Characterization of isoflavones and their conjugates in female rat urine using LC/MS/MS

Isoflavone phytoestrogens found in soybeans are the most widely studied phytochemicals in human diets and soy infant formulas. The health benefits of the isoflavones daidzein and genistein have been reported, and concerns about potential adverse effects have also been raised. However, the results of direct analysis of isoflavones and their metabolites in biological fluids after consumption of soy-containing diets are scarce. This study describes an LC/MS/MS method for the analysis of isoflavones and their metabolites in the urine of female rats fed diets made with soy protein isolate. Five isoflavones (daidzein, genistein, glycitein, dihydrodaidzein, and O-desmethylangolensin) were identified by comparison with authentic standards. Seventeen conjugates of isoflavones were characterized in the urine, the most unusual being genistein 5-glucuronide and four glucuronide conjugates of reductive metabolites of daidzein. The application of LC/MS/MS to analyze isoflavone metabolites is simple and sensitive, and appears to be an excellent method for determining the bioavailability and metabolism of food phytochemistry.     

Metabolism of oak leaf ellagitannins and urolithin production in beef cattle

Oak leaves have a high concentration of ellagitannins. These phytochemicals can be beneficial or poisonous to animals. Beef cattle are often intoxicated by oak leaf consumption, particularly after suffering feed restriction. The severity of the poisoning has recently been associated with the ruminal microbiota, as different bacterial populations were found in animals that tolerated oak leaves and in those that showed clinical and pathological signs of toxicity. Intoxication has previously been linked to the production of phenolic metabolites, particularly catechol, phloroglucinol, and resorcinol. This suggested that the microbial metabolism of ellagitannins could also be associated with its tolerance or intoxication in different animals. Therefore, it is essential to understand the metabolism of ellagitannins in cattle. Here we show that ellagitannins are metabolized in the cattle rumen to urolithins. Different urolithins were detected in ruminal fluid, feces, urine, and plasma. Oak leaf ellagitannins declined as they were converted to urolithins, mainly isourolithin A and urolithin B, by the ruminal and fecal microbiota. Urolithin aglycons were observed in rumen and feces, and glucuronide and sulfate derivatives were detected in plasma and urine. Sulfate derivatives were the main metabolites detected in plasma, while glucuronide derivatives were the main ones in urine. The main urolithins produced in cattle were isourolithin A and urolithin B. This is a relevant difference from the monogastric mammals studied previously in which urolithin A was the main metabolite produced. Low molecular weight phenolics of the benzoic, phenylacetic, and phenylpropionic groups and metabolites such as catechol, resorcinol, and related compounds were also detected. There was a large variability in the kinetics of production of these metabolites in individual animals, although they produced similar metabolites in all cases. This large variability could be associated with the large variability in the rumen and intestine microbiota that has previously been observed. Further studies are needed to demonstrate if the efficiency in the metabolism of ellagitannins by the microbiota could explain the differences observed in susceptibility to intoxication by the different animals.     

Discrimination and classification of olive tree varieties and cultivation zones by biophenol contents

The peak areas from a high-performance liquid chromatography-diode array (HPLC-DAD) analysis of biophenols extracted from olive leaves have been used as chemotaxonomic markers to construct chemometric models in order to discriminate and classify (1) 13 varieties of Olea europaea olive trees, namely, Alame?o, Arbequina, Azulillo, Chorna, Hojiblanca, Lechín, Manzanillo, Negrillo, Nevadillo, Ocal, Pierra, Sevillano, and Tempranillo, from the same cultivation zone and (2) Arbequina samples from six different geoghaphical origins, namely, Córdoba, Mallorca (north and south), Ciudad Real, Lleida, and Navarra. Models based on principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used for discrimination between samples as a function of the tree varieties and cultivation zone, whereas K nearest neighbors (KNN) and soft independent modeling of class analogy (SIMCA) models were generated to classify the samples used to validate the models into one of the groups previously established by PCA and HCA. KNN classified correctly 93 and 92% of the samples into the variety and cultivation zone, respectively; meanwhile, the SIMCA models predicted 85 and 92%, respectively.     

