Liquid Chromatography-mass Spectrometry Detection Research Articles

Non-noble-metal TiC-nanoparticle-promoted charge separation and photocatalytic degradation performance on Bi2O3 microrods: degradation pathway and mechanism investigation.

In the present work, a promising binary TiC/Bi2O3 photocatalyst was obtained via a simple hydrothermal route. In the photodegradation experiment of acid orange 7 (AO7) and tetracycline (TC) irradiated by simulated sunlight, the attachment of TiC nanoparticles on the Bi2O3 microrods leads to an obvious improvement in the photocatalytic removal properties of the Bi2O3 microrods. The optimal removal efficiency of AO7 was achieved by the 7.5%TiC/Bi2O3 sample, which results in about 91.5% dye removal within 75 min of reaction. Meanwhile, the 7.5%TiC/Bi2O3 sample also exhibits favorable photodegradation performance for the degradation of TC, leading to about ∼76.9% TC being degraded after 75 min of irradiation. More importantly, the degradation pathways of AO7 and toxicity analysis of the intermediate products were performed based on liquid chromatography-mass spectrometry detection and theoretical simulation. The superior photocatalytic behavior of the TiC/Bi2O3 composite is attributed to the effective separation and migration of photoexcited electrons and holes in the heterojunction, where the TiC nanoparticles act as an acceptor of photoexcited electrons.

Scopolamine and atropine in feeds – determination with liquid chromatography mass spectrometry

Tropane alkaloids (TAs) are naturally occurring plant toxins. Due to the fact that TA-producing plants can enter the food chain, they pose a risk for animals and human health. Therefore, sensitive analytical methods need to be developed to provide an adequate safety of feed and food. The presented method is based on liquid chromatography-mass spectrometry detection and enables the determination of scopolamine and atropine in compound feeds at a low level of contamination. Limits of quantification for scopolamine and atropine were 0.92 and 0.93 µg kg−1, respectively. Scopolamine-D3 and atropine-D3 were used for quantification. The method was successfully validated and applied to the analysis of 42 feed samples. Among investigated feeds, 67% contained at least one of the monitored alkaloids. Soybean meals were the feed materials contaminated most often, also with the highest determined concentrations of TAs, which reached 147.9 µg kg−1.

Monitoring of pharmaceuticals in aquatic biota (Procambarus clarkii) of the Doñana National Park (Spain)

In this work the presence of different pharmaceuticals at Doñana National Park (Spain) and their main entry sources (input source or entry points) have been stated over the 2011–2016 years period. Twenty-three selected pharmaceuticals (corresponding to eight therapeutic families) were evaluated in crayfish and water samples from Doñana National Park (Spain) (six sampling points selected in order to cover different possible pollution sources into and surrounding the Park). The multiresidue determination was carried out using enzymatic-microwave assisted extraction prior to high performance liquid chromatography mass spectrometry detection. Sulphonamides (sulfadiazine, sulfamerazine, sulfamethazine, and sulfamethoxazole); trimethoprim, an antibiotic that is frequently co-administered with sulfamethoxazole; amphenicols (chloramphenicol, florfenicol and thiamphenicol); fluoroquinolones (ciprofloxacin, enrofloxacin, flumequine, danofloxacin, gatifloxacin, norfloxacin, marbofloxacin and grepafloxacin); penicillins (amoxicillin); tetracyclines (chlortetracycline and oxytetracycline); non-steroidal anti-inflammatory drugs (salicylic acid and ibuprofen); beta-blocker drugs (atenolol); and antiepileptics (carbamazepine) were analysed. Ciprofloxacin, ibuprofen, salicylic acid, flumequine, and carbamazepine were detected and/or quantified at some of the selected sampling points. A clear ecotoxicological risk to the ecosystem was demonstrated from the occurrence of ciprofloxacin in samples obtained after the punctual and massive presence of people inside the Park. Furthermore, flumequine and carbamazepine have been detected in Procambarus clarkii specimens in concentrations around 30 ng g−1 and 14 ng g−1, respectively, and their occurrence in the specimens could indicate the persistence of the discharge sources. The main source of pharmaceuticals into the Park might be the livestock farming activities, and the influence of urban wastewaters from surrounding villages does not seem to be very important.

