Progress, applications, and challenges in high-throughput effect-directed analysis for toxicity driver identification - is it time for HT-EDA?

The rapid increase in the production and global use of chemicals and their mixtures has raised concerns about their potential impact on human and environmental health. With advances in analytical techniques, in particular, high-resolution mass spectrometry (HRMS), thousands of compounds and transformation products with potential adverse effects can now be detected in environmental samples. However, identifying and prioritizing the toxicity drivers among these compounds remain a significant challenge. Effect-directed analysis (EDA) emerged as an important tool to address this challenge, combining biotesting, sample fractionation, and chemical analysis to unravel toxicity drivers in complex mixtures. Traditional EDA workflows are labor-intensive and time-consuming, hindering large-scale applications. The concept of high-throughput (HT) EDA has recently gained traction as a means of accelerating these workflows. Key features of HT-EDA include the combination of microfractionation and downscaled bioassays, automation of sample preparation and biotesting, and efficient data processing workflows supported by novel computational tools. In addition to microplate-based fractionation, high-performance thin-layer chromatography (HPTLC) offers an interesting alternative to HPLC in HT-EDA. This review provides an updated perspective on the state-of-the-art in HT-EDA, and novel methods/tools that can be incorporated into HT-EDA workflows. It also discusses recent studies on HT-EDA, HT bioassays, and computational prioritization tools, along with considerations regarding HPTLC. By identifying current gaps in HT-EDA and proposing new approaches to overcome them, this review aims to bring HT-EDA a step closer to monitoring applications.

Chronic administration of grape-seed polyphenols attenuates the development of hypertension and improves other cardiometabolic risk factors associated with the metabolic syndrome in cafeteria diet-fed rats.

The effects of grape-seed polyphenols against the development of hypertension and other cardiometabolic conditions associated with the metabolic syndrome (MetS) were studied in rats fed a high-fat, high-carbohydrate diet, known as the cafeteria (CAF) diet. Two groups of Wistar rats were fed standard (STD) or CAF diets for 12 weeks. The CAF diet-fed rats were administered different doses of a low-molecular-weight grape-seed polyphenol extract (LM-GSPE) (25, 100 and 200 mg/kg per d) or vehicle daily, and the STD diet-fed rats were administered LM-GSPE (100 mg/kg per d) or vehicle using ten animals per group. Body weight (BW), waist perimeter (WP) and systolic and diastolic blood pressures (BP) by the tail-cuff method were recorded weekly. The animals were housed in metabolic chambers every 2 weeks to estimate daily food and liquid intakes and to collect faeces and urine samples. The plasma lipid profile was analysed at time 0 and on the 4th, 7th, 10th and 12th weeks of the experiment. Moreover, plasma leptin was measured at the end of the experiment. Results demonstrated that LM-GSPE, when administered with the CAF diet, attenuated the increase in BP, BW, WP and improved lipid metabolism in these animals. However, although the 25- and 100-mg/kg per d doses were sufficient to produce beneficial effects on BP and lipid metabolism, a 200-mg/kg per d dose was necessary to have an effect on BW and WP. The present findings suggest that LM-GSPE is a good candidate for a BP-lowering agent that can also ameliorate other conditions associated with the MetS.

Physico-chemical and sensory properties of marmalades made from mixtures of fruits and under-exploited Andean tubers.

BACKGROUND: This work studies the added value to the Andean tuber crops through the production of jams. The objective were: (1) to study the sensory and instrumental characteristics of dietetic marmalades made with fruits and Andean tubers; (2) to research consumer's acceptability and emotional responses; (3) to assess the relationship between sensory and instrumental variables and (4) to determine sensory, instrumental and emotional variables that influence the acceptability. RESULTS: Pearson's correlations showed that spreadability was the variable better predicted by sensory and instrumental ones. The analysis of variance showed that sourness increased with the increase of strawberry and the decrease of apple contents (P < 0.05). The acceptability increased when the strawberry proportion was higher. The sweetness-sourness balance drove the hedonic response and some emotions changed from one feeling to its corresponding opposite when the strawberry proportion reached 50 or 60. According to a partial least square 2 and a principal components analysis of sensory/acceptability/emotion data, 'typical', 'autochthonous', 'urban' and 'present' resulted in positive emotions which favored the consumer liking. CONCLUSION: Formulating marmalades with mixtures of fruits and Andean tubers will allow giving value-added to these crops. The elaboration of products using innovative raw materials will be an incentive for farmers to cultivate them. © 2017 Society of Chemical Industry.

