Chiral separation of (+)/(-)-catechin from sulfated and glucuronidated metabolites in human plasma after cocoa consumption.

Cocoa is well-known to be rich in flavan-3-ols. Previous analyses have established that alkaline treatment of cocoa beans results in epimerization of (-)-epicatechin to (-)-catechin and (+)-catechin to (+)-epicatechin. Now, the question is whether both epimers can be absorbed by the human organism. This paper describes sample preparation and an HPLC method for chiral determination of (+)/(-)-catechin from sulfated and glucuronidated metabolites in human plasma. The sample preparation includes enzymatic hydrolysis of the catechin metabolites, and solid-phase extraction (SPE). A PM-gamma-cyclodextrin column is used with a coulometric electrode-array detection (CEAD) system. The recovery of catechin ranges from 89.9 to 96.8%. The limit of detection is 5.9 ng mL(-1) for (-)-catechin and 6.8 ng mL(-1) for (+)-catechin, and the limit of quantification is 12.8 ng mL(-1) for (-)-catechin and 16.9 ng mL(-1) for (+)-catechin. The relative standard deviation of the method ranges from 0.9 to 1.5%. This method was successfully applied to human plasma after consumption of a cocoa drink. In one human self-experiment, (+)-catechin and (-)-catechin were found in human plasma, but metabolism of the two enantiomers differed.

Low Plasma Appearance of (+)-Catechin and (-)-Catechin Compared with Epicatechin after Consumption of Beverages Prepared from Nonalkalized or Alkalized Cocoa-A Randomized, Double-Blind Trial.

Flavan-3-ols are claimed to be responsible for the cardioprotective effects of cocoa. Alkalized cocoa powder (ALC), commonly used for many non-confectionary products, including beverages, provides less (+)-catechin, (-)-epicatechin, and procyanidins and more (-)-catechin than nonalkalized cocoa powder (NALC). This may affect the plasma appearance of monomeric flavan-3-ol stereoisomers after consumption of NALC vs. ALC. Within a randomized, crossover trial, 12 healthy nonsmokers ingested a milk-based cocoa beverage providing either NALC or ALC. Blood was collected before and within 6 h postconsumption. (+)-Catechin, (-)-catechin, and epicatechin were analyzed in plasma by HPLC as sum of free and glucuronidated metabolites. Pharmacokinetic parameters were obtained by a one-compartment model with nonlinear regression methods. For epicatechin in plasma, total area under the curve within 6 h postconsumption (AUC0-6h) and incremental AUC0-6h were additionally calculated by using the linear trapezoidal method. After consumption of NALC and ALC, (+)-catechin and (-)-catechin were mostly not detectable in plasma, in contrast to epicatechin. For epicatechin, total AUC0-6h was different between both treatments, but not incremental AUC0-6h. Most kinetic parameters were similar for both treatments, but they varied strongly between individuals. Thus, epicatechin is the main monomeric flavan-3-ol in plasma after cocoa consumption. Whether NALC should be preferred against ALC due to its higher (-)-epicatechin content remains unclear with regard to the results on incremental AUC0-6h. Future studies should investigate epicatechin metabolites in plasma for a period up to 24 h in a larger sample size, taking into account genetic polymorphisms in epicatechin metabolism and should consider all metabolites to understand inter-individual differences after cocoa intake.

Contamination of semi-solid dosage forms by leachables from aluminium tubes.

The objective of this study was to determine to what extent bisphenol A (BPA), bisphenol A diglycidyl ether (BADGE) and its derivatives are extractable from epoxy-based coatings of aluminium tubes for pharmaceutical use and to monitor their leaching into different kinds of semi-solid dosage forms. Migration increasing factors should be evaluated. Extraction tests using acetonitrile for 10 days at 40 degrees C turned out to be suitable to estimate the maximum amount of extractables. A plain variability in the nature and amount of extractables among tubes of different vendors (n=7) could be demonstrated. Leaching of the remnants into various semi-solid drug products (ointment, cream, gel) during storage (30 degrees C/40 degrees C) was verifiable. Leachable profiles were, apart from storage time and temperature, decisively influenced by the matrix. In particular, matrix polarity seemed to play a crucial role. Thus, the highest amount of leachables was found in isopropanol-based carbomer gel. Furthermore, in-use conditions (mechanical stress) enhanced migration significantly. In order to ensure quality and safety of semi-solid formulae, interactions between the coating material and the drug product should be thoroughly evaluated.

