Biliary excretion of amphetamine and methamphetamine in the rat

1. (14)C-labelled amphetamine and methamphetamine were injected into rats cannulated at the bile duct under thiopentone anaesthesia and the output of their metabolites in urine and bile was determined. 2. With amphetamine, 69% of the (14)C was excreted in the urine and 16% in the bile in 24h. The main metabolite in bile was the glucuronide of 4-hydroxyamphetamine. The output of unchanged amphetamine was much greater in cannulated rats than in intact rats. 3. With methamphetamine, 54% of the (14)C appeared in the urine and 18% in the bile. The main metabolite in the bile was the glucuronide of 4-hydroxynorephedrine. The output of amphetamine, a metabolite of methamphetamine, was much greater in cannulated rats than in intact rats. 4. Evidence has been obtained for the enterohepatic circulation of certain amphetamine and methamphetamine metabolites in the rat. 5. Thiopentone anaesthesia appeared to inhibit the ring hydroxylation of amphetamine administered as such or formed as a metabolite of methamphetamine.     

Quantitative profiling of bile acids in biofluids and tissues based on accurate mass high resolution LC-FT-MS: compound class targeting in a metabolomics workflow

We report a sensitive, generic method for quantitative profiling of bile acids and other endogenous metabolites in small quantities of various biological fluids and tissues. The method is based on a straightforward sample preparation, separation by reversed-phase high performance liquid-chromatography mass spectrometry (HPLC-MS) and electrospray ionisation in the negative ionisation mode (ESI-). Detection is performed in full scan using the linear ion trap Fourier transform mass spectrometer (LTQ-FTMS) generating data for many (endogenous) metabolites, not only bile acids. A validation of the method in urine, plasma and liver was performed for 17 bile acids including their taurine, sulfate and glycine conjugates. The method is linear in the 0.01-1muM range. The accuracy in human plasma ranges from 74 to 113%, in human urine 77 to 104% and in mouse liver 79 to 140%. The precision ranges from 2 to 20% for pooled samples even in studies with large number of samples (n>250). The method was successfully applied to a multi-compartmental APOE*3-Leiden mouse study, the main goal of which was to analyze the effect of increasing dietary cholesterol concentrations on hepatic cholesterol homeostasis and bile acid synthesis. Serum and liver samples from different treatment groups were profiled with the new method. Statistically significant differences between the diet groups were observed regarding total as well as individual bile acid concentrations.     

Simultaneous gas chromatographic determination of methamphetamine, amphetamine and their p-hydroxylated metabolites in plasma and urine

We report a method for the simultaneous determination of methamphetamine, amphetamine and their hydroxylated metabolites in plasma and urine samples using a GC-NPD system. The analytical procedures are: (1) adjust the sample to pH 11.5 with bicarbonate buffer, saturate with NaCl and extract with acetate; (2) back-extract the amines in the ethyl acetate fraction with 0.1 M HCl; (3) adjust the pH of the acid fraction to 11.5 and follow by extraction in ethyl acetate; (4) reduce the volume of ethyl acetate under nitrogen and derivatize the concentrate with trifluoroacetic anhydride or heptaflourobutyric anhydride before the GC analysis. The derivatives were separated on a GC-NPD system equipped with a HP-5 column of 25 m×0.32 m I.D. and a 0.52 μm film of 5% phenylmethylsilicone. The detection limit (taking a signal-to-noise ratio of 2) of heptafluorobutyl derivatives of methamphetamine and its metabolites in plasma and the trifluoroacetyl derivatives in urine was 1 ng/ml (22 pg on column). The limit of quantitation of the heptafluorobutyl derivatives in the plasma was 1 ng/ml (22 pg on column), and that of the trifluoroacetyl derivatives in urine was 20 ng/ml (73 pg on column). The between-day variation was from 0.9 to 17.4% and within-day variation from 0.9 to 8.3%. This method was used successfully in the quantitative determination of methamphetamine and its p-hydroxylated metabolites in the plasma and urine of human subjects.     

