Comparative ecotoxicological hazard assessment of beta-blockers and their human metabolites using a mode-of-action-based test battery and a QSAR approach

We analyzed nontarget effects of the beta-blockers propranolol, metoprolol, and atenolol with a screening test battery encompassing nonspecific, receptor-mediated, and reactive modes of toxic action. All beta-blockers were baseline toxicants and showed no specific effects on energy transduction nor endocrine activity in the yeast estrogen and androgen screen, and no reactive toxicity toward proteins and DNA. However, in a phytotoxicity assay based on the inhibition of the photosynthesis efficiency in green algae, all beta-blockers were 10 times more toxic than their modeled baseline toxicity. Baseline- and phytotoxicity effects increased with hydrophobicity. The beta-blockers showed concentration addition in mixture experiments, indicating a mutual specific nontarget effect on algae. Using literature data and quantitative structure-activity relationships (QSAR), we modeled the total toxic potential of mixtures of the beta-blockers and their associated human metabolites for the phytotoxicity endpoint with two scenarios. The realistic scenario (I) assumes that the metabolites lose their specific activity and act as baseline toxicants. In the worst-case scenario (II) the metabolites exhibitthe same specific mode of action as their parent drug. For scenario (II), metabolism hardly affected the overall toxicity of atenolol and metoprolol, whereas propranolol's hazard potential decreased significantly. In scenario (I), metabolism reduced the apparent EC50 of the mixture of parent drug and metabolite even further. The proposed method is a simple approach to initial hazard assessment of pharmaceuticals and can guide higher tier testing. It can be applied to other classes of pollutants, e.g., biocides, as well as to environmental transformation products of pollutants.     

Occurrence and fate of the cytostatic drugs cyclophosphamide and ifosfamide in wastewater and surface waters

The two oxazaphosphorine compounds cyclophosphamide and ifosfamide are important cytostatic drugs used in the chemotherapy of cancer and in the treatment of autoimmune diseases. Their mechanism of action, involving metabolic activation and unspecific alkylation of nucleophilic compounds, accounts for genotoxic effects described in the literature and is reason for environmental concern. The occurrence and fate of cyclophosphamide and ifosfamide were studied in wastewater treatment plants (WWTPs) and surface waters in Switzerland, using a highly sensitive analytical method based on solid-phase extraction and liquid chromatography tandem mass spectrometry. The compounds were detected in untreated and treated wastewater at concentrations of <0.3-11 ng/L, which corresponded well with concentrations predicted from consumption data and typical renal excretion rates. Weekly loads determined in influent and effluent wastewater were comparable and suggested a high persistence in WWTPs. Furthermore, no degradation was observed in activated sludge incubation experiments within 24 h at concentrations of approximately 100 ng/L. Processes that may be relevant for elimination in natural waterbodies were studied with a set of incubation experiments in the laboratory. After extrapolation to natural conditions in surface waters, a slow dark-chemical degradation (half-lives on the order of years) is the most important transformation process. Degradation by photochemically formed HO* radicals may be of some relevance only in shallow, clear, and nitrate-rich waterbodies but could be further exploited for elimination of these compounds by advanced oxidation processes, i.e., in a treatment of hospital wastewater. In surface waters, concentrations ranged from < or =50 to 170 pg/L and were thus several orders of magnitude lower than the levels at which acute ecotoxicological effects have been reported in the literature (mg/L range). However, due to a lack of studies on chronic effects on aquatic organisms and data on occurrence and effects of metabolites, a final risk assessment cannot be made.     

Significance of xenobiotic metabolism for bioaccumulation kinetics of organic chemicals in Gammarus pulex