Polyphenol Phase-II Metabolites are Detectable in Human Plasma after Ingestion of 13 C Labeled Spinach-a Pilot Intervention Trial in Young Healthy Adults.

SCOPE: After intrinsic labeling of spinach (Spinacia oleracea L., Chenopodiaceae) with 13 CO2 , we investigated if labeled polyphenol metabolites were detectable in human plasma. METHODS AND RESULT: In a pilot intervention trial, five healthy men consumed 5 g freeze-dried 13 C labeled spinach, including a total amount of 160 µmol methoxyflavonols, including 70 µmol 5,3',4'-trihydroxy-3-methoxy-6,7-methylendioxyflavone-4'-glucuronide. Plasma samples of all subjects were analyzed with regard to their 13 C/12 C ratio. Additionally, 13 C labeled metabolites of patuletin, spinacetin, and 5,3',4'-trihydroxy-3-methoxy-6,7-methylendioxyflavone (TMM) were analyzed in plasma samples in a subgroup of three subjects. TMM-glucuronide, TMM-sulfate, and spinacetin-glucuronide-sulfate, the latter as 12 C113 C16 and 13 C17 isotopologs, were tentatively identified. Plasma concentration of TMM-glucuronide and TMM-sulfate reached cmax from 19.1-54.3  and 22.5-125.5 nmol L-1 , respectively, 7-9 h post-ingestion. CONCLUSION: It seems likely that 13 C labeled TMM-glucuronide and TMM-sulfate are phase-II metabolites which were converted after colonic transformation. Variations in plasma kinetics were observed for these two metabolites and may be attributed to the individual composition of the microbiota. We conclude that 13 C labeled polyphenol metabolites are detectable and quantifiable in human plasma.

(-)-Catechin in cocoa and chocolate: occurrence and analysis of an atypical flavan-3-ol enantiomer.

Cocoa contains high levels of different flavonoids. In the present study, the enantioseparation of catechin and epicatechin in cocoa and cocoa products by chiral capillary electrophoresis (CCE) was performed. A baseline separation of the catechin and epicatechin enantiomers was achieved by using 0.1 mol x L(-1) borate buffer (pH 8.5) with 12 mmol x L(-1) (2-hydroxypropyl)-gamma-cyclodextrin as chiral selector, a fused-silica capillary with 50 cm effective length (75 microm I.D.), +18 kV applied voltage, a temperature of 20 degrees C and direct UV detection at 280 nm. To avoid comigration or coelution of other similar substances, the flavan-3-ols were isolated and purified using polyamide-solid-phase-extraction and LC-MS analysis. As expected, we found (-)-epicatechin and (+)-catechin in unfermented, dried, unroasted cocoa beans. In contrast, roasted cocoa beans and cocoa products additionally contained the atypical flavan-3-ol (-)-catechin. This is generally formed during the manufacturing process by an epimerization which converts (-)-epicatechin to its epimer (-)-catechin. High temperatures during the cocoa bean roasting process and particularly the alkalization of the cocoa powder are the main factors inducing the epimerization reaction. In addition to the analysis of cocoa and cocoa products, peak ratios were calculated for a better differentiation of the cocoa products.

Online coupling of pressurized liquid extraction, solid-phase extraction and high-performance liquid chromatography for automated analysis of proanthocyanidins in malt.

A new instrumental setup for automated extraction of solid samples by online coupling of pressurized liquid extraction, automated SPE (solid-phase extraction) and HPLC is presented. From the extraction to the chromatogram no manual sample handling is required. The application to the determination of proanthocyanidins in malt reduces time and manual work to a minimum compared to former manual methods. Twenty samples can be processed within 24 h in respect to eight samples with the manual method. Using the features of the instrumental coupling, an optimized strategy for SPE of proanthocyanidins from natural samples was developed, requiring no evaporation step, using commercial cartridges and delivering concentrated eluates. The recovery of five main malt proanthocyanidins was 97%, with a reproducibility of 5%. This new instrumental coupling is thought to reduce time and costs along with improved results for a broad range of solid sample materials.

Identification and quantification of polyphenols in carob fruits (Ceratonia siliqua L.) and derived products by HPLC-UV-ESI/MSn.