Specific assay for radiolabelled digoxin and its known apolar metabolites in biological fluids. I.

A specific assay method for radiolabelled digoxin and its known apolar metabolites in plasma, urine and saliva was developed. The assay permits the delineation of the pharmacokinetics of digoxin and its metabolites after single-dose administration of the drug to humans. Column chromatographic and solvent extraction procedures were used for the separation of apolar and polar compounds. Thin-layer chromatography was applied for the individual and specific assessment of digoxin and its apolar metabolites. Apolar and polar standards were used for quantitative assessments of all the procedures used. Accuracy and precision of the assay developed were evaluated in plasma, urine and saliva using biological samples spiked with known amounts of standards and by measuring replicates of biological samples obtained from pharmacokinetic studies with digoxin administration to humans.     

Enantioselective quantitation of the ecstasy compound (R)- and (S)-N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma and urine

An enantioselective HPLC method has been developed and validated for the stereospecific analysis of N-ethyl-3,4-methylenedioxyamphetamine (MDE) and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA). These compounds have been analyzed both from human plasma and urine after administration of 70 mg pure MDE-hydrochloride enantiomers to four subjects. The samples were prepared by hydrolysis of the o-glucuronate and sulfate conjugates using beta-glucuronidase/arylsulfatase and solid-phase extraction with a cation-exchange phase. A chiral stationary protein phase (chiral-CBH) was used for the stereoselective determination of MDE, HME and MDA in a single HPLC run using sodium dihydrogenphosphate, ethylendiaminetetraacetic acid disodium salt and isopropanol as the mobile phase (pH 6.44) and fluorimetric detection (lambda(ex) 286 nm, lambda(em) 322 nm). Moreover, a suitable internal standard (N-ethyl-3,4-methylenedioxybenzylamine) was synthesized and qualified for quantitation purposes. The method showed high recovery rates (>95%) and limits of quantitation for MDE and MDA of 5 ng/ml and for HME of 10 ng/ml. The RSDs for all working ranges of MDE, MDA and HME in plasma and urine, respectively, were less than 1.5%. After validation of the analytical methods in plasma and urine samples pharmacokinetic parameters were calculated. The plasma concentrations of (R)-MDE exceeded those of the S-enantiomer (ratio R:S of the area under the curve, 3.1) and the plasma half time of (R)-MDE was longer than that of (S)-MDE (7.9 vs. 4.0 h). In contrast, the stereochemical disposition of the MDE metabolites HME and MDA was reversed. Concentrations of the (S)-metabolites in plasma of volunteers were much higher than those of the (R)-enantiomers.     

Quantitative determination of Astragaloside IV, a natural product with cardioprotective activity, in plasma, urine and other biological samples by HPLC coupled with tandem mass spectrometry

Astragaloside IV is a novel cardioprotective agent extracted from the Chinese medical herb Astragalus membranaceus (Fisch) Bge. This agent is being developed for treatment for cardiovascular disease. Further development of Astragaloside IV will require detailed pharmacokinetic studies in preclinical animal models. Therefore, we established a sensitive and accurate high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) quantitative detection method for measurement of Astragaloside IV levels in plasma, urine as well as other biological samples including bile fluid, feces and various tissues. Extraction of Astragaloside IV from plasma and other biological samples was performed by Waters OASIS(trade mark) solid phase extraction column by washing with water and eluting with methanol, respectively. An aliquot of extracted residues was injected into LC/MS/MS system with separation by a Cosmosil C18 5 microm, 150 mm x 2.0 mm) column. Acetonitrile:water containing 5 microM NaAc (40:60, v/v) was used as a mobile phase. The eluted compounds were detected by tandem mass spectrometry. The average extraction recoveries were greater than 89% for Astragaloside IV and digoxin from plasma, while extraction recovery of Astragaloside IV and digoxin from tissues, bile fluid, urine and fece ranged from 61 to 85%, respectively. Good linearity (R2>0.9999) was observed throughout the range of 10-5000 ng/ml in 0.5 ml rat plasma and 5-5000 ng/ml in 0.5 ml dog plasma. In addition, good linearity (R2>0.9999) was also observed in urine, bile fluid, feces samples and various tissue samples. The overall accuracy of this method was 93-110% for both rat plasma and dog plasma. Intra-assay and inter-assay variabilities were less than 15.03% in plasma. The lowest quantitation limit of Astragaloside IV was 10 ng/ml in 0.5 ml rat plasma and 5 ng/ml in 0.5 ml dog plasma, respectively. Practical utility of this new LC/MS/MS method was confirmed in pilot pharmacokinetic studies in both rats and dogs following intravenous administration.     