Bioaccumulation and biotransformation are key toxicokinetic processes that modify toxicity of chemicals and sensitivity of organisms. Bioaccumulation kinetics vary greatly among organisms and chemicals; thus, we investigated the influence of biotransformation kinetics on bioaccumulation in a model aquatic invertebrate using fifteen (14)C-labeled organic xenobiotics from diverse chemical classes and physicochemical properties (1,2,3-trichlorobenzene, imidacloprid, 4,6-dinitro-o-cresol, ethylacrylate, malathion, chlorpyrifos, aldicarb, carbofuran, carbaryl, 2,4-dichlorophenol, 2,4,5-trichlorophenol, pentachlorophenol, 4-nitrobenzyl-chloride, 2,4-dichloroaniline, and sea-nine (4,5-dichloro-2-octyl-3-isothiazolone)). We detected and identified metabolites using HPLC with UV and radio-detection as well as high resolution mass spectrometry (LTQ-Orbitrap). Kinetics of uptake, biotransformation, and elimination of parent compounds and metabolites were modeled with a first-order one-compartment model. Bioaccumulation factors were calculated for parent compounds and metabolite enrichment factors for metabolites. Out of 19 detected metabolites, we identified seven by standards or accurate mass measurements and two via pathway analysis and analogies to other compounds. 1,2,3-Trichlorobenzene, imidacloprid, and 4,6-dinitro-o-cresol were not biotransformed. Dietary uptake contributed little to overall uptake. Differentiation between parent and metabolites increased accuracy of bioaccumulation parameters compared to total (14)C measurements. Biotransformation dominated toxicokinetics and strongly affected internal concentrations of parent compounds and metabolites. Many metabolites reached higher internal concentrations than their parents, characterized by large metabolite enrichment factors.     

Recent advances in environmental risk assessment of transformation products

When micropollutants degrade in the environment, they may form persistent and toxic transformation products, which should be accounted for in the environmental risk assessment of the parent compounds. Transformation products have become a topic of interest not only with regard to their formation in the environment, but also during advanced water treatment processes, where disinfection byproducts can form from benign precursors. In addition, environmental risk assessment of human and veterinary pharmaceuticals requires inclusion of human metabolites as most pharmaceuticals are not excreted into wastewater in their original form, but are extensively metabolized. All three areas have developed their independent approaches to assess the risk associated with transformation product formation including hazard identification, exposure assessment, hazard assessment including dose-response characterization, and risk characterization. This review provides an overview and defines a link among those areas, emphasizing commonalities and encouraging a common approach. We distinguish among approaches to assess transformation products of individual pollutants that are undergoing a particular transformation process, e.g., biotransformation or (photo)oxidation, and approaches with the goal of prioritizing transformation products in terms of their contribution to environmental risk. We classify existing approaches for transformation product assessment in degradation studies as exposure- or effect-driven. In the exposure-driven approach, transformation products are identified and quantified by chemical analysis followed by effect assessment. In the effect-driven approach, a reaction mixture undergoes toxicity testing. If the decrease in toxicity parallels the decrease of parent compound concentration, the transformation products are considered to be irrelevant, and only when toxicity increases or the decrease is not proportional to the parent compound concentration are the TPs identified. For prioritization of transformation products in terms of their contribution to overall environmental risk, we integrate existing research into a coherent model-based, risk-driven framework. In the proposed framework, read-across from data of the parent compound to the transformation products is emphasized, but limitations to this approach are also discussed. Most prominently, we demonstrate how effect data for parent compounds can be used in combination with analysis of toxicophore structures and bioconcentration potential to facilitate transformation product effect assessment.     

Estimation of residue depletion of cyadox and its marker residue in edible tissues of pigs using physiologically based pharmacokinetic modelling

Physiologically based pharmacokinetic (PBPK) models are powerful tools to predict tissue distribution and depletion of veterinary drugs in food animals. However, most models only simulate the pharmacokinetics of the parent drug without considering their metabolites. In this study, a PBPK model was developed to simultaneously describe the depletion in pigs of the food animal antimicrobial agent cyadox (CYA), and its marker residue 1,4-bisdesoxycyadox (BDCYA). The CYA and BDCYA sub-models included blood, liver, kidney, gastrointestinal tract, muscle, fat and other organ compartments. Extent of plasma-protein binding, renal clearance and tissue-plasma partition coefficients of BDCYA were measured experimentally. The model was calibrated with the reported pharmacokinetic and residue depletion data from pigs dosed by oral gavage with CYA for five consecutive days, and then extrapolated to exposure in feed for two months. The model was validated with 14 consecutive day feed administration data. This PBPK model accurately simulated CYA and BDCYA in four edible tissues at 24–120 h after both oral exposure and 2-month feed administration. There was only slight overestimation of CYA in muscle and BDCYA in kidney at earlier time points (6–12 h) when dosed in feed. Monte Carlo analysis revealed excellent agreement between the estimated concentration distributions and observed data. The present model could be used for tissue residue monitoring of CYA and BDCYA in food animals, and provides a foundation for developing PBPK models to predict residue depletion of both parent drugs and their metabolites in food animals.     