Discrimination of three Pegaga (Centella) varieties and determination of growth-lighting effects on metabolites content based on the chemometry of 1H nuclear magnetic resonance spectroscopy

The metabolites of three species of Apiaceae, also known as Pegaga, were analyzed utilizing (1)H NMR spectroscopy and multivariate data analysis. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) resolved the species, Centella asiatica, Hydrocotyle bonariensis, and Hydrocotyle sibthorpioides, into three clusters. The saponins, asiaticoside and madecassoside, along with chlorogenic acids were the metabolites that contributed most to the separation. Furthermore, the effects of growth-lighting condition to metabolite contents were also investigated. The extracts of C. asiatica grown in full-day light exposure exhibited a stronger radical scavenging activity and contained more triterpenes (asiaticoside and madecassoside), flavonoids, and chlorogenic acids as compared to plants grown in 50% shade. This study established the potential of using a combination of (1)H NMR spectroscopy and multivariate data analyses in differentiating three closely related species and the effects of growth lighting, based on their metabolite contents and identification of the markers contributing to their differences.     

Identification of rat urinary and fecal metabolites of a new herbicide, pyribenzoxim

Rats were treated with pyribenzoxim (O-[2,6-bis[(4,6-dimethoxy-2-pyrimidinyl)oxy]benzoyl]oxime), a new herbicide, to investigate the related metabolites in urine and feces. Metabolites were identified using LC/MS (electrospray ionization) and GC/MS (electron impact ionization) following the relatively simple and rapid extraction and purification procedures. Three metabolites were identified in urine either from oral gavage or intravenous (iv) injection. They were benzophenone oxime (BO), benzophenone oxime glucuronide (BOG), and 2-hydroxy-6-(4,6-dimethoxypyrimidin-2-yloxy)benzoic acid (HDB). Benzophenone oxime was present in larger quantity than BOG and HDB in urine from oral treatment, while the case was opposite in urine from iv treatment. Glucuronide conjugate was confirmed unambiguously by enzyme hydrolysis. 2,6-Bis(4,6-dimethoxypyrimidin-2-yloxy)benzoic acid (KIH-2023) and benzophenone were identified in feces. Benzophenone was confirmed by GC/MS and HPLC/DAD since LC/MS could not produce an ESI spectrum. On the basis of the results obtained, a metabolic map of pyribenzoxim is proposed.     

Comparison of leaf and fruit metabolism in two tomato (Solanum lycopersicum L.) genotypes varying in total soluble solids

Sink and source activity in two tomato (Solanum lycopersicum L.) genotypes that vary in fruit Brix were investigated to identify differences that potentially underscore this trait. Solara (Brix 9%) accumulated almost twice the glucose, fructose, and sucrose in ripe fruit and had a higher horticultural yield (25% greater) compared to Moneymaker (Brix 5%). 14C-glucose feeding suggested large disparities in sucrose metabolism in ripe fruit between genotypes. Biochemical pathways in the leaf adjacent to a fruiting truss at night were also analyzed since in many species, this is the period when leaf reserves are mobilized to feed the plant. Surprisingly, leaf metabolism, i.e., starch and sugar content, the levels of polar metabolites assayed by GC-TOF MS and 14CO2-pulse-chase fluxes in detached leaves, did not change between the day and night in either genotype. Solara has a higher morphological source-to-sink ratio, and this may contribute to higher Brix in that genotype.     

Biochemical profiling and chemometric analysis of seventeen UK-grown black currant cultivars

Black currant fruits are recognized as being an important dietary source of health-related compounds, such as anthocyanins and ascorbic acid. In the present study, the biochemical composition (viz., nonstructural carbohydrates, individual anthocyanins, total anthocyanins, total phenolics, and organic acids, including ascorbic acid) from 17 UK-grown black currant cultivars was analyzed. Berry composition was significantly affected by genotype. Nonstructural carbohydrates ranged from 85.09 to 179.92 mg g(-1) on a fresh weight (FW) basis, while concentration for organic acids ranged from 36.56 to 73.35 mg g(-1) FW. Relative concentrations of cyanidin 3-glucoside, cyanidin 3-rutinoside, delphinidin 3-glucoside and delphinidin 3-rutinoside were 3.1-7.9%, 35.4-47.0%, 7.6-12.5% and 36.9-50.9%, respectively. Differences in the biochemical profile among cultivars were emphasized by principal component analysis (PCA) and hierarchical cluster analysis (HCA). PCA was able to discriminate between cultivars, especially on the basis of health-related compounds. Initial exploration revealed that individual anthocyanins, total phenolics, and ascorbic acid could be used to characterize and classify different cultivars. HCA showed that the biochemical composition of the different cultivars was related to parentage information.     