Determination of metoclopramide and two of its metabolites using a sensitive and selective gas chromatographic—mass spectrometric assay

A modified gas chromatographic—mass spectrometric (GC—MS) assay has been developed to quantitate metoclopramide (MCP) and two of its metabolites [monodeethylated-MCP (mdMCP), dideethylated-MCP (ddMCP)] in the plasma, bile and urine of sheep. The heptafluorobutyryl derivatives of the compounds were formed and quantitated using electron-impact ionization in the selected-ion monitoring mode (MCP, m/z 86, 380; mdMCP, m/z 380 and ddMCP, m/z 380). No interference was observed from endogenous compounds following the extraction of various biological fluids obtained from non-pregnant sheep. Sample preparation has been simplified and the method is more selective and sensitive (2 fold) than our previous assay using electron-capture detection. The limit of quantitation for MCP, mdMCP and ddMCP was 1 ng/ml in plasma, urine and bile, requiring 0.5 ml of sample. This represents 2.5 pg of the analytes at the detector. The standard curves were linear over a working range of 1–40 ng/ml. Absolute recoveries in plasma ranged from 76.5–94.7%, 79.2–96.8%, 80.3–102.2% for MCP, mdMCP and ddMCP, respectively. In urine, recoveries ranged from 56.5–87.8%, 61.5–87.5%, 62.6–90.2% for MCP, mdMCP and ddMCP, respectively. Recoveries in bile ranged from 83.5–100.9%, 78.5–90.5%, 66.9–79.2% for MCP, mdMCP and ddMCP, respectively. Overall intra-day precision ranged from 2.9% for MCP in plasma to 12.6% for mdMCP in bile. Overall inter-day precision ranged from 5.9% for MCP in urine to 14.9% for ddMCP in bile. Bias was the greatest at the 1 ng/ml concentration in all biological fluids ranging from a low of 2.4% for mdMCP in plasma to a high of 11.9% for ddMCP in urine. Applicability of the assay for pharmacokinetic studies of MCP, mdMCP and ddMCP in the plasma and urine of a non-pregnant ewe is demonstrated.     

Quantification of 3,4-methylenedioxymetamphetamine and its metabolites in plasma and urine by gas chromatography with nitrogen–phosphorus detection

A gas chromatographic method with nitrogen–phosphorus detection involving a solid–liquid extraction phase was developed and validated for the simultaneous quantification of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) in plasma. A modification of this method was validated for the analysis of MDMA, MDA, 4-hydroxy-3-methoxymethamphetamine (HMMA) and, 4-hydroxy-3-methoxyamphetamine (HMA) in urine. Under the analytical conditions described, the limits of detection in plasma and urine were less than 1.6 μg/l and 47 μg/l, respectively, for all the compounds studied. Good linearity was observed in the concentration range evaluated in plasma (5–400 μg/l) and urine (100–2000 μg/l) for all compounds tested. The recoveries obtained from plasma were 85.1% and 91.6% for MDMA and MDA, respectively. Urine recoveries were higher than 90% for MDMA and MDA, 74% for HMMA, and 64% for HMA. Methods have been successfully used in the assessment of plasma and urine concentrations of MDMA and its main metabolites in samples from clinical studies in healthy volunteers.     

