Predictors of Urinary 3-Phenoxybenzoic Acid Levels in 50 North Carolina Adults.

Limited data are available on the non-chemical stressors that impact adult exposures to pyrethroid insecticides based on urinary biomonitoring. The urinary metabolite, 3-phenoxybenzoic acid (3-PBA), is commonly used to assess human exposure to a number of pyrethroids. In a further analysis of published study data, we quantified urinary 3-PBA levels of 50 adults over a single, 24-h sampling period and examined the associations between the biomarker measurements and selected non-chemical stressors (demographic, lifestyle, and dietary factors). A convenience sample of 50 adults was recruited in North Carolina in 2009-2011. Participants collected individual urine voids (up to 11) and filled out activity, food, and pesticide use diaries over a 24-h sampling period. Urine voids (n = 326) were analyzed for 3-PBA concentrations using high-performance liquid chromatography-tandem mass spectrometry. 3-PBA was detected in 98% of the 24-h composited urine samples. The geometric mean urinary 3-PBA level was 1.68 ng/mL in adults. Time spent outside (p = 0.0006) was a highly significant predictor of natural log-transformed (ln) urinary 3-PBA levels, while consumption of coffee (p = 0.007) and breads (p = 0.019) and ln creatinine levels (p = 0.037) were significant predictors of urinary 3-PBA levels. In conclusion, we identified specific factors that substantially increased adult exposures to pyrethroids in their everyday environments.

The profiling and identification of the absorbed constituents and metabolites of Paeoniae Radix Rubra decoction in rat plasma and urine by the HPLC-DAD-ESI-IT-TOF-MS(n) technique: a novel strategy for the systematic screening and identification of absorbed constituents and metabolites from traditional Chinese medicines.

Paeoniae Radix Rubra (PRR, the dried roots of Paeonia lactiflora) is a commonly used traditional Chinese medicine (TCM). A clear understanding of the absorption and metabolism of TCMs is very important in their rational clinical use and pharmacological research. To find more of the absorbed constituents and metabolites of TCMs, a novel strategy was proposed. This strategy was characterized by the following: the establishment and utilization of the databases of parent compounds, known metabolites and characteristic neutral losses; the comparison of base peak chromatograms and ClogPs; and the use of the HPLC-DAD-ESI-IT-TOF-MS(n) technique. This strategy was first applied to screen and identify the absorbed constituents and metabolites of PRR decoction and paeoniflorin in rats. In total, 13 new absorbed constituents and 90 new metabolites of PRR decoction were detected. Among these metabolites, the structures of 70 metabolites were identified, and the conjugation types and structure skeletons of the other 20 metabolites were preliminarily determined. Moreover, 35 new metabolites of some constituents of PRR, i.e., 22 new metabolites of paeoniflorin, 10 new metabolites of gallic acid-related compounds, 1 new metabolite of (epi)catechin-related compounds, and 2 new metabolites of other compounds, were reported for the first time. The results also indicated that (epi)catechin-related compounds, gallic acid-related compounds and paeoniflorin were the main precursors of these metabolites. Phase I reactions (dehydroxylation, decarboxylation, dehydrogenation) and phase II reactions (sulfation, glucuronidation and methylation) were observed as the main metabolic pathways of PRR. According to the literature, the 11 absorbed constituents and 11 metabolites have various bioactivities. This study is the first to explore the absorption and metabolism of PRR decoction, and the result also is a notable improvement in the discovery of paeoniflorin metabolites in vivo. These findings enhance our understanding of the metabolism and Effective forms (the truly active structures) of PRR decoction and paeoniflorin.

An investigation of the antidepressant action of xiaoyaosan in rats using ultra performance liquid chromatography-mass spectrometry combined with metabonomics.

A rapid, highly sensitive, and selective method was applied in a non-invasive way to investigate the antidepressant action of Xiaoyaosan (XYS) using ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and chemometrics. Many significantly altered metabolites were used to explain the mechanism. Venlafaxine HCl and fluoxetine HCl were used as chemical positive control drugs with a relatively clear mechanism of action to evaluate the efficiency and to predict the mechanism of action of XYS. Urine obtained from rats subjected to chronic unpredictable mild stress (CUMS) was analyzed by UPLC-MS. Distinct changes in the pattern of metabolites in the rat urine after CUMS production and drug intervention were observed using partial least squares-discriminant analysis. The results of behavioral tests and multivariate analysis showed that CUMS was successfully reproduced, and a moderate-dose XYS produced significant therapeutic effects in the rodent model, equivalent to those of the positive control drugs, venlafaxine HCl and fluoxetine HCl. Metabolites with significant changes induced by CUMS were identified, and 17 biomarker candidates for stress and drug intervention were identified. The therapeutic effect of XYS on depression may involve regulation of the dysfunctions of energy metabolism, amino acid metabolism, and gut microflora changes. Metabonomic methods are valuable tools for measuring efficacy and mechanisms of action in the study of traditional Chinese medicines.

