High-performance liquid chromatographic method for the determination of sorafenib in human serum and peritoneal fluid.

PURPOSE: Sorafenib is recommended for therapy of advanced hepatocellular carcinoma and renal cell carcinoma. Preclinical data indicate a relation between dose and antitumor efficacy. In clinical trials, adverse events improve after dose reduction suggesting a dose-dependent toxicity. Given dose has a direct impact on the drug serum concentration, but the latter also can be influenced by multiple factors, including interaction and metabolisation. To enable the investigation of concentration-related effects, an easy and sensitive assay for sorafenib drug monitoring is essential. METHODS: A high-performance liquid chromatography (HPLC) analysis involving an extraction with diethyl ether followed by separation on a Pinnacle™ DB C18 column and quantitation by UV absorbance at 260 nm was established. Sorafenib concentrations in samples of serum and peritoneal fluid have been determined. RESULTS: The assay was validated for serum samples and is linear over the concentration range of 100-5,000 ng/ml with a determination coefficient of >0.999. The limit of detection is 0.25 ng/ml. The intra- and inter-day coefficients of variation were below 3.03%. Sorafenib recovery in spiked probes of peritoneal fluid was above 85%. Sorafenib concentrations in 44 serum samples and 14 probes of peritoneal fluid have been determined with a mean of 3,328 and 1,380 ng/ml, respectively (standard deviation 2,267 and 659 ng/ml). CONCLUSIONS: A sensitive and selective HPLC method for the determination of sorafenib in human serum was developed and also verified for peritoneal fluid. This method provides a useful tool for pharmacokinetic investigations as well as for therapeutic drug monitoring of sorafenib.

Intestinal transit and systemic metabolism of apple polyphenols.

BACKGROUND: Apples are the most widely consumed fruits in Germany and various other countries. Positive health effects of apple-derived polyphenols in vivo depend on their absorption, metabolism, distribution, and elimination from the body after consumption. Data on the metabolism of these polyphenols in humans are scarce. In order to study the intestinal transit and metabolism of apple polyphenols in humans, a variety of experiments were carried out. METHODS: Polyphenols were incubated with saliva (for 5 min), simulated gastric or duodenal juice (4 or 10 h, respectively), or rat hepatocytes (4 h) under aerobic conditions, and with ileostomy fluid under aerobic conditions for 10 h. The polyphenol profile in human serum (8 h later) and renal elimination in urine (24 h later) were also investigated after consumption of 1 L apple juice. Polyphenols and their metabolites were identified and quantified by high-performance liquid chromatography with diode array detection (HPLC-DAD), HPLC-electrospray ionization-tandem mass spectrometry (ESI-MS/MS), and gas chromatography (GC)-MS. RESULTS: In the presence of native saliva or ileostomy fluid, ß-glycosides of phloretin and quercetin were hydrolyzed, to varying degrees depending on the sugar moiety, and to much lesser degrees in the presence of antibiotics. In the gastric milieu, almost complete degradation of procyanidin B(2) to (-)-epicatechin was observed. In the presence of artificial duodenal juice flavan-3-ol epimerization occurred. Quercetin was completely converted to phloroglucinol, 3,4-dihydroxybenzoic acid, and 2,4,6-trihydroxybenzoic acid. Formation of isomeric products of hydroxycinnamic acid esters and their corresponding methyl esters was also observed, and similar results were obtained after incubation with rat hepatocytes. Products of phase II metabolism, two phloretin O-glucuronides and eight (methyl) quercetin O-glucuronides, were identified in the hepatocyte samples. Following enzymatic hydrolysis, 5-caffeoylquinic acid, 4-p-coumaroylquinic acid, caffeic acid, (-)-epicatechin, phloretin, and quercetin were recovered in both serum and urine (5.3% and 3.5% of the amounts consumed, respectively). In addition, 19.5% of the polyphenols consumed were identified in the urine in the form of hydroxylated phenolic and hippuric acids. CONCLUSION: The findings relating to the absorption, metabolism, and systemic availability of polyphenols in vivo should contribute to our understanding of their biological effects, and the characterization of newly formed metabolites should facilitate further studies.

Simultaneous determination of voriconazole and posaconazole concentrations in human plasma by high-performance liquid chromatography.

A simple, sensitive, and selective high-performance liquid chromatographic method for the simultaneous determination of voriconazole and posaconazole concentrations in human plasma was developed and validated. Quantitative recovery following liquid-liquid extraction with diethyl ether was achieved. Linearity ranged from 0.10 to 20.0 microg/ml for voriconazole and from 0.05 to 10.0 microg/ml for posaconazole. The intra- and interday coefficients of variation were less than 8.5%, and the lower limits of quantitation were <0.05 microg/ml.