链霉菌生物活性物质分离纯化技术研究进展

放线菌尤其是链霉菌是微生物生物活性天然产物的主要产生菌,链霉菌属的很多菌能产生多种抗生素、抗肿瘤药物及酶等重要活性代谢产物,具有广泛的商业和医用开发价值。本文对链霉菌产生的生物活性物质分离纯化方法进行了综述,并对发展趋势进行了展望。      

Pesticide nonextractable residue formation in soil: insights from inverse modeling of degradation time series

Formation of soil nonextractable residues (NER) is central to the fate and persistence of pesticides. To investigate pools and extent of NER formation, an established inverse modeling approach for pesticide soil degradation time series was evaluated with a Monte Carlo Markov Chain (MCMC) sampling procedure. It was found that only half of 73 pesticide degradation time series from a homogeneous soil source allowed for well-behaved identification of kinetic parameters with a four-pool model containing a parent compound, a metabolite, a volatile, and a NER pool. A subsequent simulation indeed confirmed distinct parameter combinations of low identifiability. Taking the resulting uncertainties into account, several conclusions regarding NER formation and its impact on persistence assessment could nonetheless be drawn. First, rate constants for transformation of parent compounds to metabolites were correlated to those for transformation of parent compounds to NER, leading to degradation half-lives (DegT50) typically not being larger than disappearance half-lives (DT50) by more than a factor of 2. Second, estimated rate constants were used to evaluate NER formation over time. This showed that NER formation, particularly through the metabolite pool, may be grossly underestimated when using standard incubation periods. It further showed that amounts and uncertainties in (i) total NER, (ii) NER formed from the parent pool, and (iii) NER formed from the metabolite pool vary considerably among data sets at t→∞, with no clear dominance between (ii) and (iii). However, compounds containing aromatic amine moieties were found to form significantly more total NER when extrapolating to t→∞ than the other compounds studied. Overall, our study stresses the general need for assessing uncertainties, identifiability issues, and resulting biases when using inverse modeling of degradation time series for evaluating persistence and NER formation.     

Tea: A new perspective on health benefits

Although among the five main types of teas (white, green, oolong, black and pu-erh), black tea is the most consumed worldwide, an impressive number of scientific publications have been focused on green tea and its major compounds, flavan-3-ols (“catechins”). However, besides flavan-3-ols, there are other compounds present in tea that could be accounted as potential bioactive compounds. Therefore, the objective of the present review is to provide a new perspective on the health benefits associated with tea consumption by critically analyzing the available literature on the potential tea bioactive compounds and the current level of scientific evidence for these health benefits. Until now the exact mechanisms of action or compounds responsible for the health benefits associated with tea consumption have only been poorly investigated. It is important to consider that tea compounds will be extensively metabolized to different metabolites that will, most likely, be the compounds circulating in blood and potentially reaching the different sites of action.     

Development of a physiologically based pharmacokinetic model for flunixin in cattle (Bos taurus)

Frequent violation of flunixin residues in tissues from cattle has been attributed to non-compliance with the USFDA-approved route of administration and withdrawal time. However, the effect of administration route and physiological differences among animals on tissue depletion has not been determined. The objective of this work was to develop a physiologically based pharmacokinetic (PBPK) model to predict plasma, liver and milk concentrations of flunixin in cattle following intravenous (i.v.), intramuscular (i.m.) or subcutaneous (s.c.) administration for use as a tool to determine factors that may affect the withdrawal time. The PBPK model included blood flow-limited distribution in all tissues and elimination in the liver, kidney and milk. Regeneration of parent flunixin due to enterohepatic recirculation and hydrolysis of conjugated metabolites was incorporated in the liver compartment. Values for physiological parameters were obtained from the literature, and partition coefficients for all tissues but liver and kidney were derived empirically. Liver and kidney partition coefficients and elimination parameters were estimated for 14 pharmacokinetic studies (including five crossover studies) from the literature or government sources in which flunixin was administered i.v., i.m. or s.c. Model simulations compared well with data for the matrices following all routes of administration. Influential model parameters included those that may be age or disease-dependent, such as clearance and rate of milk production. Based on the model, route of administration would not affect the estimated days to reach the tolerance concentration (0.125 mg kg^?1) in the liver of treated cattle. The majority of USDA-reported violative residues in liver were below the upper uncertainty predictions based on estimated parameters, which suggests the need to consider variability due to disease and age in establishing withdrawal intervals for drugs used in food animals. The model predicted that extravascular routes of administration prolonged flunixin concentrations in milk, which could result in violative milk residues in treated cattle.     