Acute administration of single oral dose of grape seed polyphenols restores blood pressure in a rat model of metabolic syndrome: role of nitric oxide and prostacyclin.

PURPOSE: The aims of this study were to evaluate the antihypertensive effectiveness of different doses of grape seed polyphenols in cafeteria diet-fed hypertensive rats (CHRs) and to establish the mechanism involved in the blood pressure (BP) lowering effect of these compounds in this experimental model of metabolic syndrome (MS). METHODS: Male 8-week-old Wistar rats were fed cafeteria or standard (ST) diet for 10 weeks. After this, the antihypertensive effect of a single oral administration of a polyphenol grape seed extract (GSPE) was tested at different doses (250, 375 and 500 mg/kg) in CHRs. BP was recorded before and 2, 4, 6, 8, 24 and 48 h post-administration. The hypotensive effect of GSPE was also proved in ST diet-fed rats. Additionally, in other experiment, CHRs were orally administered 375 mg/kg GSPE. Four hours post-administration, the rats were intraperitoneally administrated 30 mg/kg NG-nitro-L-arginine methyl ester (L-NAME) or 5 mg/kg indomethacin [inhibitors of nitric oxide (NO) and prostacyclin synthesis, respectively]. BP was recorded initially and 6 h post-administration. RESULTS: GSPE produced a decrease in SBP and DBP, the most effective dose (375 mg/kg) showing an antihypertensive effect in CHRs similar to the drug captopril, and did not affect BP of ST diet-fed rats. The antihypertensive effect was completely abolished by L-NAME and partially inhibited by indomethacin. CONCLUSIONS: GSPE acts as an antihypertensive agent in a rat model of hypertension associated with MS. The change in endothelium-derived NO availability is one of the mechanisms involved in the antihypertensive effect of GSPE in CHRs. Additionally, endothelial prostacyclin contributes to the effect of GSPE on arterial BP.

Mixture Risk Assessment of Complex Real-Life Mixtures-The PANORAMIX Project.

Humans are involuntarily exposed to hundreds of chemicals that either contaminate our environment and food or are added intentionally to our daily products. These complex mixtures of chemicals may pose a risk to human health. One of the goals of the European Union's Green Deal and zero-pollution ambition for a toxic-free environment is to tackle the existent gaps in chemical mixture risk assessment by providing scientific grounds that support the implementation of adequate regulatory measures within the EU. We suggest dealing with this challenge by: (1) characterising 'real-life' chemical mixtures and determining to what extent they are transferred from the environment to humans via food and water, and from the mother to the foetus; (2) establishing a high-throughput whole-mixture-based in vitro strategy for screening of real-life complex mixtures of organic chemicals extracted from humans using integrated chemical profiling (suspect screening) together with effect-directed analysis; (3) evaluating which human blood levels of chemical mixtures might be of concern for children's development; and (4) developing a web-based, ready-to-use interface that integrates hazard and exposure data to enable component-based mixture risk estimation. These concepts form the basis of the Green Deal project PANORAMIX, whose ultimate goal is to progress mixture risk assessment of chemicals.

International Consensus Document on Obstructive Sleep Apnea. / Documento internacional de consenso sobre apnea obstructiva del sueño.

The main aim of this international consensus document on obstructive sleep apnea is to provide guidelines based on a critical analysis of the latest literature to help health professionals make the best decisions in the care of adult patients with this disease. The expert working group was formed primarily of 17 scientific societies and 56 specialists from a wide geographical area (including the participation of 4 international societies), an expert in methodology, and a documentalist from the Iberoamerican Cochrane Center. The document consists of a main section containing the most significant innovations and a series of online manuscripts that report the systematic literature searches performed for each section of the international consensus document. This document does not discuss pediatric patients or the management of patients receiving chronic non-invasive mechanical ventilation (these topics will be addressed in separate consensus documents).

Enzyme-Assisted Extraction to Obtain Phenolic-Enriched Wine Lees with Enhanced Bioactivity in Hypertensive Rats.