Metabolites in contact with the rat digestive tract after ingestion of a phenolic-rich dietary fiber matrix

Grape antioxidant dietary fiber (GADF) is a phenolic-rich dietary fiber matrix. The aim of this work was to determine which phenolic compounds come into contact with colonic epithelial tissue after the ingestion of GADF. By use of HPLC-ESI-MS/MS techniques phenolic metabolites were detected in feces, cecal content, and colonic tissue from rats. Free (epi)catechin (EC) was detected in all three sources, and more than 20 conjugated metabolites of EC were also detected in feces. Fourteen microbially derived phenolic metabolites were also identified in feces, cecal content, and/or colonic tissue. These results show that during transit along the digestive tract, proanthocyanidin oligomers and polymers are depolymerized into EC units. After ingestion of GADF, free EC and its conjugates, as well as free and conjugated microbially derived phenolic metabolites, come into contact with the intestine epithelium for more than 24 h and may be partly responsible for the positive influence of GADF on gut health.     

Simultaneous characterization of bile acid, sterols, and determination of acylglycerides in feces from soluble cellulose-fed hamsters using HPLC with evaporative light-scattering detection and APCI-MS

The rapid rise in obesity-related diseases has increased interest in oral and dietary agents that disrupt fat metabolism, resulting in the excretion of dietary lipids in the feces. In this study, a rapid and convenient liquid chromatography method to comprehensively analyze fecal lipids in a single injection was developed. An evaporative light-scattering detector (ELSD) for routine analysis or atmosphere pressure chemical ionization tandem mass spectrometry [(+)APCI-MS/MS] for structural confirmation and peak purity was used. The method was applied to characterize lipid components of feces from hamsters fed high-fat diets with either 5% microcrystalline cellulose or 5% hydroxypropyl methylcellulose (HPMC) fibers, to test the effect of HPMC on lipid metabolism. HPMC is a nonfermentable, soluble cellulose fiber. The fecal lipid components identified using this method includes two secondary bile acids, deoxycholic acid, lithocholic acid, and neutral sterols including cholesterol, coprostanol, stigmastanol, and sitosterol. The profile of fecal lipid components was compared between two groups. It was found that the bile acid excretion was increased 2-fold in HPMC-fed hamsters. More interestingly, diacylglycerides and triacylglycerides were detected in feces from hamsters on HPMC-included high-fat diets. We believe that this is the first report of excretion of acylglycerides following neutral soluble fiber feeding.     

Metabolite profiling using (1)H NMR spectroscopy for quality assessment of green tea, Camellia sinensis (L.)

A set of 191 green teas from different countries was collected and analyzed by (1)H NMR. It was proposed to establish if the teas could be discriminated according to the country of origin or with respect to quality. Both principal component analysis (PCA) and cluster analysis were applied to the data. Some separation of Chinese and non-Chinese teas was observed. The present results did not allow allocation of samples to individual countries, but cluster analysis suggested that it might be possible with an augmented sample set. The PCA did show a separation between the Longjing type (highest quality Chinese tea) and most other Chinese teas and indicated some metabolites that could be responsible for the difference. Longjing teas showed higher levels of theanine, gallic acid, caffeine, epigallocatechin gallate, and epicatechin gallate and lower levels of epigallocatechin when compared with other teas. These compounds have been mentioned previously in connection with quality, but it was also shown that higher levels of theogallin (5-galloyl quinic acid), theobromine, 2-O-(beta-l-arabinopyranosyl)-myo-inositol and some minor sugar-containing compounds were found in Longjing teas while higher levels of fatty acids and sucrose were found in the other teas. These new markers could prove to be useful for the authentication of bulk tea.     