Liquid Chromatography Mass Spectrometry Detection of Antibiotic Agents in Sputum from Persons with Cystic Fibrosis.

Antibiotic therapy is expected to impact host microbial communities considerably, yet many studies focused on microbiome and health are often confounded by limited information about antibiotic exposure. Given that antibiotics have diverse pharmacokinetic and antimicrobial properties, investigating the type and concentration of these agents in specific host specimens would provide much needed insight into their impact on the microbes therein. Here, we developed liquid chromatography mass spectrometry (LC-MS) methods to detect 18 antibiotic agents in sputum from persons with cystic fibrosis. Antibiotic spike-in control samples were used to compare three liquid extraction methods on the Waters Acquity Quattro Premier XE. Extraction with dithiothreitol captured the most antibiotics and was used to detect antibiotics in sputum samples from 11 people with cystic fibrosis, with results being compared to the individuals' self-reported antibiotic use. For the sputum samples, two LC-MS assays were used; the Quattro Premier detected nanomolar or micromolar concentrations of 16 antibiotics, whereas the Xevo TQ-XS detected all 18 antibiotics, most at subnanomolar levels. In 45% of tested sputum samples (71/158), at least one antibiotic that was not reported by the subject was detected by both LC-MS methods, a discordance largely explained by the thrice weekly administration and long half-life of azithromycin. For ∼37% of samples, antibiotics reported as taken by the individual were not detected by either instrument. Our results provide an approach for detecting a variety of antibiotics at the site of infection, thereby providing a means to include antibiotic usage data into microbiome studies.

Determination of Synthetic Cathinones in Urine and Oral Fluid by Liquid Chromatography High-Resolution Mass Spectrometry and Low-Resolution Mass Spectrometry: A Method Comparison

Synthetic cathinones have become very popular recreational drugs. Therefore, determining them in biological samples is now a matter of concern. In recent years, different methods that have been developed can determine these drugs at low-concentration levels. In general, liquid chromatography mass spectrometry detection plays an important role in these methods and the trend is to use low-resolution and high-resolution mass spectrometry. In this article, for the first time, we compare these two analyzers using an Orbitrap and a triple quadrupole mass spectrometer in order to determine a group of synthetic cathinones in urine and oral fluid samples. For this comparison, we evaluated and compared different parameters: Method detection and quantification limits, linearity, apparent recoveries, matrix effect, repeatability (intra-day), reproducibility (inter-day), and accuracy. Similar results were obtained for the two analyzers for the apparent recoveries and matrix effect. However, triple quadrupole showed higher sensitivity compared to Orbitrap for both urine and oral fluid samples. The quantification limits in urine and the detection limits in saliva were two times lower for triple quadrupole. Finally, when blind samples were analyzed to study the accuracy, similar results were obtained for both analyzers.

Determination of organophosphorus pesticides in wastewater samples using vortex-assisted dispersive liquid–liquid microextraction with liquid chromatography–mass spectrometry

A simple and rapid method for determination of azinphos-methyl, parathion-methyl and ethoprofos, group of organophosphorus pesticides in wastewater matrices, is presented. A chemometric approach for the optimisation of vortex-assisted dispersive liquid–liquid microextraction experimental conditions prior to liquid chromatography–mass spectrometry detection was applied. In this method, a high-density organic solvent (chloroform) was used as the extractant, with acetone as the disperser solvent. Vortexing was applied prior to centrifugation for phase separation of the organic phase (sedimented layer of extractant) and the aqueous layer. A two-level full factorial design (24) was employed initially for the screening process, and final optimisation of the significant parameters was performed using response surface methodology based on central composite design. The method performance characteristics investigated included linear dynamic range (LDR, 5–100 µg L−1) with a good correlation coefficient (> 0.999). The method precision expressed as intra-day and inter-day relative standard deviation (%RSD) was in the range of 7.8–8.2% and 8.1–9.4%, respectively. The influence of matrix was found to be negligible with recoveries ranging from 99.9 to 106.7%. The proposed method was then applied in real wastewater samples. Extraction recoveries performed at two spiking levels (25 and 100 µg L−1) in untreated (influent) and treated (effluent) wastewater matrices ranged between 94.95 and 119.47%.

G Protein-Coupled Estrogen Receptor Production Using an Escherichia coli Cell-Free Expression System.