Determination of atrazine and its metabolites in mouse urine and plasma by LC-MS analysis

Atrazine is a herbicide widely used on agricultural commodities. Existing analytical methods to analyze atrazine and its metabolites in biological matrices have various drawbacks. Thus, further development of such methods will be needed to correlate the growing number of toxicological effects associated with atrazine exposure with the concentrations of this compound and its metabolites in plasma, urine, and tissues. The purpose of this study was to develop a broad and sensitive LC-MS method for the analysis of atrazine and its metabolites in mouse urine and plasma. We were able to simultaneously measure atrazine and its major mammalian metabolites, which include didealkyl atrazine, desisopropyl atrazine, desethyl atrazine, atrazine-glutathione conjugate, and atrazine-mercapturate, using preparation procedures that used small sample volumes of plasma and urine (0.25 and 0.5 ml, respectively). Furthermore, derivatization of analytes prior to analysis was unnecessary. This method was used to analyze plasma and urine samples following single in vivo oral exposures of a limited number of mice to atrazine (doses, 5-250 mg/kg body weight) to demonstrate the utility of this LC-MS method. The data obtained from this study suggest that atrazine is rapidly metabolized in mice. Didealkyl atrazine was the most abundant metabolite detected in the urine and plasma samples (approximately 1000 microM in 24-h urine and approximately 100 microM in plasma following the highest dose of atrazine), with lesser quantities of mono N-dealkylated metabolites and thio conjugates of atrazine observed. We also used this methodology in a preliminary study of cytochrome P450-catalyzed metabolism of atrazine in vitro. The results obtained in this study suggest that this method will be a useful tool for the determination of atrazine and its metabolites in future pharmacokinetic studies and for the subsequent development and refinement of biologically based models of atrazine disposition.     

Chlorpheniramine : I. Rapid quantitative analysis of chlorpheniramine in plasma,saliva and urine by high-performance liquid chromatography

A method was developed for the rapid quantitative analysis of chlorpheniramine in plasma, saliva and urine using high-performance liquid chromatography. A diethyl ether or hexane extract of the alkalinized biological samples was extracted with dilute acid which was chromatographed on a reversed-phase column using mixtures of acetonitrile and ammonium phosphate buffer as the mobile phase. Ultraviolet absorption at 254 nm was monitored for the detection and brompheniramine was employed as the internal standard for the quantitation. The effects of buffer, pH, and acetonitrile concentration in the mobile phase on the chromatographic separation were investigated. A mobile phase 20% acetonitrile in 0.0075 M phosphate buffer at a flow-rate of 2 ml/min was used for the assays of plasma and saliva samples. A similar mobile phase was used for urine samples. The drug and internal standard were eluted at retention volumes of less than 17 ml. The method can also be used to quantify two metabolites, didesmethyl- and desmethylchlorpheniramine, in the urine. The method can accurately measure chlorpheniramine levels down to 2 ng/ml in plasma or saliva using 1 ml of sample, and should be adequate for biopharmaceutical and pharmacokinetic studies. Various precautions for using the assay are discussed.     

Gas chromatographic determination of novel valproyl taurinamide derivatives in mouse and dog plasma

Valproyl taurinamides are a novel group of compounds that possess anticonvulsant activity. In this study a gas chromatographic micromethod was developed for the quantification of selected valproyl taurinamides and some of their metabolites in biological samples. Valproyl taurinamide (VTD), N-methyl valproyl taurinamide (M-VTD), N,N-dimethyl valproyl taurinamide (DM-VTD) and N-isopropyl valproyl taurinamide (I-VTD) were analyzed in mouse and dog plasma and in dog urine using gas chromatography. Flame ionization detection and mass spectrometric detection were compared. The plasma samples were prepared by solid-phase extraction using C(18) cartridges. The urine samples were prepared by liquid-liquid extraction. The sample volume used was 100 microl of dog plasma, 50 microl of mouse plasma and 20 microl of dog or mouse urine. The quantification range of the method was 1.5-50 mg/l in dog plasma (VTD only), 2.5-250 mg/l in mouse plasma (0.7-90 pmol injected) and 0.04-2 mg/ml in dog urine (VTD only). The inter-day precision in plasma and urine samples was around 10% for all quantified concentrations except LOQ (15-20%). The accuracy for all four compounds was between 90 and 110% within the entire concentration range. The developed method was suitable for quantification of a series of CNS-active valproyl taurineamide derivatives in biological samples at relevant in vivo concentrations.     