Metabolism of 7-fluoro-6-(3,4,5,6-tetrahydrophthalimido)-4- (2-propynyl)-2H-1,4-benzoxazin-3(4H)-one (S-53482) in rat. 1. Identification of a sulfonic acid type conjugate

To examine the metabolic fate of 7-fluoro-6-(3,4,5, 6-tetrahydrophthalimido)-4-(2-propynyl)-2H-1,4-benzoxazin-3( 4H)-one (S-53482), rats were given a single oral dose of [phenyl-(14)C]-S-53482 at 1 (low) or 100 (high) mg/kg. The radiocarbon was almost completely eliminated within 7 days after administration in both groups. (14)C recoveries (expressed as percentages relative to the dosed (14)C) in feces and urine were 56-72 and 31-43%, respectively, for the low dose and 78-85 and 13-23%, respectively, for the high dose. S-53482 and seven metabolites were identified in urine and feces. Six of them were purified by several chromatographic techniques and identified by spectroanalyses (NMR and MS). Alcohol derivatives and an acetoanilide derivative were isolated from urine. Three sulfonic acid conjugates having a sulfonic acid group incorporated into the double bond of the 3,4,5,6-tetrahydrophthalimide moiety were isolated from feces. On the basis of the metabolites identified in this study, the metabolic pathways of S-53482 in rats are proposed.     

Colorimetric sensor array for soft drink analysis

Fourteen commercial soft drinks have been analyzed using colorimetric sensor arrays made from a set of 25 chemically responsive dyes printed on a hydrophobic membrane. Digital imaging of the dye array before and after immersion provides a color change profile as a unique fingerprint for each specific analyte. The digital data library generated was analyzed with statistical and chemometric methods, including principal component analysis (PCA) and hierarchical clustering analysis (HCA). Facile identification of all of the soft drinks was readily achieved using comparison of the color change profiles or a PCA score plot. Using a HCA dendrogram, the misclassification rate was <2%, and even very similar sodas were easily differentiated. In addition, the monitoring of soft drinks as they degas or upon dilution also proved to be possible. This work demonstrates the potential of our colorimetric sensor array technology for quality assurance/control applications of sodas and perhaps other beverages as well.     

Colorimetric sensor arrays for the analysis of beers: a feasibility study

Eighteen commercial beers have been analyzed in both liquid and gas phases using colorimetric sensor arrays made from selected chemically responsive dyes printed on a hydrophobic membrane. Digital imaging of the dye array before and after exposure to the complex analytes in either the liquid phase or the head-gas provides a color change profile as a unique fingerprint for the specific analyte. The digital data libraries generated were analyzed using statistical and chemometric methods, including principal component analysis (PCA) and hierarchical clustering analysis (HCA). In either liquid- or gas-phase experiments, facile identification of specific beers was achieved using comparison of the color change profiles; using HCA statistical analysis the error rate of identification was <3%. Differentiation between even very similar beers proved to be straightforward. In addition, differentiation of pristine beer from the effects of watering or decarbonation proved to be possible. These results suggest that colorimetric sensor arrays may prove to be useful for quality assurance/quality control applications of beers and perhaps other beverages.     

Anthocyanin absorption and antioxidant status in pigs

The effect of a simultaneous intake of food or flavonoids on anthocyanins absorption and antioxidant status in pigs was investigated. Twelve male pigs at 27.1 +/- 0.7 kg BW fitted with jugular venous cannulae were maintained in individual metabolic crates. The animals were each given one of three dietary treatments in random order: blackcurrant powder (BC) to give a dose of 100 mg total ACNs/kg BW mixed either with water and sugar (Diet A), cereal (Weet-Bix), milk, and sugar (Diet B), or cereal, milk, sugar, and an additional flavonol (rutin, approximately 100 mg/kg BW) (Diet C). The four major anthocyanins of BC, delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-glucoside, and cyanidin-3-rutinoside, were identified and quantified by HPLC-PDA in all three diets. In the pig plasma, four peaks with a reversed pattern to those of anthocyanins in the BC extract were detected. The total amount of anthocyanins absorbed was not significantly different between the three diets, but the rate of absorption and subsequent decline was slower following administration of diet B and C than diet A. All three diets increased antioxidant capacity when measured by the FRAP assay but not when measured by the ORAC and non-protein ORAC assay. However, the increase was delayed and did not appear until 4 h after ingestion, at a time when plasma anthocyanin levels had returned to baseline. The present study demonstrates that the simultaneous intake of food or other flavonoids delays the absorption profile for anthocyanins. Our results also suggest that the increase in antioxidant capacity is not due to dietary anthocyanins but may be due to metabolites that result from anthocyanin consumption.     