Heterologous expression of the G protein-coupled estrogen receptor (GPER) comes with a suite of challenges intrinsic to membrane proteins. This receptor's low expression levels and tendency to form insoluble aggregates in Escherichia coli and yeast make it a difficult receptor-target to study. In this unit, we detail steps to produce monomeric GPER using a precipitation-based cell-free system. We provide information on the DNA construct for expression, the pipetting scheme for the reaction supplements to generate a master mix, and the cell-free reaction setup. In the last portion of this unit, we outline steps for solubilization and purification, and we provide a viable method for qualitatively observing functionality by liquid chromatography-mass spectrometry detection. © 2019 by John Wiley & Sons, Inc.

Phytochelatin Characterization in Commonly Consumed Plant Foods Using Mass Spectrometry-based Metabolomics (P18-122-19)

ObjectivesPhytochelatins (PyCs) are a group of metal-binding compounds formed by plants which could impact bioavailability of essential and toxic metals in the human diet. Liquid chromatography mass spectrometry (LC-MS)-based metabolomics can characterize a diversity of compounds from complex matrices. The aim of this project was to determine chromatographic characteristics of PyCs and their mass spectral signatures using authentic standards and determine if two types of PyCs (PyC2-Gly and PyC3-Gly) can be detected in commonly consumed plant foods and validated using chromatographic characteristics and ion dissociation mass spectrometry (MS/MS). MethodsWe analyzed PyC2-Gly and PyC3-Gly standards using LC-MS (C18 column; 10 minute analysis, positive ionization mode) at 5 different concentrations with 3 sample preparation methods to determine the common adducts, retention times, and optimal sample preparation methods. The most common adducts were selected for MS/MS to determine the characteristic fragmentation patterns. Onion, carrot, tomato, and whole wheat flour samples were analyzed with LC-MS/MS using the optimized sample preparation method and determined chromatographic and ion dissociation characteristics from the PyC standards analysis. ResultsFor both PyC standards, the most common adducts were identified as follows: M + H > M + Na > M +K > 2M + H > 2M + Na = 2M + K > M + 2H and metabolite features had a retention time of 42 seconds. The fragmentation patterns for PyC2-Gly and PyC3-Gly for the M + H adduct (m/z 538.1272 and m/z 770.1790) were determined by MS/MS. The results show that fragmentation of m/z 538.1272 yielded m/z 231.0433, 334.0525, and 409.0844, and m/z 770.1790 yielded m/z 231.0433, 308.0911, and 641.1358. PyC2-Gly was detected in all 4 food types with a fragmentation pattern matching the PyC2-Gly standard while PyC3-Gly was not detected in these food samples. ConclusionsThis project defines chromatographic and mass spectrometry characteristics of PyC2-Gly and PyC3-Gly. Using these characteristics, we were able to validate PyC2-Gly in 4 plant foods. As PyCs may impact metal bioavailability from the diet, optimizing LC-MS detection methods for PyCs in plant foods facilitates future characterization of PyCs in the human diet. Funding SourcesNational Institute of Environmental Health Sciences.

Ricin-like proteins from the castor plant do not influence liquid chromatography-mass spectrometry detection of ricin in forensically relevant samples

The toxic protein ricin (also known as RCA60), found in the seed of the castor plant (Ricinus communis) is frequently encountered in law enforcement investigations. The ability to detect ricin by analyzing its proteolytic (tryptic) peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is well established. However, ricin is just one member of a family of proteins in R. communis with closely related amino acid sequences, including R. communis agglutinin I (RCA120) and other ricin-like proteins (RLPs). Inferring the presence of ricin from its constituent peptides requires an understanding of the specificity, or uniqueness to ricin, of each peptide. Here we describe the set of ricin-derived tryptic peptides that can serve to uniquely identify ricin in distinction to closely-related RLPs and to proteins from other species. Other ricin-derived peptide sequences occur only in the castor plant, and still others are shared with unrelated species. We also characterized the occurrence and relative abundance of ricin and related proteins in an assortment of forensically relevant crude castor seed preparations. We find that whereas ricin and RCA120 are abundant in castor seed extracts, other RLPs are not represented by abundant unique peptides. Therefore, the detection of peptides shared between ricin and RLPs (other than RCA120) in crude castor seed extracts most likely reflects the presence of ricin in the sample.