Model experiments mimicking the human intestinal transit and metabolism of D-galacturonic acid and amidated pectin.

In order to study the human intestinal transit and metabolism of D-galacturonic acid and amidated pectin a number of model experiments were carried out. Both substrates were incubated under aerobic conditions at 37 degrees C using saliva (2 min) and simulated gastric juice (4 h). Under anaerobic conditions the substrates were incubated at 37 degrees C using human ileostomy and colostomy fluids, each obtained from three different donors, for 10 and for 24 h, respectively. D-Galacturonic acid, SCFA (acetic acid, propionic acid, and butyric acid), as well as methanol were analyzed photometrically after carbazole reaction, GC-flame ionization detection (GC-FID), and headspace solid-phase microextraction GC/MS (HS-SPME-GC/MS), respectively. D-Galacturonic acid and amidated pectin were found to be stable during incubations with saliva and simulated gastric juice, whereas both substrates underwent degradation in the course of human ileostomy and colostomy fluid incubations. D-Galacturonic acid was practically completely decomposed within 10 h and SCFA, with acetic acid as the major representative, were formed up to 98% of the incubated substrate in colostomy effluent. The amidated pectin was only degraded in part, revealing stable amounts of 22-35% and 3-17% in ileostomy (after 10 h) and colostomy fluid (after 24 h), respectively. SCFA were generated up to 59% of the applied amidated pectin. In parallel, 19-60% and 52-67% of the available methyl ester groups were cleaved in the course of incubations with ileostomy and colostomy fluids, respectively. The results demonstrate for the first time that D-galacturonic acid and amidated pectin are stable in human saliva and simulated gastric juice. The degradation of both compounds during incubation with ileostomy effluent is highlighted, providing evidence for a considerable metabolic potential of the small intestine.

Intervention with cloudy apple juice results in altered biological activities of ileostomy samples collected from individual volunteers.

BACKGROUND: Apple juice is considered to be an important component of the healthy diet, with anticancer activities in colon cancer animal models and key ingredients have numerous chemoprotective activities in human colon cells in vitro. AIM OF THE STUDY: Since only little is known on comparable activities in the human colon in vivo, here a pilot study was performed to assess related mechanisms caused by ileostomy samples from volunteers that had consumed apple juice. METHODS: Ileostomy samples were collected after intervention (0-8 h) with cloudy apple juice (1 l). They were characterized analytically for major apple polyphenols and biologically in HT29 colon cells for their potential to cause genotoxic damage, protect from the genotoxic insult by hydrogen peroxide (H(2)O(2)) and modulate the expression of GSTT2, an enzyme related to antioxidative defence against different peroxides. RESULTS: The analytical determination of polyphenols in the ileostomy samples revealed that the majority of the compounds were recovered in the samples collected 2 h after intervention. The comparison of genotoxic effects of samples before intervention and 2 h after intervention revealed a considerable variation of genotoxic response, but there was a trend for reduced genotoxicity in three of eight persons (P) after intervention. Samples collected at 2 h protected HT29 cells from genotoxic damage by H(2)O(2) (for 4 of 8 persons), resulted in an increased GSTT2 expression (for 2 of 6 persons) and of GSTT2 promotor activity (2 of 6 persons). CONCLUSIONS: The intervention with apple juice results in bioavailable concentrations of related polyphenols in the gut lumen, which could contribute to reduced genotoxicity, enhanced antigenotoxicity and favorable modulation of GSTT2 gene expression in some individuals. The pilot study for the first time used this combination of faecal biomarkers which in larger cohorts may either reveal overall significant alterations of chemoprotection or may be used to identify individuals which could particularly benefit from a personalized nutrition.

Polyphenols are intensively metabolized in the human gastrointestinal tract after apple juice consumption.