The antihypertensive effect of the soluble fraction of wine lees (WL) from Cabernet variety grapes was recently reported by our group. This blood pressure (BP)-lowering effect was attributed to the presence of flavanols and anthocyanins. In this context, phenolic-enriched wine lees (PWL) could potentially exhibit a stronger bioactivity. Therefore, the aim of this study was to obtain a soluble fraction of WL with increased phenolic content and evaluate its functionality. The PWL were obtained using an enzyme-assisted extraction based on the hydrolysis of WL proteins with Flavourzyme®. They contained 57.20% more total phenolic compounds than WL, with anthocyanins and flavanols being the largest families present. In addition, PWL also showed greater angiotensin-converting enzyme inhibitory and antioxidant activities. Finally, the antihypertensive activity of the PWL was evaluated in spontaneously hypertensive rats. A single dose of 5 mL/kg body weight of PWL showed a greater BP-lowering effect than the one shown by WL. Moreover, this antihypertensive effect was more prolonged than the one produced by the antihypertensive drug Captopril. These results demonstrate that enzymatic protein hydrolysis is a useful method to maximize the extraction of phenolic compounds from WL and to obtain extracts with enhanced functionalities.

ACE Inhibitory and Antihypertensive Activities of Wine Lees and Relationship among Bioactivity and Phenolic Profile.

Wine lees (WL) are by-products generated in the winemaking process. The aim of this study was to investigate the angiotensin-converting enzyme inhibitory (ACEi) activity, and the blood pressure (BP) lowering effect of WL from individual grape varieties. The relationship among their activities and phenolic profiles was also studied. Three WL, from Cabernet, Mazuela, and Garnacha grape varieties, were firstly selected based on their ACEi properties. Their phenolic profiles were fully characterized by UHPLC-ESI-Q-TOF-MS. Then, their potential antihypertensive effects were evaluated in spontaneously hypertensive rats (SHR). BP was recorded before and after their oral administrations (2, 4, 6, 8, 24, and 48 h) at a dose of 5 mL/kg bw. Cabernet WL (CWL) exhibited a potent antihypertensive activity, similar to that obtained with the drug Captopril. This BP-lowering effect was related to the high amount of anthocyanins and flavanols present in these lees. In addition, a potential hypotensive effect of CWL was discarded in normotensive Wistar-Kyoto rats. Finally, the ACEi and antihypertensive activities of CWL coming from a different harvest were confirmed. Our results suggest the potential of CWL for controlling arterial BP, opening the door to commercial use within the wine industry.

Virgin olive oil (unfiltered) extract contains peptides and possesses ACE inhibitory and antihypertensive activity.

BACKGROUND & AIMS: The peptide and protein composition of olive oil is mostly unknown and the few studies available have not focused on the study of its low molecular weight peptides. We hypothesised that olive oil could naturally contain low molecular weight peptides with antihypertensive effect. METHODS: We produced virgin olive oil (unfiltered, var. Picual) and obtained a water-soluble peptide extract. We fractionated the peptide extract by FPLC and studied its angiotensin converting enzyme (ACE) inhibitory activity. We studied the antihypertensive effect of olive oil peptides on the systolic blood pressure (SBP) and diastolic blood pressure (DBP) using an animal model of hypertension (spontaneously hypertensive rats, SHR). The animals were randomly distributed into 3 study groups (n = 8 per group) and received an oral dose of olive oil peptides (0.425 mg/kg of BW), or a dose of Captopril (50 mg/kg of BW) or water. SBP and DBP were registered in the rats before administration and a at 2, 4, 6, 8, 24 and 48 h post-administration of the corresponding dose. RESULTS: The peptide extract and FPLC purified fractions possessed angiotensin converting enzyme (ACE) inhibitory activity. Acute oral administration of olive oil water-soluble extract produced an average blood pressure reduction of 10 mmHg at 4 h (P < 0.01) and reached a maximum antihypertensive effect of 20 mmHg at 6 h, compared with baseline. CONCLUSION: Unfiltered virgin olive oil contains peptides and a water-soluble extract obtained from this oil possesses ACE inhibitory activity and in vivo antihypertensive effect.

Novel Antihypertensive Peptides Derived from Chicken Foot Proteins.