Analysis of trace microcystins in vegetables using matrix solid-phase dispersion followed by high performance liquid chromatography triple-quadrupole mass spectrometry detection

A selective and sensitive method for the simultaneous determination of five common microcystins (MC-LR, -RR, -YR, -LW, -LF) in various vegetables was established using Matrix Solid-Phase Dispersion (MSPD) followed by high performance liquid chromatography-mass spectrometry detection. To optimize the pretreatment procedure for extracting the microcystins from three vegetable matrices (tomato, cucumber and spinacia, which represented different colors), Box-Behnken design was employed to optimize the main factors affecting the extraction efficiency, including sample/dispersant mass ratio, as well as the proportion mixture dispersant and the volumes of solvent. Based on the optimum conditions, the intra-day and inter-day variability for each microcystins in all vegetable samples were less than 8.6%, with the recoveries ranging from 71.9% to 96.5%. The limits of detection and quantitation of the method were 13.0μg/kg (dw) and 43.0μg/kg (dw), respectively. The established method was successfully applied to the analysis of MCs in vegetable samples which were collected by Lake Taihu. The procedure promising to be relative small sample size and short pretreatment time for the assay that can be used for monitoring MCs concentrations in vegetables.

Development of chemical isotope labeling liquid chromatography mass spectrometry for silkworm hemolymph metabolomics

Silkworm (Bombyx mori) is a very useful target insect for evaluation of endocrine disruptor chemicals (EDCs) due to mature breeding techniques, complete endocrine system and broad basic knowledge on developmental biology. Comparative metabolomics of silkworms with and without EDC exposure offers another dimension of studying EDCs. In this work, we report a workflow on metabolomic profiling of silkworm hemolymph based on high-performance chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) and demonstrate its application in studying the metabolic changes associated with the pesticide dichlorodiphenyltrichloroethane (DDT) exposure in silkworm. Hemolymph samples were taken from mature silkworms after growing on diet that contained DDT at four different concentrations (1, 0.1, 0.01, 0.001 ppm) as well as on diet without DDT as controls. They were subjected to differential 12C-/13C-dansyl labeling of the amine/phenol submetabolome, LC-UV quantification of the total amount of labeled metabolites for sample normalization, and LC-MS detection and relative quantification of individual metabolites in comparative samples. The total concentration of labeled metabolites did not show any significant change between four DDT-treatment groups and one control group. Multivariate statistical analysis of the metabolome data set showed that there was a distinct metabolomic separation between the five groups. Out of the 2044 detected peak pairs, 338 and 1471 metabolites have been putatively identified against the HMDB database and the EML library, respectively. 65 metabolites were identified by the dansyl library searching based on the accurate mass and retention time. Among the 65 identified metabolites, 33 positive metabolites had changes of greater than 1.20-fold or less than 0.83-fold in one or more groups with p-value of smaller than 0.05. Several useful biomarkers including serine, methionine, tryptophan, asymmetric dimethylarginine, N-Methyl-D-aspartic and tyrosine were identified. The changes of these biomarkers were likely due to the disruption of the endocrine system of silkworm by DDT. This work illustrates that the method of CIL LC-MS is useful to generate quantitative submetabolome profiles from a small volume of silkworm hemolymph with much higher coverage than conventional LC-MS methods, thereby facilitating the discovery of potential metabolite biomarkers related to EDC or other chemical exposure.

Determination of α-tocopherol in cereal grains by use of saponification coupled with ionic-liquid-based dispersive liquid–liquid microextraction before high-performance liquid chromatography-mass spectrometry

A selective and low organic-solvent-consuming method of sample preparation combined with high-performance liquid chromatography-mass spectrometry detection is introduced for the analysis of α-tocopherol in cereal grains.

Liquid chromatography–fluorescence and liquid chromatography–mass spectrometry detection of tryptophan degradation products of a recombinant monoclonal antibody

Light exposure is one of several conditions used to study the degradation pathways of recombinant monoclonal antibodies. Tryptophan is of particular interest among the 20 amino acids because it is the most photosensitive. Tryptophan degradation forms several products, including an even stronger photosensitizer and several reactive oxygen species. The current study reports a specific peptide mapping procedure to monitor tryptophan degradation. Instead of monitoring peptides using UV 214 nm, fluorescence detection with an excitation wavelength of 295 nm and an emission wavelength of 350 nm was used to enable specific detection of tryptophan-containing peptides. Peaks that decreased in area over time are likely to contain susceptible tryptophan residues. This observation can allow further liquid chromatography–mass spectrometry (LC–MS) analysis to focus only on those peaks to confirm tryptophan degradation products. After confirmation of tryptophan degradation, susceptibility of tryptophan residues can be compared based on the peak area decrease.