Liquid chromatographic-mass spectrometric determination of haloperidol and its metabolites in human plasma and urine

Haloperidol and its two metabolites, reduced haloperidol and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP) in human plasma and urine were analyzed by HPLC-MS using a new polymer column (MSpak GF-310), which enabled direct injection of crude biological samples without pretreatment. Recoveries of haloperidol and reduced haloperidol spiked into plasma were 64.4-76.1% and 46.8-50.2%, respectively; those for urine were 87.3-99.4% and 94.2-98.5%, respectively; those of CPHP for both samples were not less than 92.7%. The regression equations for haloperidol, reduced haloperidol and CPHP showed good linearity in the ranges of 10-800, 15-800 and 400-800 ng/ml, respectively, for both plasma and urine. Their detection limits were 5, 10 and 300 ng/ml, respectively, for both samples. Thus, the present method was sensitive enough for detection and determination of high therapeutic and toxic levels for haloperidol and its metabolites present in biological samples.     

Determination of amphetamines in human urine by headspace solid-phase microextraction and gas chromatography

Solid-phase microextraction (SPME) is under investigation for its usefulness in the determination of a widening variety of volatile and semivolatile analytes in biological fluids and materials. Semivolatiles are increasingly under study as analytical targets, and difficulties with small partition coefficients and long equilibration times have been identified. Amphetamines were selected as semivolatiles exhibiting these limitations and methods to optimize their determination were investigated. A 100- micro m polydimethylsiloxane (PDMS)-coated SPME fiber was used for the extraction of the amphetamines from human urine. Amphetamine determination was made using gas chromatography (GC) with flame-ionization detection (FID). Temperature, time and salt saturation were optimized to obtain consistent extraction. A simple procedure for the analysis of amphetamine (AMP) and methamphetamine (MA) in urine was developed and another for 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-methamphetamine (MDMA) and 3,4-methylenedioxy-N-ethylamphetamine (MDEA) using headspace solid-phase microextraction (HS-SPME) and GC-FID. Higher recoveries were obtained for amphetamine (19.5-47%) and methamphetamine (20-38.1%) than MDA (5.1-6.6%), MDMA (7-9.6%) and MDEA (5.4-9.6%).     

Simultaneous gas chromatographic determination of lorcainide hydrochloride and three of its principal metabolites in biological samples

A method is described for the determination of the antiarrhythmic drug lorcainide hydrochloride and its three main metabolites in plasma, urine, faeces and tissues from man and animals. The procedure involves the extraction of the parent drug, its metabolites and the internal standard from the biological materials at different alkaline pH values, back-extraction into sulphuric acid and re-extraction into the organic phase (heptane—isoamyl alcohol). After silylation of the different phenolic and the N-dealkylated metabolites, analyses were carried out by automated gas—liquid chromatography with electron-capture detection. The method has a sensitivity limit of 5 ng for lorcainide, and 10–20 ng for the various metabolites, per millilitre of plasma.The method was applied to urine, faeces, plasma and tissue samples from man and animals. It was also suitable for automatic sample analysis.     