Polyphenols are secondary plant compounds showing anticarcinogenic effects both in vitro and in animal experiments and may thus reduce the risk of colorectal cancer in man. The identification of polyphenol metabolites formed via their passage through the small intestine of healthy ileostomy subjects after apple juice consumption is presented. Identification and quantification of polyphenols and their metabolites were performed using HPLC-DAD as well as HPLC-ESI-MS/MS. Total procyanidin content (TPA) was measured, and additionally the mean degree of polymerization (DPm) of the procyanidins was determined in the apple juice and ileostomy effluents. As products of polyphenol metabolism, D-(-)-quinic acid and methyl esters of caffeic acid and p-coumaric acid are liberated from the corresponding hydroxycinnamic acid esters. 1-Caffeoylquinic acid and 3-caffeoylquinic acid were determined as products of isomerization. Phloretin 2'-O-glucoside (phloridzin) and phloretin 2'-O-xyloglucoside were metabolized into the corresponding aglycons phloretin and phloretin 2'-O-glucuronide and all were found in the ileostomy effluent. Ninety percent of the consumed procyanidins were recovered in the ileostomy effluent and therefore would reach the colon under physiologic circumstances. The DP m was reduced (DP m of apple juice=5.7) and varied depending on the time point of excretion. The gastrointestinal passage seems to play an important role in the colonic availability of apple polyphenols.

Human intestinal hydrolysis of phenol glycosides - a study with quercetin and p-nitrophenol glycosides using ileostomy fluid.

In order to study the influence of sugar moiety, aglycon structure and microflora concentration on the human ileal hydrolysis of phenol glycosides, various quercetin and p-nitrophenol glycosides were incubated under anaerobic conditions (37 degrees C for 0, 0.5, 1, 2, 4, 6, 8, 10 and 24 h) with ileostomy fluids from three different donors. The glycosides, i.e. beta-D-glucopyranosides, beta-D-galactopyranosides, alpha-L-arabinofuranosides, beta-D-xylopyranosides and alpha-L-rhamnopyranosides as well as the liberated aglycones were identified by HPLC-DAD and HPLC-ESI-MS/MS. Among the quercetin glycosides under study, the 3-O-beta-D-glucopyranoside showed with 0.22 micromol/h the highest hydrolysis rate, followed by the 3-O-beta-D-galactopyranoside, the 3-O-beta-D-xylopyranoside and the 3-O-alpha-L-arabinofuranoside (0.04 and each 0.03 micromol/h, respectively). Quercetin 3-O-alpha-L-rhamnopyranoside was found to be stable for the entire incubation period. Using quercetin 3-O-beta-D-glucopyranoside as a representative example, linear hydrolysis rate was observed from 75 to 2500 microL ileostomy fluid corresponding to its microflora content (log 0.68 up to 21.9 colony forming units). Studies performed in the presence of antibiotics did not reveal any hydrolysis. The p-nitrophenol glycosides were hydrolyzed faster than the corresponding quercetin glycosides. The hydrolysis rate decreased from the beta-D-glucopyranoside (0.41 micromol/h), to the beta-D-galactopyranoside (0.21 micromol/h), the beta-D-xylopyranoside (0.12 micromol/h), the alpha-L-arabinofuranoside (0.09 micromol/h) to the alpha-L-rhamnopyranoside (0.06 micromol/h). These results demonstrate that the human ileal hydrolysis of phenol glycosides depends on the sugar and the aglycon structure as well as the microflora.

Studies on apple and blueberry fruit constituents: do the polyphenols reach the colon after ingestion?

The aim of our studies was to determine the amount of polyphenols reaching the colon after oral intake of apple juice and blueberries. After a polyphenol-free diet healthy ileostomy volunteers consumed a polyphenol-rich cloudy apple juice while others consumed anthocyanin-rich blueberries. Ileostomy effluent was collected and polyphenols were identified using HPLC-DAD as well as HPLC-ESI-MS/MS; quantification was performed with HPLC-DAD. Most of the orally administered apple polyphenols were absorbed from or metabolized in the small intestine. Between 0 and 33% of the oral dose was recovered in the ileostomy bags with a maximum of excretion after 2 h. A higher amount of the blueberry anthocyanins under study (up to 85%, depending on the sugar moiety) were determined in the ileostomy bags and therefore would reach the colon under physiological circumstances. Such structure-related availability has to be considered when polyphenols are used in model systems to study potential preventive effects in colorectal diseases.

Colonic availability of apple polyphenols--a study in ileostomy subjects.