Liquid-liquid extraction and liquid chromatography-mass spectrometry detection of curcuminoids from bacterial culture medium.

Liquid chromatography-mass spectrometry (LC-MS) has been used to detect polyphenolic curcuminoids found in turmeric but studies of metabolism by bacterial and mammalian cells in vitro are compromised by poor recovery from the culture medium. We report a liquid-liquid extraction procedure with ethyl acetate and use LC-MS to quantify extracted curcuminoids. Ethyl acetate allows recoveries of ∼ 80-86% of curcuminoids from the bacterial growth medium, bacterial cell lysate and combined bacterial cell and growth medium matrices; a clear improvement over acetonitrile where recoveries were ∼ 25-66%. This optimised method will enable studies of curcuminoid metabolism and may be applicable to other hydrophobic polyphenolic compounds.

Multiresidue analysis of pesticides in peanuts using modified QuEChERS sample preparation and liquid chromatography-mass spectrometry detection

A multiclass method has been optimized and validated for the simultaneous determination of 113 pesticide residues belonging to several classes in peanuts.

Relative binding affinities of integrin antagonists by equilibrium dialysis and liquid chromatography-mass spectrometry.

The integrin αvβ6 is a potential target for treatment of idiopathic pulmonary fibrosis (IPF). Equilibrium dialysis (ED) was investigated for its ability to report ligand binding in an αvβ6 inhibitor screening assay. As a preliminary experiment, an established peptidomimetic inhibitor of the integrin was dialyzed against αvβ6, and the fraction bound (f b) and percentage saturation determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Quantitation of the inhibitor in the two chambers of the ED cartridge revealed an uneven distribution in the presence of αvβ6, corresponding to near saturation binding to the protein (93 ± 3%), while the control (without integrin) showed an equal partitioning of the inhibitor on either side of the dialysis membrane. A competitive ED assay with a 12 component mixture of antagonists was conducted, and the results compared with an established cell adhesion assay for quantifying αvβ6 inhibition of individual antagonists. Compounds clustered into three groupings: those with pIC 50 values between ca. 5.0 and 5.5, which possessed ED f b values indistinguishable from the controls, those with pIC 50s of 6.5 ± 0.2, which exhibited detectable integrin binding (f b 13-25%) in the ED assay, and a single compound of pIC 50 7.2 possessing an f b value of 38%. A good correlation between ED-derived f b and pIC 50 was observed despite the two assays utilizing quite different outputs. These results demonstrate that ED with LC-MS detection shows promise as a rapid αvβ6 integrin antagonist screening assay for mixtures of putative ligands.

Highly Sensitive Liquid Chromatography-Mass Spectrometry Detection of Microcystins with Molecularly Imprinted Polymer Extraction from Complicated Aqueous Ecosystems

In the present study, a liquid chromatography-mass spectrometry (LC-MS) method has been developed and validated to monitor traces of microcystins (MCs) in lake and marine waters. The molecularly imprinted polymer (MIP) formulated with itaconic acid as the functional monomer and ethylene glycol dimethacrylate as the crosslinking monomer has been used to selectively enrich MCs from the aqueous solutions. The extraction capacity and selectivity of MIP was higher when comparison with conventionally used resin XAD and powdered activated carbon (PAC). The MIP showed an outstanding selectivity for microcystin-LR (MC-LR) in a mixture of MCs from aqueous solutions in the pH range 6-9. The LC-MS analysis of MCs after MIP extraction showed an excellent linearity in the working range (R2=0.998) with high repeatability (RSD%, <6.3) and recoveries above 90%. Interference of dissolved ions and solution pH on MCs trace quantification in the lake and marine water samples were quantified. The limits of quantification (LOQ) and lower limit of detection (LOD) for the MC-LR were 10 and 1 ng L-1, respectively, which satisfies the strictest World Health Organization standard for MC-LR in drinking water (1 ng mL-1). The proposed analytical approach is simple, efficient and comparable with the detection limit of the traditional and expensive ELISA method of MCs analysis.