SCOPE: Chicken foot proteins have recently been demonstrated by the group to be a great source of hydrolysates with antihypertensive properties. The aim of this study was to isolate and identify angiotensin I-converting enzyme inhibitory (ACEI) peptides from chicken foot hydrolysate Hpp11 and to test their antihypertensive properties. METHODS AND RESULTS: Peptides are separated into fractions according to their molecular size and hydrophobicity by ultrafiltration and RP-HPLC, respectively. Subsequent peptide identification in the two fractions that present the highest ACEI activities is carried out by HPLC-MS. Ten of the identified peptides are synthesized and five of them show ACEI (IC50 ) values lower than 100 µm. The antihypertensive effects of these ACEI peptides after oral administration is evaluated in spontaneously hypertensive rats. The peptides AVFQHNCQE and QVGPLIGRYCG exhibit antihypertensive activity when administered at an oral dose of 10 mg kg-1 body weight. The maximal decrease in systolic blood pressure is recorded 6 h after their administration (-25.07 ± 4.21 and -10.94 ± 1.96 mmHg, respectively). CONCLUSION: These results suggest that AVFQHNCQE and QVGPLIGRYCG could be used as functional ingredients with antihypertensive effects, although it would be necessary to perform bioavailability and clinical studies to demonstrate their efficiency in humans.

Predictors of long-term adherence to continuous positive airway pressure in patients with obstructive sleep apnea and cardiovascular disease.

STUDY OBJECTIVES: Poor adherence to continuous positive airway pressure (CPAP) commonly affects therapeutic response in obstructive sleep apnea (OSA). We aimed to determine predictors of adherence to CPAP among participants of the Sleep Apnea and cardioVascular Endpoints (SAVE) trial. METHODS: SAVE was an international, randomized, open trial of CPAP plus usual care versus usual care (UC) alone in participants (45-75 years) with co-occurring moderate-to-severe OSA (≥12 episodes/h of ≥4% oxygen desaturation) and established cardiovascular (CV) disease. Baseline sociodemographic, health and lifestyle factors, OSA symptoms, and 1-month change in daytime sleepiness, as well as CPAP side effects and adherence (during sham screening, titration week, and in the first month), were entered in univariate linear regression analyses to identify predictors of CPAP adherence at 24 months. Variables with p <0.2 were assessed for inclusion in a multivariate linear mixed model with country, age, and sex included a priori and site as a random effect. RESULTS: Significant univariate predictors of adherence at 24 months in 1,121 participants included: early adherence measures, improvement in daytime sleepiness at 1 month, fixed CPAP pressure, some measures of OSA severity, cardiovascular disease history, breathing pauses, and very loud snoring. While observed adherence varied between countries, adherence during sham screening, initial titration, and the first month of treatment retained independent predictive value in the multivariate model along with fixed CPAP pressure and very loud snoring. CONCLUSIONS: Early CPAP adherence had the greatest predictive value for identifying those at highest risk of non-adherence to long-term CPAP therapy. CLINICAL TRIAL REGISTRATION: SAVE is registered with clinicaltrials.gov (NCT00738179).

Flavanol plasma bioavailability is affected by metabolic syndrome in rats.

Flavanols, which exert several health benefits, are metabolized after ingestion. Factors such as the host physiological condition could affect the metabolism and bioavailability of flavanols, influencing their bioactivities. This study aimed to qualitatively evaluate whether a pathological state influenced flavanol plasma bioavailability. Standard and cafeteria (CAF) diet fed rats, a robust model of metabolic syndrome (MeS), were administered 1000mg/kg of flavanol enriched grape seed polyphenol extract (GSPE). Flavanols and their metabolites were quantified by HPLC-MS/MS in plasma before and at 2, 4, 7, 24, and 48h after GSPE ingestion. Results showed that in CAF administered rats the maximum time of plasma flavanol concentration was delayed and these animals presented higher levels of plasma phase-II metabolites as well as altered microbial metabolites. In conclusion, this study demonstrated that MeS pathological state modified flavanol bioavailability, supporting the hypothesis that flavanol metabolism, and therefore flavanol functionality, depend on the organism's state of health.

Rat health status affects bioavailability, target tissue levels, and bioactivity of grape seed flavanols.