Detection of ketamine and its metabolites in urine by ultra high pressure liquid chromatography-tandem mass spectrometry

Current analytical methods used for screening drugs and their metabolites in biological samples from victims of drug-facilitated sexual assault (DFSA) or other vulnerable groups can lack sufficient sensitivity. The application of liquid chromatography, employing small particle sizes, with tandem mass spectrometry (MS/MS) is likely to offer the sensitivity required for detecting candidate drugs and/or their metabolites in urine, as demonstrated here for ketamine. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) was performed following extraction of urine (4 mL) using mixed-mode (cation and C8) solid-phase cartridges. Only 20 microL of the 250 microL extract was injected, leaving sufficient volume for other assays important in DFSA cases. Three ion transitions were chosen for confirmatory purposes. As ketamine and norketamine (including their stable isotopes) are available as reference standards, the assay was additionally validated for quantification purposes to study elimination of the drug and primary metabolite following a small oral dose of ketamine (50 mg) in 6 volunteers. Dehydronorketamine, a secondary metabolite, was also analyzed qualitatively to determine whether monitoring could improve retrospective detection of administration. The detection limit for ketamine and norketamine was 0.03 ng/mL and 0.05 ng/mL, respectively, and these compounds could be confirmed in urine for up to 5 and 6 days, respectively. Dehydronorketamine was confirmed up to 10 days, providing a very broad window of detection.     

An efficient method for the purification and quantification of a galactose-specific lectin from vegetative tissues of Dolichos lablab

The differences among individual bile acids (BAs) in eliciting different physiological and pathological responses are largely unknown because of the lack of valid and simple analytical methods for the quantification of individual BAs and their taurine and glycine conjugates. Therefore, a simple and sensitive LC-MS/MS method for the simultaneous quantification of 6 major BAs, their glycine, and taurine conjugates in mouse liver, bile, plasma, and urine was developed and validated. One-step sample preparation using solid-phase extraction (for bile and urine) or protein precipitation (for plasma and liver) was used to extract BAs. This method is valid and sensitive with a limit of quantification ranging from 10 to 40 ng/ml for the various analytes, has a large dynamic range (2500), and a short run time (20 min). Detailed BA profiles were obtained from mouse liver, plasma, bile, and urine using this method. Muricholic acid (MCA) and cholic acid (CA) taurine conjugates constituted more than 90% of BAs in liver and bile. BA concentrations in liver were about 300-fold higher than in plasma, and about 180-fold higher in bile than in liver. In summary, a reliable and simple LC-MS/MS method to quantify major BAs and their metabolites was developed and applied to quantify BAs in mouse tissues and fluids.     

Enterohepatic circulation of N-acetyl-leukotriene E4

N-Acetyl-leukotriene E4, the end product of leukotriene C4 metabolism in the mercapturic acid pathway, was rapidly eliminated from the blood circulation into the bile of rats. Part of the N-acetyl-leukotriene E4 secreted from bile into the intestine underwent enterohepatic circulation. Leukotriene absorption occurred from the small intestine and from the colon. Biliary and urinary excretion within 5.5 h amounted to 15 and 2%, respectively, of the intraduodenally administered N-acetyl- 3H leukotriene E4 in animals anesthetized with ketamine. HPLC analyses indicated that 35% of the biliary radioactivity corresponded to unchanged N-acetyl-3H leukotriene E4, while 65% in bile and 100% in urine were polar metabolites. Enterohepatic circulation extends the biological half-life of N-acetyl-leukotriene E4.     

Direct injection LC-MS/MS method for identification and quantification of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine in urine drug testing