Quantitative determination of hippuric and benzoic acids in urine by LC-MS/MS using surrogate standards.

An LC-MS/MS method for simultaneous determination of hippuric acid (HA) and benzoic acid (BA) in monkey urine after direct injection was developed. Since HA and BA are endogenous compounds in urine, surrogate standards ((13)C(6)-hippuric and (13)C(6)-benzoic acid) were employed to generate calibration curves. l-Phenylalanine-ring-D5 served as an internal standard. Multiple reaction monitoring in the negative ionization mode with an APCI source was used for detection of all components in the assay. The developed method is intended for determination of HA and BA in the range of 0.25-250 and 0.1-100microg/ml, respectively. Weighted (1/x) quadratic regression (r(2)>0.99) was used to generate calibration curves. Precision and accuracy of the method were assessed by analyzing 3 quality control samples (concentrations at low, medium, and high range of calibration curve) prepared in monkey urine. Stability for 48h at room temperature and after 3 freeze-thaw cycles was also evaluated. The proposed method was successfully utilized for analysis of urine samples from female monkeys following the administration of everninomicin alone and in combination with gentamicin. The concentrations of endogenous HA and BA were calculated based on the peak area ratio of the analyte to the internal standard using a regression equation for corresponding surrogate standard.

The metabolic fate of red wine and grape juice polyphenols in humans assessed by metabolomics.

The metabolic impact of polyphenol-rich red wine and grape juice consumption in humans was studied using a metabolomics approach. Fifty-eight men and women participated in a placebo-controlled, double-crossover study in which they consumed during a period of 4 wk, either a polyphenol-rich 2:1 dry mix of red wine and red grape juice extracts (MIX) or only a grape juice extract (GJX). Twenty-four-hour urine samples were collected after each intervention. (1)H NMR spectroscopy was applied for global metabolite profiling, while GC-MS was used for focused profiling of urinary phenolic acids. Urine metabolic profiles after intake of both polyphenol-rich extracts were significantly differentiated from placebo using multilevel partial least squares discriminant analysis. A significant 35% increase in hippuric acid excretion (p<0.001) in urine was measured after the MIX consumption as) or only a red grape juice dry extract (GJX). 24-h urine samples were collected after each intervention. 1H-NMR spectroscopy was applied for global metabolite profiling, while gas chromatography-mass spectrometry (GC-MS) was used for focused profiling of urinary phenolic acids. Urine metabolic profiles after intake of both polyphenol-rich extracts were significantly differentiated from placebo using multilevel partial least squares discriminant analysis (ML-PLS-DA). A significant 35% increase in hippuric acid excretion (p<0.001) in urine was measured after the MIX consumption compared with placebo, whereas no change was found after GJX consumption. GC-MS-based metabolomics of urine allowed identification of 18 different phenolic acids, which were significantly elevated following intake of either extract. Syringic acid, 3- and 4-hydroxyhippuric acid and 4-hydroxymandelic acid were the strongest urinary markers for both extracts. MIX and GJX consumption had a slightly different effect on the excreted phenolic acid profile and on endogenous metabolite excretion, possibly reflecting their different polyphenol composition.

Clinical applications of urinary organic acids. Part I: Detoxification markers.

Modern instrumentation allows the measurement of organic acids in urine in their physiological concentration ranges. Eight of the compounds that are reported can serve as markers for specific toxicant exposure or detoxification challenges. Xylene exposure causes elevation of 2-methylhippurate, and orotic acid elevation reveals ammonia challenge that exceeds the capacity of the urea cycle. General hepatic detoxification stimulation by natural compounds, drugs, or xenobiotic compounds causes elevated levels of glucaric acid. Abnormalities of alpha-hydroxybutyrate, pyroglutamate, and sulfate can indicate up-regulated glutathione biosynthesis, impaired reformation of glutathione in the gamma-glutamyl cycle, and depleted total body glutathione status, respectively. Patterns of these compounds measured in a simple overnight urine specimen help to identify focal areas of clinical concern and monitor patient responses to detoxification interventions.

Simultaneous determination of albiflorin and paeoniflorin in rat urine by solid-phase extraction and high-performance liquid chromatography following oral administration of Si-Wu decoction.