Metabolism of N-[(R)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (Delaus, S-2900) and its isomer, N-[(S)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (S-2900S), in rats. 1. Identification of metabolites in feces and urine

Rats were orally dosed with a 1:1 diastereomixture of N-[(R)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (Delaus, S-2900) and N-[(S)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (S-2900S), both labeled with 14C, at 200 mg/kg/day for 5 consecutive days, and 16 metabolites in urine and feces were purified by a combination of several chromatographic techniques. The chemical structures of all isolated metabolites were identified by spectroanalyses (NMR and MS). Several of them were unique decyanated and/or cyclic compounds (lactone, imide, cyclic amide, cyclic imino ether forms). Major biotransformation reactions of the mixture of S-2900 and S-2900S in rats are proposed on the basis of the metabolites identified in this study.     

Identification of three novel polyphenolic compounds, origanine A-C, with unique skeleton from Origanum vulgare L. using the hyphenated LC-DAD-SPE-NMR/MS methods

Identification of new compounds especially those with new skeletons from plant kingdom has long been a vital aspect for understanding phytochemistry, plant metabolisms and discovering new bioactive compounds. In this study, we identified and isolated three novel polyphenolic compounds, origanine A-C, from a well-researched plant Origanum vulgare L. using the hyphenated LC-DAD-SPE-NMR/MS methods. Based on the combined information from UV-visible, accurate mass and 2D NMR spectra together with computational calculations, we found that these compounds all had a novel skeleton of cyclohexenetetracarboxylic acids attached with some well-known bioactive moieties including 3,4-dihydroxyphenyl, 4-(β-d-glucopyranosyloxy)benzyl alcohol (gastrodin), and 3-(3,4-dihydroxyphenyl)lactic acid (danshensu) residues. These findings provided crucial information to fill the gaps in our knowledge in terms of the plant secondary metabolism. This study also indicated the necessity for further research in plant secondary metabolism for even well-studied plants and demonstrated the powerfulness of the hyphenated LC-DAD-SPE-NMR/MS methods for comprehensive analysis of plant metabolites in particular for discovering new natural compounds.     

Extreme variability of steroid profiles in cow feces and pig slurries at the regional scale: implications for the use of steroids to specify fecal pollution sources in waters

Thirty-five samples of cow feces (cowpat and cow manure) and pig slurries subjected to different treatment processes and different storage times before land spreading were extracted and analyzed by gas chromatography-mass spectrometry to determine their fecal stanol profiles. The fresh pig slurry data presented here increase considerably the classical range of values obtained for steroid ratios, resulting in an overlap with the range for cow feces. These results lead to the inability to distinguish species source of feces on the basis of steroid ratios alone. The cause of these differences is not known, although it appears likely to be related to differences in the metabolism of animals in relation to their age and/or variations in diet, rather than to secondary mechanisms of steroid degradation during storage or/and treatment of the feces. Nevertheless, the specificity of steroids to serve as a tool to differentiate cow feces from pig slurries is restored by considering the fecal stanol profile, notably, the six most diagnostic stanol compounds, which are 5β-cholestan-3β-ol (coprostanol), 5β-cholestan-3α-ol (epicoprostanol), 24-methyl-5α-cholestan-3β-ol (campestanol), 24-ethyl-5α-cholestan-3β-ol (sitostanol), 24-ethyl-5β-cholestan-3β-ol (24-ethylcoprostanol), and 24-ethyl-5β-cholestan-3α-ol (24-ethylepicoprostanol). In this study, chemometric analysis of the fingerprint of these six stanols using principal components analysis (PCA) distinguished pig slurries from cow feces. The application of PCA to the stanol profiles, as developed in this study, could be a promising tool for identifying the animal source in fecal contamination of waters.     