SCOPE: Studying the flavanol metabolism is essential to identify bioactive compounds, as beneficial effects of flavanols have been attributed to their metabolic products. However, host-related factors, including pathological conditions, may affect flavanol metabolism and, thus, their bioactivity. This study aims to elucidate whether hypertension affects grape seed flavanol metabolism, influencing their bioactivity in relation to hypertension. METHODS AND RESULTS: Grape seed flavanols' effect on blood pressure (BP) was studied in spontaneously hypertensive rats (SHR) and healthy Wistar rats 6 h after grape seed extract administration (375 mg/kg). Animals were then sacrificed, and plasma bioavailability and aorta distribution of flavanol metabolites were studied by HPLC-MS/MS in both the groups. Grape seed flavanols were only able to decrease BP in SHR. Plasma total flavanol metabolites showed similar levels, being the difference noticed in specific metabolites' concentrations. Specifically, microbial metabolites showed quantitative and qualitative differences between both health states. Moreover, aorta total concentrations were found decreased in SHR. Interestingly, flavanol microbial metabolites were specifically increased SHR aortas, showing qualitative differences in small phenolic forms. CONCLUSION: This study demonstrates important differences in bioactivity and target tissue metabolite levels between healthy and diseased rats, indicating potential metabolites responsible of the anti-hypertensive effect.

Gender-related similarities and differences in the body distribution of grape seed flavanols in rats.

SCOPE: Dietary flavanols produce beneficial health effects, and once absorbed, they are recognized as xenobiotics and undergo phase-II enzymatic detoxification. Flavanols health-promoting properties are mainly attributed to their metabolic products. This work aimed to elucidate whether rats of the opposite sex exhibited differences in the metabolism and distribution of ingested flavanols. METHODS AND RESULTS: Acute doses of grape seed polyphenols were administered to male and female rats. After 1, 2 and 4 h, plasma, liver, mesenteric white adipose tissue (MWAT), brain and hypothalamus flavanol metabolites were quantified by HPLC-MS/MS. Results indicated important sex-related quantitative differences in plasma and brain. Moreover, remarkable sex-related differences in the distributions and types of flavanol metabolites were also observed between liver and brain. CONCLUSIONS: This study demonstrated that sex differentially influences the metabolism and distribution of flavanols throughout the bodies of rats, which may affect the physiological bioactivities of flavanols between males and females.

Age related differences in the plasma kinetics of flavanols in rats.

Dietary flavanols produce beneficial health effects; once absorbed, they are recognized as xenobiotics and undergo Phase-II enzymatic detoxification. However, flavanols with a degree of polymerization greater than 2 reach the colon, where they are subjected to microbial metabolism and can be further absorbed and undergo Phase-II reactions. In this sense, flavanols' health-promoting properties are mainly attributed to their metabolic products. Several age-related physiological changes have been evidenced, and it is known that flavanols' bioavailability is affected by internal factors. Therefore, this study aimed to elucidate whether animals of different ages, specifically young and adult rats, exhibit differences in their flavanol metabolism and plasma bioavailability. To accomplish this, an acute dose of a grape seed polyphenol extract was administered to male rats; after 2, 4, 7, 24 and 48 h, flavanols and their Phase-II and microbial metabolites were quantified by HPLC-ESI-MS/MS in plasma. The results indicated important age-related quantitative differences in plasma flavanol metabolites. Interestingly, adult rats presented a remarkable reduction in flavanol absorption and Phase-II flavanol metabolism. Consequently, microbial-derived flavanol metabolism is triggered by higher flavanol affluence in the colonic tract. Furthermore, young rats presented a faster metabolic profile than adult rats. Hence, our results indicate that the physiological bioactivities of flavanols may depend on age.

Lack of tissue accumulation of grape seed flavanols after daily long-term administration in healthy and cafeteria-diet obese rats.

After ingestion flavanols are metabolized by phase-II enzymes and the microbiota and are distributed throughout the body depending on several factors. Herein we aim to evaluate whether flavanols are tissue-accumulated after the long-term administration of a grape seed polyphenol extract (GSPE) in rats and to study if compounds present in tissues differ in a cafeteria-diet obesity state. For that, plasma, liver, mesenteric white adipose tissue (MWAT), brain, and aorta flavanol metabolites from standard chow-diet-fed (ST) and cafeteria-diet-fed (CAF) rats were analyzed by high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) 21 h after the last 12-week-daily GSPE (100 mg/kg) dosage. Results showed that long-term GSPE intake did not trigger a flavanol tissue accumulation, indicating a clearance of products at each daily dosage. Therefore, results suggest that polyphenol benefits in a disease state would be due to a daily pulsatile effect. Moreover, obesity induced by diet also influences the metabolism and bioavailability of flavanols in rats.

The blood pressure effect and related plasma levels of flavan-3-ols in spontaneously hypertensive rats.