Population Growth Rate Responses of Ceriodaphnia dubia to Ternary Mixtures of Specific Acting Chemicals: Pharmacological versus Ecotoxicological Modes of Action

When considering joint toxic apical effects at higher levels of biological organization, such as the growth of populations, the so-called pharmacological mode of action that relies on toxicological mechanistic effects on molecular target sites may not be relevant. Such effects on population growth rate will depend on the extent to which juvenile and adult survival rates and production rates (juvenile developmental rates and reproduction) are affected by toxic exposure and also by the sensitivity of population growth rates to life-history changes. In such cases, the ecotoxicological mode of action, defined as the crucial life-history trait processes and/or xenobiotic-life-history trait interactions underlying a toxicological effect on population growth rate, should be considered. Life-table response experiments with the crustacean Ceriodaphnia dubia exposed to single and ternary mixtures of nine compounds were conducted to test the hypothesis that joint effects on population growth rates could be predicted from the mixture constituent ecotoxicological mode of action. Joint effects of mixtures containing pharmacologically dissimilar compounds (cadmium, λ-cyhalothrin, and chlorpyrifos) that differentially affected life-history traits contributing to population growth rates were accurately predicted by the independent-action concept. Conversely, the concentration-addition concept accurately predicted joint effects of two different mixtures: one containing pharmacologically similar acting pyrethroids that also affected similarly life-history traits, the other one that included pharmacologically dissimilar compounds (3,4-dichloroaniline, sodium bromide, and fenoxycarb) acting mainly on reproduction rates. These results indicate that when assessing combined effects on population growth rate responses, selection of mixture toxicity conceptual models based on the ecotoxicological mode of action of mixture constituents provided more accurate predictions than those based on the pharmacological mode of action.     

The key importance of soy isoflavone bioavailability to understanding health benefits

Research over the past two decades has provided significant epidemiological and other evidence for the health benefits of the consumption of soy-based foods. A large number of dietary intervention studies have examined the effects of soy isoflavones on risk factors for cardiovascular disease and hormone-dependent cancers. However, these report large variability in outcome measures, very limited reproducibility between studies, and in some cases, controversy between the results of clinical trials using dietary soy or soy protein and isoflavone supplementation. This highlights a major gap in our understanding of soy isoflavone uptake, metabolism, distribution, and overall bioavailability. There are many potential factors that may influence bioavailability and a better knowledge is necessary to rationalize the inconsistencies in the intervention and clinical studies. This review focuses attention on our current state of knowledge in this area and highlights the importance of metabolism of the parent soy isoflavones and the critical role of gut microbiota on the bioavailability of these compounds and their metabolites.     

Can degradation products be used as documentation for natural attenuation of phenoxy acids in groundwater?

In situ indicators of degradation are important tools in the demonstration of natural attenuation. A literature survey on the production history of phenoxy acids and degradation pathways has shown that metabolites of phenoxy acid herbicides also are impurities in the herbicide products, making the bare presence of these compounds useless as in situ indicators. These impurities can make up more than 30% of the herbicides. Degradation of phenoxy acids was demonstrated in microcosm experiments using groundwater and sediment contaminated with MCPP, dichlorprop, and related compounds such as other phenoxypropionic acids and chlorophenols. Field observations at two phenoxy acid-contaminated sites showed the occurrence of several impurities including metabolites in the groundwater. Neither the microcosm experiments nor the field observations verified that metabolites were actually produced or accumulated in situ. However, it was demonstrated that the impurity/parent herbicide ratios can be useful in situ indicators of degradation.     

2017~2019年昆明市市售食用鱼中几种违禁药物分析

目的 了解2017~2019年昆明市市售食用鱼中违禁药物残留状况。方法 2017~2019年随机采集市售食用鱼142份, 按照《国家食品污染和有害因素风险监测工作手册》检测违禁药物残留, 几种违禁药物都不得检出。结果 142件样品中违禁药物添加检出62件, 检出率39.4%(56件)。孔雀石绿和隐形孔雀石绿, 硝基呋喃代谢物, 喹乙醇及其代谢物检出率分别为2.8%(4件), 28.2%(40件), 8.4%(12件), 检出率最高的是硝基呋喃代谢物。违禁药物检出率最高的是鳝鱼, 其次是泥鳅。结论 昆明市市售食用鱼存在不同程度的违禁药物使用情况, 建议相关部门加大监管力度。     