Influence of pro- and prebiotics on gastric, duodenal and colonic bioaccessibility of the mycotoxin beauvericin

Beauvericin (BEA) is a bioactive compound produced by the secondary metabolism of several Fusarium strains and known to have various biological activities. This study investigates the influence of several dietary fibers (galactomanan, glucomannan, citrus fiber, bamboo fiber, carrot fiber, pie fiber, β-glucan, xilan, and cellulose) and probiotic strains (Lactobacillus animalis, Lb. casei, Lb. casei, Lb. plantarum, Lb. rhuminis, Lb. casei casei, Bifidobacterium breve, Bf. Adolescents, Bf. bifidum, Corynebacterium vitaeruminis, Streptococcus faecalis, Eubacterium crispatus, and Saccharomyces cerevisiae) on the minor Fusarium mycotoxin BEA bioaccessibility employing a model solution. The bioaccessibility was determined using a simulated gastrointestinal digestion that mimics the physiological conditions of the digestive tract until the colonic compartment. The determination of BEA in the intestinal fluids was carried out by liquid chromatography–mass spectrometry detection (LC–MS). The reduction of BEA bioaccessibility in the experiments carried out using the prebiotic compounds ranged from 60 to 80%, whereas in the trials carried out using the probiotic strains the bioaccessibility observed ranged from 30 to 85%. A BEA degradation product produced by colonic fermentation was identified using the technique of LC–MS-LIT.

Gas and Liquid Chromatography-Mass Spectrometry Detection of the Urinary Metabolites of UR-144 and Its Major Pyrolysis Product

Studies on the pyrolysis of the synthetic cannabinoid agonist UR-144 ((1-pentyl-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone) have shown that its major pyrolysis product is a tetramethylcyclopropane ring-opened alkene. Considering that smoking is a common way of ingesting synthetic cannabimimetics, the presence of the metabolites of this pyrolysis product would be expected in biological fluids. Using GC-MS and LC-MS-MS methods, a series of phase I metabolites of UR-144 and its pyrolysis product were detected in the urine samples from patients admitted to hospital with suspected drug intoxication. The metabolites were tentatively identified as the products of mono-hydroxylation, di-hydroxylation, mono-hydroxylation with formation of the carbonyl group on the N-alkyl chain, carboxylation and N-dealkylation with mono-hydroxylation. In the case of the UR-144 pyrolysis product, metabolites with hydration of the aliphatic double bond were also identified. The parent compounds were detected as trace amounts in some urine samples, and the hydrated derivative of the UR-144 pyrolysis product was detected in the majority of samples. The detection of mono-hydroxylated metabolites of UR-144 (LC-MS-MS) and mono-hydroxylated/with hydration metabolites of the UR-144 pyrolysis product (GC-MS) was found to be the most useful method of establishing UR-144 ingestion.

Influence of different soluble dietary fibers on the bioaccessibility of the minor Fusarium mycotoxin beauvericin

Beauvericin (BEA) is a bioactive compound produced by the secondary metabolism of several Fusarium strains and is known to have various biological activities.This study investigated the bioaccessibility of the BEA tested in concentrations of 5 and 25mg/L, in a model solution and in wheat crispy breads elaborated with different natural binding compounds as the soluble alimentary dietary fibers β-1,3 glucan, chitosan low molecular weight (L.M.W.), chitosan medium molecular weight (M.M.W.), fructooligosaccharides (FOS), galattomannan, inulin and pectin, added at concentrations of 1% and 5%. The bioaccessibility was determinated by employing a simulated gastrointestinal digestion that simulates the physiologic conditions of the digestive tract until the colonic compartment. The determination of BEA in the intestinal fluids was carried out by liquid chromatography–mass spectrometry detection (LC–MS). The mean BEA bioaccessibility data in the model solutions ranged from 31.8% of the samples treated with only the duodenal digestion until 54.0% of the samples processed including the colonic fermentation, whereas in the alimentary system composed by the wheat crispy breads produced with different fiber concentration the duodenal and the duodenal+colonic BEA bioaccessibility resulted in 1.9% and 27.0% respectively.