The polyphenolic patterns of carob pods (Ceratonia siliqua L.) and derived products were identified and quantified using high-performance liquid chromatography-UV absorption-electrospray ion trap mass spectrometry after pressurized liquid extraction and solid-phase extraction. In carob fiber, 41 individual phenolic compounds could be identified. In addition, spectrophotometric quantification using the Folin-Ciocalteu and vanillin assays was performed, and the antioxidative activity was determined as the 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. Carob pods contain 448 mg/kg extractable polyphenols comprising gallic acid, hydrolyzable and condensed tannins, flavonol-glycosides, and traces of isoflavonoids. Among the products investigated, carob fiber, a carob pod preparation rich in insoluble dietary fiber (total polyphenol content = 4142 mg/kg), shows the highest concentrations in flavonol-glycosides and hydrolyzable tannins, whereas roasted carob products contain the highest levels of gallic acid. The production process seems to have an important influence on the polyphenolic patterns and quantities in carob products.

Temperature influences epimerization and composition of flavanol monomers, dimers and trimers during cocoa bean roasting.

Cocoa consumption is suggested to promote many health benefits, since cocoa is a rich source of flavanols; but amounts and profiles of flavanols depend strongly on the bean type, origin and manufacturing process. Roasting is known as a crucial step in technical treatment of cocoa, which leads to flavanol losses and modifications, especially the epimerization of (-)-epicatechin to (-)-catechin. This study monitors the influence of cocoa bean roasting on the composition of flavanol monomers to trimers, with special focus on epimerization, which was quantified for procyanidin dimers, and also observed for trimers for the first time. Five dimeric and two trimeric potential epimerization products were detected and the extent of epimerization during cocoa roasting was shown to be a function of temperature. The data also showed remarkable variations in the change of flavanol content. The quantified flavanols decreased about 50% in Java beans and increased about 30% in Ivory Coast beans, despite being roasted under equal conditions.

Intrinsic isotopic 13C labelling of polyphenols.

The intrinsic isotopic labelling of plants with (13)CO2 is an effective method to generate highly labelled compounds using photosynthesis and avoiding labour-intensive complex organic syntheses. In this study, the intrinsic isotopic labelling of polyphenols in parsley, spinach and peppermint is shown for the first time. The plants were grown in an atmosphere where (12)CO2 was replaced by (13)CO2, in order to generate highly labelled compounds. The total content of (13)C as well as the individual polyphenols were analysed by Isotopic Ratio-MS and HPLC-Iontrap-MS(n). After 34 days of plant growth under (13)CO2, degree of labelling was found to be higher than 90 atom% (13)C for most polyphenols, predominantly consisting of highly and fully labelled isotopomers; the total plant material contained more than 88 atom% (13)C. Such highly labelled compounds can be used in future studies to dissect both metabolism and bioavailability of polyphenols in humans.

Comparing procyanidins in selected Vaccinium species by UHPLC-MS(2) with regard to authenticity and health effects.

Cranberry procyanidins have been associated with an effect against urinary tract infections (UTI) for decades, and European health claims are requested. This study compares the procyanidin profiles and concentrations of American cranberry ( Vaccinium macrocarpon Ait.), European cranberry ( Vaccinium oxycoccus L.), and lingonberry ( Vaccinium vitis-idaea L.) analyzed using ultrahigh-performance liquid chromatoraphy coupled to a triple-quadrupole mass spectrometer with electrospray interface (UHPLC-MS(2)). Concentrations of A-type trimers, procyanidin A2, catechin, epicatechin, and B-type dimers and trimers have been evaluated and compared for the first time in the three berries. The data clearly show remarkable differences in the procyanidin profiles and concentrations, especially the lack of A-type trimers in V. oxycoccus; thus, the effectiveness against UTI may vary among the Vaccinium species. These differences can be used to prove authenticity.

A shortcut from plasma to chromatographic analysis: straightforward and fast sample preparation for analysis of green tea catechins in human plasma.

This paper describes a new and straightforward method for determination of the green tea catechins epicatechin, epicatechin gallate, epigallocatechin, and epigallocatechin gallate in human plasma. Sample preparation includes addition only of dimethylformamide and trichloroacetic acid. After centrifugation, the supernatant can be injected into the HPLC. If required, the glucuronides and sulphates of the catechins can be enzymatically hydrolysed before extraction. Recovery ranges from 92.9 to 98.2%; limits of detection, from 2.4 to 5.0 ng/mL; and relative standard deviations, from 3.1 to 8.6%. Twelve samples can be processed within 45 min, and are then ready to be injected into the HPLC. The method was successfully applied to human plasma. This method is suitable for studies on absorption, bioavailability, and kinetics of green tea catechins in plasma. Since manual work and time consumption are minimal, the procedure is especially useful for large numbers of samples.

Influence of postharvest processing and storage on the content of phenolic acids and flavonoids in foods.