The power of hyphenated chromatography/time-of-flight mass spectrometry in public health laboratories

Laboratories devoted to the public health field have to face the analysis of a large number of organic contaminants/residues in many different types of samples. Analytical techniques applied in this field are normally focused on quantification of a limited number of analytes. At present, most of these techniques are based on gas chromatography (GC) or liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). Using these techniques only analyte-specific information is acquired, and many other compounds that might be present in the samples would be ignored. In this paper, we explore the potential of time-of-flight (TOF) MS hyphenated to GC or LC to provide additional information, highly useful in this field. Thus, all positives reported by standard reference targeted LC-MS/MS methods were unequivocally confirmed by LC-QTOF MS. Only 61% of positives reported by targeted GC-MS/MS could be confirmed by GC-TOF MS, which was due to its lower sensitivity as nonconfirmations corresponded to analytes that were present at very low concentrations. In addition, the use of TOF MS allowed searching for additional compounds in large-scope screening methodologies. In this way, different contaminants/residues not included in either LC or GC tandem MS analyses were detected. This was the case of the insecticide thiacloprid, the plant growth regulator paclobutrazol, the fungicide prochloraz, or the UV filter benzophenone, among others. Finally, elucidation of unknowns was another of the possibilities offered by TOF MS thanks to the accurate-mass full-acquisition data available when using this technique.     

Absorption, disposition, metabolism, and excretion of [3-(14)C]caffeic acid in rats

Male Sprague-Dawley rats ingested 140 × 10(6) dpm of [3-(14)C]trans-caffeic acid, and over the ensuing 72 h period, body tissues, plasma, urine, and feces were collected and the overall levels of radioactivity determined. Where sufficient radioactivity had accumulated, samples were analyzed by HPLC with online radioactivity and tandem mass spectrometric detection. Nine labeled compounds were identified, the substrate and its cis isomer, 3'-O- and 4'-O-sulfates and glucuronides of caffeic acid, 4'-O-sulfates and glucuronides of ferulic acid, and isoferulic acid-4'-O-sulfate. Four unidentified metabolites were also detected. After passing down the gastrointestinal tract, the majority of the radiolabeled metabolites were excreted in urine with minimal accumulation in plasma. Only relatively small amounts of an unidentified (14)C-labeled metabolite were expelled in feces. There was little or no accumulation of radioactivity in body tissues, including the brain. The overall recovery of radioactivity 72 h after ingestion of [3-(14)C]caffeic acid was ～80% of intake.     

Metabolism of 7-fluoro-6-(3,4,5,6-tetrahydrophthalimido)-4- (2-propynyl)-2H-1,4-benzoxazin-3(4H)-one (S-53482, flumioxazin) in the rat: II. Identification of reduced metabolites

On single oral administration of (14)C-S-53482 [7-fluoro-6-(3,4,5, 6-tetrahydrophthalimido)-4-(2-propynyl)-2H-1,4-benzoxazin-3( 4H)-one, Flumioxazin] labeled at the 1- and 2-positions of tetrahydrophthaloyl group to rats at 1 (low dose) or 100 (high dose) mg/kg, the radiocarbon was almost completely eliminated within 7 days after administration in both groups with generally very low residual (14)C tissue levels. The predominant excretion route was via the feces. The major fecal and urinary metabolites involved reduction or sulfonic acid addition reactions at the 1,2-double bond of the 3,4,5,6-tetrahydrophthalimide moiety and hydroxylation of the cyclohexene or cyclohexane ring. One urinary and four fecal metabolites were identified using chromatographic techniques and spectroanalyses (NMR and MS). Three of five identified metabolites were unique forms, reduced at the 1,2-double bond of the 3,4,5, 6-tetrahydrophthalimide moiety. On the basis of the metabolites identified in this study, the metabolic pathways of S-53482 in rats are proposed. To specify tissues forming reduced metabolites, an in vitro study was conducted. Reduction was found to take place in red blood cells.