Development of a multi-component Damage Assessment Model (MDAM) for time-dependent mixture toxicity with toxicokinetic interactions

A new mixture toxicity model was developed to predict the time-dependent toxicity of a mixture with toxicokinetic interactions directed specifically toward addressing biotransformation. The Damage Assessment Model (DAM), a toxicokinetic-toxicodynamic model that describes and predicts the time-dependent toxicity of a single compound, was extended to a multicomponent model for mixture toxicity. The model assumes that cumulative damage from the parent compound, metabolites, and/or a biotransformation inhibitor are additive, and the sum of the cumulative damage determines mixture toxicity. Since incorporation of the damage addition hypothesis into the DAM was equivalent to an independent action model for mixture toxicity, it was applied to describe the combined effect of mixture components with potentially dissimilar modes of action. From the multicomponent DAM, a time-dependent toxic unit model was derived and applied to determine the toxic units of mixture components. This model suggests a series of experimental designs required to assess the role of biotransformation in the toxicity of metabolized organic compounds and a data analysis method to separately estimate toxicodynamic parameters forthe parent compound and metabolites.     

Detection, accumulation and distribution of nitrofuran residues in egg yolk, albumen and shell.

Nitrofuran antibiotics have been banned for use in food-producing animals in many countries, including the European Union, owing to the threat they pose to human health. Research continues into the accumulation of these drugs in animal tissues and into the appropriate methods for their detection. In this study, an LC-MS/MS method is presented for the detection of the parent compounds, furazolidone, nitrofurantoin, furaltadone and nitrofurazone, in eggs. The parent compounds are first extracted into ethyl acetate, fats are removed by partition between acetonitrile and hexane, and the concentrated sample is analysed by LC-MS/MS. Decision limits (CCalpha) for the parents were < or =1 microg kg-1 for all four compounds. Within-day and between-day CVs are well within the limits stated in Commission Decision 2002/657/EC. The method provides an alternative to the testing of side-chain metabolites in eggs, which is particularly important in the case of nitrofurazone, where semicarbazide contamination of food has been attributed to sources other than nitrofurazone use. This method was used together with a method for the detection of the side-chain metabolite compounds, 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-1,3-oxazolidin-2-one (AMOZ), 1-amino-hydantoin (AHD) and semicarbazide (SEM), to study the accumulation and distribution of nitrofurans in eggs. Eggs were collected from four groups of hens that had been treated with one of the nitrofurans at a feed concentration of 300 mg kg-1 for 1 week. Parent compounds and metabolites were found in the yolk, albumen and shell. Albumen/yolk ratios for the parent compounds were 0.7, 0.82, 0.83 and 0.31 for furazolidone, furaltadone, nitrofurantoin and nitrofurazone, respectively. Ratios for the side-chain metabolites were 1.02, 1.06, 0.83 and 0.55 for AOZ, AMOZ, AHD and SEM, respectively. However, 50% of the total SEM residues were found in eggshell. This may be significant if eggshell products reach the consumer.     

Absorption,metabolism, and excretion of green tea flavan‐3‐ols in humans with an ileostomy

Green tea containing 634 μmol of flavan‐3‐ols was ingested by human subjects with an ileostomy. Ileal fluid, plasma, and urine collected 0–24 h after ingestion were analysed by HPLC‐MS. The ileal fluid contained 70% of the ingested flavan‐3‐ols in the form of parent compounds (33%) and 23 metabolites (37%). The main metabolites effluxed back into the lumen of the small intestine were O‐linked sulphates and methyl‐sulphates of (epi)catechin and (epi)gallocatechin. Thus, in subjects with a functioning colon substantial quantities of flavan‐3‐ols would pass from the small to the large intestine. Plasma contained 16 metabolites, principally methylated, sulphated, and glucuronidated conjugates of (epi)catechin and (epi)gallocatechin, exhibiting 101–256 nM peak plasma concentration and the time to reach peak plasma concentration ranging from 0.8 to 2.2 h. Plasma pharmacokinetic profiles were similar to those obtained with healthy subjects, indicating that flavan‐3‐ol absorption occurs in the small intestine. Ileostomists had earlier plasma time to reach peak plasma concentration values than subjects with an intact colon, indicating the absence of an ileal brake. Urine contained 18 metabolites of (epi)catechin and (epi)gallocatechin in amounts corresponding to 6.8±0.6% of total flavan‐3‐ol intake. However, excretion of (epi)catechin metabolites was equivalent to 27% of the ingested (?)‐epicatechin and (+)‐catechin.     