Nutrition is thought to play an essential role in the pathogenesis of inflammatory and malignant gastrointestinal diseases. It is well known that plant ingredients such as polyphenols and flavonoids show anticarcinogenic effects both in vitro and in animal experiments, and may thus reduce the risk of colorectal cancer in man. The aim of the study was to determine the amount of polyphenols reaching the colon after oral intake of apple juice. After consumption of a polyphenol-free diet 11 healthy ileostomy volunteers drank 1 L of a polyphenol-rich cloudy apple juice. Ileostomy effluent was collected immediately before and 1, 2, 4, 6, and 8 h after consumption of apple juice. A broad spectrum of polyphenols was identified using HPLC-diode array detection (HPLC-DAD) as well as HPLC-ESI-MS/MS; quantitation was performed with HPLC-DAD. Most of the orally administered apple polyphenols were absorbed from or metabolized in the small intestine. Between 0 and 33% of the oral dose was recovered in the ileostomy bags with a maximum of excretion after 2 h. Phloretin glucuronide as product of polyphenol metabolism was detected in the ileostomy effluent. The present results show that most of the apple juice polyphenols are absorbed in the small intestine. Minor amounts of unmetabolized polyphenols are recovered in the ileostomy effluent, which would reach the colon under physiologic circumstances. These data have to be considered when polyphenols are used in model systems to show preventive effects in colorectal carcinogenesis.

Determination of the 2H/1H and 15N/14N ratios of Alkylpyrazines from coffee beans (Coffea arabica L. and Coffea canephoravar. robusta) by isotope ratio mass spectrometry.

The delta15N(AIR) and delta2H(VSMOW) data for several alkylpyrazines formed during the roasting process of coffee are reported. Samples of commercially available roasted (n = 9) as well as self-roasted (n = 8) coffee beans (Coffea arabica L. and Coffea canephora var. robusta) of different origins were investigated. By use of extracts prepared by simultaneous distillation extraction (SDE) and subsequently fractionated by liquid chromatography on silica gel, on-line capillary gas chromatography-isotope ratio mass spectrometry was employed in the combustion (C) and pyrolysis (P) modes (HRGC-C/P-IRMS) to determine the delta15N(AIR) and delta2H(VSMOW) values, respectively. In addition to the constituents of coffee beans, data for commercial synthetic alkylpyrazines and substances declared to be "natural" were determined. The delta15N(AIR) data for coffee alkylpyrazines under study-2-ethyl-5-methylpyrazine (1) and 2-ethyl-6-methylpyrazine (2) (measured as sum 1/2), 2-ethyl-3-methylpyrazine (3), 2-methylpyrazine (4), 2,5-dimethylpyrazine (5) and 2,6-dimethylpyrazine (6) (measured as sum 5/6), and 2,3-dimethylpyrazine (7), as well as 2,3,5-trimethylpyrazine (8)-varied in the range from +8.3 to -10.2 per thousand, thus revealing their biogeneration from amino acids (delta15N(AIR) ranging from +8 per thousand to -10 per thousand). The delta2H(VSMOW) values were determined in the range from -5 per thousand to -127 per thousand. Owing to the analytical differentiation observed between coffee alkylpyrazines and synthetic/"natural" samples of 3, 4, and 7, authenticity assessment of coffee-flavored products seems to be promising, provided that extended data will be available in the future. In the literature, there were no IRMS data available for the alkylpyrazines (1-8) under study.

Polyphenol profiles of apple juices.

Focusing on 17 constituents, the polyphenol profiles of juices freshly made from various dessert (n = 4) and cider apple cultivars (n = 7) as well as commercially available apple juices (n = 24) were investigated using high-performance liquid chromatography-photodiode array detection (HPLC-DAD) and (HPLC)-electrospray ionization-tandem mass spectrometry (ESI(neg)-MS/MS) analyses. Significant differences in the total polyphenol content as well as the profiles of the apple cultivars under study were observed. For dessert apples the total polyphenol content ranged from 154 to 178 mg/L, whereas for 'old' German cider apple cultivars 261-970 mg/L were determined. Boskoop showed the highest (970 mg/L) and Granny Smith the lowest (154 mg/L) polyphenol content of the freshly prepared samples under study. Hydroxycinnamic acids, with chlorogenic acid as dominating constituent, ranged from 57 to 68 mg/L as well as from 134-593 mg/L in juices made from dessert apples and that from cider apples, respectively. Dessert apple juices showed lower contents of dihydrochalcones (10-35 mg/L) and flavan-3-ols (50-95 mg/L) compared to that of cider apples (34-171 mg/L and 70-393 mg/L, respectively). Quercetin and its derivatives were found from 0.4-4 mg/L and 0.4-27 mg/L in juices made from dessert apples and that of cider apples, respectively. Compared with freshly made juices, lower contents of polyphenols were determined in the commercial samples under study. Amounts ranging from 110-459 mg/L, dominated by chlorogenic acid with concentrations from 53-217 mg/L, were determined. Information about cultivar-typical apple polyphenol content and profile is important for bioactivity studies and, consequently, essential for the development of consumer-relevant products with particular nutritional functionalities.