The review is based on the evaluation of electronically collated data published between 2002 to June 2006. It is based on 325 references dealing with the following subclasses of phenolic compounds: hydroxycinnamic and hydroxybenzoic acids, chalcones, flavanones, flavones, flavonols, monomeric flavanols and anthocyanins. Only publications dealing directly with the effects of storage and postharvest processing on the phenolic acid and flavonoid contents of foods were considered. The expectation that the structural diversity even within each subgroup, and the number of different procedures and of different parameters would make finding homogenous tendencies unlikely, has, in most instances, been confirmed. By adding a database Excel table combined with a focused and unified evaluation, specific additional information was rendered accessible and concise. It holds true for most of the subclasses in question that the effect of storage and food processing on the polyphenol content is negligible in comparison to the differences between different varieties of plants. Variety dependence must always be considered, for all classes of compounds.

The influence of postharvest processing and storage of foodstuffs on the bioavailability of flavonoids and phenolic acids.

Postharvest processing and storage not only influence the content and composition of flavonoids and phenolic acids in foodstuffs, thereby altering the amount of potentially bioavailable bioactive compounds, but can also modify their chemical form. Moreover, due to the intensive metabolism during absorption, the metabolites circulating in blood differ from the parent compounds found in food. Thus, it is difficult to predict potential in vivo effects of phenolic compounds merely by their contents in foodstuffs. Their specific bioavailability needs to be determined. This review considers studies regarding the bioavailability of flavonoids and phenolic acids from foodstuffs that meet the following criteria: providing actual concentrations of flavonoids and phenolic acids in blood plasma, body tissues, or urine, comparing differently stored or processed foods (excluding studies that use supplements or pure substances), and considering the high interindividual variability by repeated measurements in the same individuals. Only a few studies meet all of these criteria. In conclusion, processing and storage of food can have either positive or negative effects on the bioavailability of flavonoids and phenolic acids because these treatments may not only change the content, but also the chemical form of these compounds.

Stereoisomeric separation of flavanones and flavanone-7-O-glycosides by capillary electrophoresis and determination of interconversion barriers.

The stereoisomeric separation of several flavanones and flavanone-7-O-glycosides has been achieved with capillary electrophoresis by adding native cyclodextrins or cyclodextrin derivatives to the background electrolyte. As an alternative method, micellar electrokinetic chromatography with sodium cholate as a chiral surfactant has been used for the epimeric separation of two flavanone-7-O-glycosides. The effect of buffer systems containing mixtures of cyclodextrin with either sodium dodecyl sulfate or sodium cholate upon the chiral recognition of flavanones and flavanone-7-O-glycosides as well as the variation of the background electrolyte (concentration of buffer and surfactant, pH value, organic modifier), and its influence on the resolution factor Rs was investigated. Temperature- and pH-dependent enantiomerization or epimerization barriers of several flavanones (naringenin, homoeriodictyol) and flavanone-7-O-glycosides (naringin, neohesperidin, prunin, narirutin) in basic media (pH values of 9-11) have been observed. Interconversion profiles featuring characteristic plateau formation of the elution pattern were observed at high pH and evaluated with the simulation software ChromWin to determine rate constants k(T) and Eyring activation parameters, DeltaG#(T), DeltaH#, and DeltaS#.

Chiral separation of diastereomeric flavanone-7-O-glycosides in citrus by capillary electrophoresis.

The 2S- and 2R-diastereomers of major flavanone-7-O-glycosides found in sweet orange (Citrus sinensis), mandarine (Citrus deliciosa), grapefruit (Citrus paradisi), lemon (Citrus limon), and sour or bitter orange juice (Citrus aurantium) were separated for the first time by chiral capillary electrophoresis (CE) employing various buffers with combined chiral selectors. Native cyclodextrins (CDs), neutral and charged CD derivatives were examined as chiral additives to the background electrolyte (BGE). Separation efficiency has not proved satisfactory with one single CD as chiral selector in the buffer, a full and simultaneous separation could often be achieved only by using combined buffer with two different CDs. Chiral separation of major flavanones in sweet orange, mandarine and grapefruit juices raised more difficulties than in lemon and sour orange juices as narirutin will not readily build complexes with most CDs. Diastereomeric flavanones of mature and immature grapefruits were compared and some differences were found: naringin showed different diastereomeric ratio and 2S-prunin appeared only in immature grapefruit. Marmalade was also examined by chiral CE. Its major flavanones corresponded to flavanone pattern of mixed sour and sweet oranges.