Hydroxy-PCBs, methoxy-PCBs and hydroxy-methoxy-PCBs: metabolites of polychlorinated biphenyls formed in vitro by tobacco cells

While the metabolism of polychlorinated biphenyls (PCBs) in plant cells is a rarely studied field, hydroxy-PCBs have been detected in several studies involving the use of various plant species. The ability of the tobacco (Nicotiana tabacum) callus culture WSC-38 to metabolize six dichlorobiphenyls under aseptic conditions was studied, and the resulting PCB metabolites were analyzed. WSC-38 cultures were cultivated with individual dichlorinated PCB congeners. The metabolites were identified based on mass spectra characteristics after gas chromatography separation. In addition, metabolites of PCB 9 (2,5-dichlorobiphenyl) were identified by comparing their retention characteristics with the available standards. In most cases at least two hydroxy-PCBs were produced from each parent PCB. Methoxy-PCBs and hydroxy-methoxy-PCBs were other groups of metabolites produced. To the best of our knowledge, ours is the first report to determine the presence of methoxy- and hydroxy-methoxy-metabolites of PCBs in plants. The role of the O-methyltransferases (OMTs) in the methylation of hydroxy-PCBs is discussed. As methoxy-metabolites of acetophenone were found among our samples, we posit that the OMTs responsible for the methylation of these compounds are also involved in the metabolism of PCBs in cultures of WSC-38.     

Multiclass screening in urine by comprehensive two-dimensional liquid chromatography time of flight mass spectrometry for residues of sulphonamides,beta-agonists and steroids

Nowadays routine residue monitoring involves the analysis of many compounds from different classes, mainly in urine. In the past two decades, developments heavily focused on the use of mass spectrometers (MS) and faster and more sensitive MS detectors have reached the market. However, chromatographic separation (CS) was rather ignored and the cognate developments in CS were not in line. As a result, residue analysis did not improve to the extent anticipated. CS by LC x LC is a promising technique and will enable a further increase in the range of compounds and compound classes that can be detected in a single run. In the present study, a self-built LC x LC system, using a 10 port valve, was connected to a single quadrupole MS with electrospray interface. Standards containing a mixture of sulphonamides, β-agonists and (steroid) hormones, 53 compounds, in total, were analysed. Results demonstrated that these compounds were well separated and could be detected at low levels in urine, i.e. limit of detection (LOD) from 1 µg L^?1 for most β-agonists to 10 µg L^?1 for some sulphonamides and most hormones. To enhance the sensitivity, optimisation was performed on an advanced commercial LC x LC system connected to a full scan accurate MS. This ultimately resulted in a fast high throughput untargeted method, including a simple sample clean-up in a 96-well format, for the analysis of urine samples.     

Identification of nobiletin metabolites in mouse urine

Nobiletin, a major component of citrus polymethoxyflavones, has many potential significant health benefits. While the biological activities of nobiletin have been widely reported, its in vitro and in vivo metabolic fate has been rarely studied. To explore the biotransformation mechanism of nobiletin we conducted an investigation into its metabolic profile in mouse urine, by various analytical techniques. Due to sample amount limitations for isolating and characterizing an individual metabolite, two possible nobiletin metabolites were prepared in a similar multi-step organic synthetic route: 3'-hydroxy-5,6,7,8,4'-pentamethoxyflavone (3'-demethylnobiletin) and 4'-hydroxy-5,6,7,8,3'-pentamethoxyflavone (4'-demethylnobiletin). Normal phase (silica gel) and C(18) reverse phase chromatography, as well as liquid chromatography-mass spectrometry-mass spectrometry, were employed in the separation of 3'-demethylnobiletin and 4'-demethylnobiletin, however, without success due to the structural similarities of these mono-demethylated nobiletins. Using a chiral packed column eluted under supercritical fluid chromatography (SFC) conditions, a clear separation was achieved. Thus, by comparing the SFC profiles of metabolite mixtures with the synthesized standard compounds, the major nobiletin metabolite of mouse urine is identified as 4'-demethylnobiletin, whereas 3'-demethylnobiletin is a minor metabolite. In this study, the concentration of 4'-demethylnobiletin in mouse urine is 28.9 microg/mL.