) platform. All steps of the µSPE-BI-LOV were implemented by computer programming, rendering enhanced precision on time and flow events. Several parameters, including the type of sorbent, volume and composition of the conditioning solution, washing solution, and eluent composition, were evaluated to improve the extraction efficiency. The best results were obtained with a hydrophilic-lipophilic balanced mixed-mode sorbent, decorated with sulfonic acid groups (Oasis MCX), and 99% acetonitrile-water (50:50, v/v)-1% ammonium hydroxide as eluent. Chromatographic separation was performed using a BEH amide column coupled to MS/MS detection in positive ionization mode. Good linearity was achieved (R2 > 0.998) for TXA concentrations in urine ranging from 300 to 3000 ng mL-1, with LOD and LOQ of 30 and 65 ng mL-1, respectively. Dilution integrity was observed for dilution factors up to 20,000 times, providing the extension of the upper limit of quantification to 12 mg mL-1. The method was validated according to international guidelines and successfully applied to urine samples collected during scoliosis surgery of pediatric patients treated with TXA.

Source identification of amphetamine-like stimulants in Spanish wastewater through enantiomeric profiling.

Amphetamine (AMP), methamphetamine (MAMP) and 3,4-methylenedioxymethamphetamine (MDMA) occur in wastewater not only as a result of illicit consumption, but also, in some cases, from prescription drug use or by direct drug disposal into the sewage system. Enantiomeric profiling of these chiral drugs could give more insight into the origin of their occurrence. In this manuscript, a new analytical methodology for the enantiomeric analysis of amphetamine-like substances in wastewater has been developed. The method consists of a solid-phase extraction (SPE) followed by liquid chromatography-triple quadrupole-tandem mass spectrometry (LC-MS/MS), which showed low quantification limits in the 2.4-5.5 ng L-1 range. The LC-MS/MS method was first applied to characterize a total of 38 solid street drug samples anonymously provided by consumers. The results of these analysis showed that AMP and MDMA trafficked into Spain are synthesized as racemate, while MAMP is exclusively produced as the S(+)-enantiomer. Then, the analytical method was employed to analyse urban wastewater samples collected from the wastewater treatment plants (WWTPs) of five different cities in 2018 and 2019. Consumption estimated through normalized population loads in wastewater showed an increased pattern of AMP use in the Basque Country. Furthermore, the enantiomeric profiling of wastewater samples was contrasted to lisdexamfetamine (LIS) and selegiline (SEL) prescription figures, two pharmaceuticals which metabolize to S(+)-AMP, and to R(-)-AMP and R(-)-MAMP, respectively. From this analysis, and considering uncertainties derived from metabolism and adherence to treatment, it was concluded that LIS is a relevant source of AMP in those cases with low wastewater loads, i.e. up to a maximum of 60% of AMP detected in wastewater in some samples could originate from LIS prescription, while SEL does not represent a significant source of AMP nor MAMP. Finally, removal efficiencies could be evaluated for the WWTP (serving ca. 860,000 inhabitants) with higher AMP influent concentrations. The removal of AMP was satisfactory with rates higher than 99%, whereas MDMA showed an average removal of approximately 60%, accompanied by an enrichment of R(-)-MDMA.

Evidence of high bioaccessibility of gadolinium-contrast agents in natural waters after human oral uptake.

Considering the large occurrence of anthropogenic Gd concentrations in natural waters, its continuous usage increase in technology developments and products and the lack of data on potential Gd human exposure due to ingestion of contaminated waters, it is urgently needed to understand how gadolinium contrast agents (Gd-CAs) reacts in the human digestive system. Here, we aimed to identify through in vitro bioaccessibility tests whether Gd-CAs can be potentially assimilated by humans after oral uptake and if there is a significant difference between contrast agents. We also roughly estimated the potential bioaccessibility of anthropogenic Gd for tap waters worldwide. Gd-CAs are highly bioaccessible (77 to 112%). The macrocyclic complexes pose the highest potential risk, because there are more stable than linear complexes in the gastrointestinal tract and, as such, tend to remain in solution and thus might bring Gd at the intestinal barrier making it potentially bioavailable. The estimated range of potential intake of Gd varied from 13 to 4839 µg in a lifespan of 70 years. The high bioaccessibility of anthropogenic Gd in tap waters calls for appropriate actions to develop better practices to treat wastewater contaminated by Gd-CAs in order to safeguard population and ecosystem health.

An automatic flow-through system for exploration of the human bioaccessibility of endocrine disrupting compounds from microplastics.

This article reports on the first attempt towards investigating the leaching rates in the human gastrointestinal (GI) tract of plastic-borne contaminants that can be ingested accidentally using physiologically relevant body fluids. Oral bioaccessibility under fasted and fed states was determined in dynamic mode exploiting an automatic flow setup. The flow system is able to mimic the fast uptake of the released species from the polymeric matrix by absorption in the human digestive system by the in-line removal of the leached species. Complex GI extractants based on the Unified Bioaccessibility Method (UBM, fasted state) and Versantvoort test (fed-state) were brought through a microplastic-loaded metal microcolumn for semi-continuous leaching of plasticizers (phthalic acid ester congeners) and monomer/antioxidant species (bisphenol A, BPA) followed by in-line solid-phase extraction and clean-up of GI extracts prior to liquid chromatography analysis. The temporal extraction profiles were fitted to a first-order kinetic model for the estimation of maximum bioaccessibility pools and apparent leaching rates. Among all studied contaminants, only BPA, dimethylphthalate and diethylphthalate were appreciably released under dynamic GI conditions from high-density polyethylene pellets (average size of 110 µm), with average bioaccessibility values spanning from 51 to 84% and 48 to 87% for UBM and Versantvoort methods, respectively. No statistically significant differences in oral bioaccessibility pools were found under fed- and fasted-state dynamic extractions. The apparent kinetic constants under the fed state were greater by ≥30% as a consequence of the effect of the larger amounts of bile salts and digestive enzymes in the Versantvoort test on the leaching rates. The estimated average daily intake, in which bioaccessibility data are contemplated, indicated that plastic materials exceeding 0.3% (w/w) BPA might pose real risks to human health.