We studied the short-term antihypertensive effect of flavan-3-ols (-)-epicatechin, (+)-catechin and (-)-catechin, in spontaneously hypertensive rats (SHR). Plasma metabolites and the corresponding plasma antioxidant capacity were determined. All the assayed flavan-3-ols decreased systolic blood pressure (SBP) in SHR. Their antihypertensive effects were less pronounced than that of Captopril (50 mg kg(-1)) and were not shown in normotensive Wistar-Kyoto rats. 6 mg kg(-1) (-)-epicatechin caused the maximum decrease in SBP. The maximum effects of the catechin monomers were observed post-administration of 0.5 mg kg(-1) of flavan-3-ols, (-)-catechin being the least effective among the three assayed compounds. Glucuronide and methyl glucuronide metabolites were obtained in the flavan-3-ol treated SHR, but it was not possible to relate the antihypertensive effect of the assayed flavan-3-ols with a concrete plasma metabolite or with their antioxidant effect. In conclusion, the studied flavan-3-ols could be responsible for the antihypertensive effect of cocoa products.

Tissue distribution of rat flavanol metabolites at different doses.

Flavanols are metabolized in the small intestine and the liver to produce their glucuronidated, sulfated or methylated conjugates that can be body distributed or excreted in the urine. However, the intake of large amounts of flavanols is not directly related to their bioavailability. This study aims to investigate the administered dose dependence of flavanols' conjugation and body distribution. In this study, different doses of a grape seed proanthocyanidin extract (GSPE; 125, 250, 375 and 1000 mg/kg) were orally administered to male Wistar rats. Tissues were collected 2h after GSPE administration. Flavanols were quantified by HPLC-MS/MS. Results show that the majority of GSPE metabolites are located in the kidney, followed by the liver. Lower concentrations were found in mesenteric white adipose tissue (MWAT) and the brain. Moreover, flavanol metabolites followed a tissue-specific distribution pattern independent of dosage. In the kidney, glucuronidated metabolites were the most abundant; however, in the liver, it was mainly methyl-glucuronidated metabolites. In MWAT, free flavanols were dominant, and methylated metabolites were dominant in the brain. Concentration within a tissue was dependent on the administered dose. In conclusion, flavanol metabolites follow a tissue-specific distribution pattern and only the tissue concentration of flavanol metabolites is dependent on the administered dose.

Regulation of vascular endothelial genes by dietary flavonoids: structure-expression relationship studies and the role of the transcription factor KLF-2.

Physiological concentrations (1 µM) of 15 flavonoids were evaluated in human umbilical vein endothelial cells in the presence of hydrogen peroxide (H2O2) for their ability to affect endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) expression in order to establish the structural basis of their bioactivity. Flavonoid effects on eNOS transcription factor Krüpple like factor-2 (KLF-2) expression were also evaluated. All studied flavonoids appeared to be effective compounds for counteracting the oxidative stress-induced effects on vascular gene expression, indicating that flavonoids are an excellent source of functional endothelial regulator products. Notably, the more effective flavonoids for KLF-2 up-regulation resulted in the highest values for eNOS expression, showing that the increment of eNOS expression would take place through KLF-2 induction. Structure-activity relationship studies showed that the combinations of substructures on flavonoid skeleton that regulate eNOS expression are made up of the following elements: glycosylation and hydroxylation of C-ring, double bond C2=C3 at C-ring, methoxylation and hydroxylation of B-ring, ketone group in C4 at C-ring and glycosylation in C7 of A-ring, while flavonoid features involved in the reduction of vasoconstrictor ET-1 expression are as follows: double bond C2=C3 at C-ring glycosylation in C7 of A-ring and ketone group in C4 of C-ring.

A rapid method to determine colonic microbial metabolites derived from grape flavanols in rat plasma by liquid chromatography-tandem mass spectrometry.

This study describes the development and validation of a liquid chromatography-mass spectrometry method for determination of a large number of flavanol colonic derivatives in biological samples. The method was validated with rat plasma after the intake of grape seed flavanols. The minimum plasma volume necessary to maintain good recovery values within the range of 83-110% for all of the standards was determined by micro solid-phase extraction (µ-SPE). In total, 16 commercial standards were used to measure 30 different phenolic compounds present at low concentration levels (micromolar). The chromatographic method enabled reliable quantification of plasma colonic flavanol derivatives with low limits of detection and quantification, achieving values of 0.03 nM and 0.10 nM, respectively. The developed method can be readily applied to determine all of the flavanol metabolites that are most likely responsible for the majority of biological effects of poorly absorbed flavanols.