A method based on direct injection of diluted urine for the identification and quantification of amphetamine, methamphetamine, 3,4-methylenedioxymetamphetamine and 3,4-methylenedioxyamphetamine in human urine by electrospray ionisation liquid chromatography-tandem mass spectrometry was validated for use as a confirmation procedure in urine drug testing. Two deuterium labelled analogues, amphetamine-D5 and 3,4-methylenedioxymetamphetamine-D5, were used as internal standards. Twenty microliter aliquots of urine were mixed with 80 microL internal standard solution in autosampler vials and 10 microL was injected. The chromatographic system consisted of a 2.0 mmx100 mm C18 column and the gradient elution buffers used acetonitrile and 25 mmol/L formic acid. Two product ions produced from the protonated molecules were monitored in the selected reaction monitoring mode. The intra- and inter-assay variability (coefficient of variation) was between 5 and 16% for all analytes at 200 and 6000 ng/mL levels. Ion suppression occurred early after injection but did not affect the identification and quantification of the analytes in authentic urine samples. The method was further validated by comparison with a reference gas chromatographic-mass spectrometric method using 479 authentic urine samples. The two methods agreed almost completely (99.8%) regarding identified analytes when applying a 150 ng/mL reporting limit. Four deviating results were observed for 3,4-methylenedioxymethamphetamine and this was due to uncertainty in quantification around the reporting limit. For the quantitative results the slope of the regression lines were between 0.9769 and 1.0146, with correlation coefficients>0.9339. We conclude that the presented liquid chromatographic-tandem mass spectrometric method is robust and reliable, and suitable for use as a confirmation method in urine drug testing for amphetamines.     

Red wine anthocyanins are rapidly absorbed in humans and affect monocyte chemoattractant protein 1 levels and antioxidant capacity of plasma

Epidemiological studies suggest that a moderate consumption of anthocyanins may be associated with protection against coronary heart disease. The main dietary sources of anthocyanins include red-coloured fruits and red wine. Although dietary anthocyanins comprise a diverse mixture of molecules, little is known how structural diversity relates to their bioavailability and biological function. The aim of the present study was to evaluate the absorption and metabolism of the 3-monoglucosides of delphinidin, cyanidin, petunidin, peonidin and malvidin in humans and to examine both the effect of consuming a red wine extract on plasma antioxidant status and on monocyte chemoattractant protein 1 production in healthy human subjects. After a 12-h overnight fast, seven healthy volunteers received 12 g of an anthocyanin extract and provided 13 blood samples in the 24 h following the test meal. Furthermore, urine was collected during this 24-h period. Anthocyanins were detected in their intact form in both plasma and urine samples. Other anthocyanin metabolites could also be detected in plasma and urine and were identified as glucuronides of peonidin and malvidin. Anthocyanins and their metabolites appeared in plasma about 30 min after ingestion of the test meal and reached their maximum value around 1.6 h later for glucosides and 2.5 h for glucuronides. Total urinary excretion of red wine anthocyanins was 0.05+/-0.01% of the administered dose within 24 h. About 94% of the excreted anthocyanins was found in urine within 6 h. In spite of the low concentration of anthocyanins found in plasma, an increase in the antioxidant capacity and a decrease in MCP-1 circulating levels in plasma were observed.     

Urinary metabolites of flavonoids and hydroxycinnamic acids in humans after application of a crude extract from Equisetum arvense.

Flavonoids and hydroxycinnamic acids are polyphenolic compounds present in our daily diet in form of tea and vegetables as well as in herbal remedies used in phytomedicine. A wide range of in-vitro activities, in particular their antioxidant properties, have been studied intensively. However, in-vivo-data on absorption, bioavailability and metabolism after oral intake are scarce and contradictory. In order to examine the metabolism and renal excretion of these compounds a standardized extract from horsetail (Equisetum arvense) was administered to 11 volunteers following a flavonoid-free diet for 8 d. 24 h urine samples were collected and analyzed by HPLC-DAD. The putative quercetin metabolites, 3,4-dihydroxyphenylacetic acid or 3,4-dihydroxytoluene could not be detected in urine in any sample. The endogenous amount of homovanillic acid, generally regarded as one of the main quercetin metabolites, was 4 +/- 1 mg/d and did not increase significantly. However, hippuric acid, the glycine conjugate of benzoic acid, increased twofold after drug intake. Thus, the degradation to benzoic acid derivatives rather than phenylacetic acid derivatives seems to be a predominant route of metabolism. The results of this pilot study give rise to additional, substantial pharmacokinetic investigations in humans.