A high performance liquid chromatographic method (HPLC), together with solid phase extraction (SPE), was developed for simultaneous determination of albiflorin and paeoniflorin in rat urine after oral administration of Si-Wu decoction. The samples were pretreated with solid phase extraction using Extract-Cleantrade mark cartridges. Analysis of the extract was performed on a reversed-phase C18 column and a mobile phase made up of acetonitrile and 0.03% formic acid (17:83, v/v). UV detection was set at 230 nm. The assay was linear over the range 2.625-52.50 mg/mL for albiflorin and 3.875-77.50 microg/mL for paeoniflorin. The average percentage recoveries of three spiked urines were 97.01 +/- 3.32 and 102.32 +/- 6.97 for albiflorin and paeoniflorin, respectively. The intra-day precision (RSD) ranged from 0.21 to 1.79% at concentrations of 4.20, 10.50, 26.25 and 39.375 microg/mL of albiflorin and 0.12 to 2.92% at concentrations of 3.875, 10.85, 23.25 and 58.125 microg/mL of paeoniflorin, and inter-day precision (RSD) was from 1.02 to 1.86% for albiflorin and 0.94 to 3.30% for paeoniflorin, at the same four concentrations. This method was applied in order to analyze albiflorin and paeoniflorin in rat urine following oral administration of traditional Chinese medicinal preparation of Si-Wu decoction.

Ibuprofen interference in the determination of 3-phenoxybenzoic acid in urine.

Pyrethroid insecticides are widely used in agriculture and private households. Analysis of urine for pyrethroid metabolites is one way to detect human exposure to these insecticides and is carried out regularly as part of the Occupational and Environmental Medicine Monitoring Program recommended by the Deutsche Forschungsgemeinschaft (DFG). Samples are analyzed using GC-MS (selected ion monitoring) following acid hydrolysis, solid phase extraction, esterification with methanol/sulfuric acid, and liquid-liquid extraction. The metabolite, 3-phenoxybenzoic acid (3-PBA), can be derived from several pyrethroids and is, therefore, a useful diagnostic analyte; however, the presence of the over-the-counter drug, ibuprofen ((R,S)-2-(4-isobutylphenyl)propionic acid), interferes with this determination, even after the ingestion of only one 200-mg tablet. The interfering analyte is carboxy-ibuprofen which is not removed by the cleanup step. Experimental work shows that it takes two days for most of the ibuprofen to clear the body before 3-PBA can reliably be determined in urine.

Catechin metabolites after intake of green tea infusions.

Green tea contains relatively large amounts of catechins, that have been recognized to be efficient free-radical scavengers. In spite of a largely described antioxidant effect, the metabolic fate of catechins in humans has been scarcely studied. An infusion of green tea (about 400 mg of catechins) was given to healthy volunteers; plasma and urine samples were collected for 5 h and 2 days, respectively. Epigallocatechin gallate and epicatechin gallate were detected in plasma samples, reaching the maximum concentration (2 microM) at 2 h. Urine samples collected at 6-48 h contained detectable amounts of final catechin metabolites, including 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3-methoxy-4-hydroxy-hippuric acid and 3-methoxy-4-hydroxybenzoic acid (vanillic acid). The total content of these metabolites averaged 60 mg. The levels of free plasma catechins account only partly for the increased (approximately +20%) total radical-trapping antioxidant parameter (TRAP) detected after green tea intake. Catechin conjugates (glucuronide and sulphate) and metabolites may add further contribution and explain the measured TRAP increase.

Identification of Gingko biloba flavonol metabolites after oral administration to humans.

An extract of Ginkgo biloba leaves (EGb) was given to healthy volunteers. Urine samples were collected for 3 days, and blood samples were withdrawn every 30 min for 5 h. The samples were purified through SPE C18 cartridges and analyzed by reversed-phase LC-diode array detection for the presence of EGb metabolites. Only urine samples contained detectable amounts of substituted benzoic acids, i.e., 4-hydroxybenzoic acid conjugate, 4-hydroxyhippuric acid, 3-methoxy-4-hydroxyhippuric acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, hippuric acid and 3-methoxy-4-hydroxybenzoic acid (vanillic acid). In contrast to rats no phenylacetic acid or phenylpropionic acid derivatives were found in urine, thus indicating that in humans a more extensive metabolism takes place. As for rats the metabolites found in human urines accounted for less than 30% of the flavonoids given. The same procedure was applied to blood samples, and no metabolites could be detected.

Determination of benzoic acid and hippuric acid in human plasma and urine by high-performance liquid chromatography.

For patients with inborn errors of urea synthesis, oral administration of sodium benzoate is the usual treatment to increase the nitrogen excretion. Thus, monitoring hippuric acid and benzoic acid simultaneously in human biological fluids is considered to be clinically important. We developed a simple and accurate high-performance liquid chromatographic method for the simultaneous determination of hippuric acid and benzoic acid in human plasma and urine. This method requires no extraction step. Aliquots of urine and plasma are added to a solution of internal standard (o-chlorobenzoic acid) in acetonitrile and directly injected onto a reversed-phase column using an acidic (pH 2.7) eluent and ultraviolet detection at 235 nm. The preliminary plasma concentration-time and urinary excretion rate-time profiles of hippuric acid and benzoic acid from a healthy subject receiving small, medium and large doses of sodium benzoate are reported.