Doping control analysis of four JWH-250 metabolites in equine urine by liquid chromatography-tandem mass spectrometry.

JWH-250 is a synthetic cannabinoid. Its use is prohibited in equine sport according to the Association of Racing Commissioners International (ARCI) and the Fédération Équestre Internationale (FEI). A doping control method to confirm the presence of four JWH-250 metabolites (JWH-250 4-OH-pentyl, JWH-250 5-OH-pentyl, JWH-250 5-OH-indole, and JWH-250 N-pentanoic acid) in equine urine was developed and validated. Urine samples were treated with acetonitrile and evaporated to concentrate the analytes prior to the analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The chromatographic separation was carried out using a Phenomenex Lux® 3 µm AMP column (150 x 3.0 mm). A triple quadrupole mass spectrometer was used for detection of the analytes in positive mode electrospray ionization using multiple reaction monitoring (MRM). The limits of detection, quantification, and confirmation for these metabolites were 25, 50, and 50 pg/mL, respectively. The linear dynamic range of quantification was 50-10000 pg/mL. Enzymatic hydrolysis indicated that JWH-250 4-OH-pentyl, JWH-250 5-OH-pentyl, and JWH-250 5-OH indole are highly conjugated whereas JWH-250 N-pentanoic acid is not conjugated. Relative retention time and product ion intensity ratios were employed as the criteria to confirm the presence of these metabolites in equine urine. The method was successfully applied to post-race urine samples collected from horses suspected of being exposed to JWH-250. All four JWH-250 metabolites were confirmed in these samples, demonstrating the method applicability for equine doping control analysis.

Metabolic fate and detectability of the new psychoactive substances 2-(4-bromo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25B-NBOMe) and 2-(4-chloro-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25C-NBOMe) in human and rat urine by GC-MS, LC-MSn, and LC-HR-MS/MS approaches.

25B-NBOMe and 25C-NBOMe are potent 5-HT2A receptor agonists that have been associated with inducing hallucinogenic effects in drug users and severe intoxications. This paper describes the identification of their metabolites in rat and human urine by liquid chromatography (LC)-high resolution (HR)-MS/MS, the comparison of metabolite formation in vitro and in vivo and in different species, the general involvement of human cytochrome-P450 (CYP) isoenzymes on their metabolism steps, and their detectability by standard urine screening approaches (SUSAs) using GC-MS, LC-MSn, or LC-HR-MS/MS. Both NBOMe derivatives were mainly metabolized by O-demethylation, O,O-bis-demethylation, hydroxylation, and combinations as well as by glucuronidation and sulfation of the main phase I metabolites. For 25B-NBOMe, 66 metabolites could be identified and 69 for 25C-NBOMe. After application of low doses of both substances to rats, they were detectable mainly via their metabolites by both LC-based SUSAs. In case of acute intoxication, it was possible to detect 25B-NBOMe and its metabolites in an authentic human urine sample when using the GC-MS SUSA in addition to the LC-based SUSAs. Initial CYP activity screening revealed the involvement of CYP1A2 and CYP3A4 in hydroxylation and CYP2C9 and CYP2C19 in O-demethylation. The presented study demonstrated that 25B-NBOMe and 25C-NBOMe were extensively metabolized and detectable by both LC-based SUSAs.

Urinary Excretion Profiles of 2,5-Dimethoxy-4-alkylthiophenethylamine Analogs in Rats.

The urinary metabolic profiles of three hallucinogenic 2,5-dimethoxy-4-alkylthiophenethylamine analogs: 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2), 2,5-dimethoxy-4-isopropylthiophenethylamine (2C-T-4), and 2,5-dimethoxy-4-propylthiophenethylamine (2C-T-7), were investigated in rats. For each drug, four male Sprague-Dawley rats were orally administered 10 mg/kg of 2C-T-2, 2C-T-4, or 2C-T-7, and urine was collected 0-24 and 24-48 h after administration. The urine samples were processed by liquid-liquid extraction, and the extracts were analyzed by liquid chromatography/mass spectrometry to quantify the metabolites. The metabolic patterns of these drugs were different: for 2C-T-7, the principal metabolite was the ß-hydroxylated-N-acetylated-sulfoxide, whereas for 2C-T-2 and 2C-T-4 the major metabolites were the N-acetylated-sulfoxide and S-methylated-N-acetylated-sulfoxide, respectively.

In-syringe reversed dispersive liquid-liquid microextraction for the evaluation of three important bioactive compounds of basil, tarragon and fennel in human plasma and urine samples.

In the present study, an efficient and environmental friendly method (called in-syringe reversed dispersive liquid-liquid microextraction (IS-R-DLLME)) was developed to extract three important components (i.e. para-anisaldehyde, trans-anethole and its isomer estragole) simultaneously in different plant extracts (basil, fennel and tarragon), human plasma and urine samples prior their determination using high-performance liquid chromatography. The importance of choosing these plant extracts as samples is emanating from the dual roles of their bioactive compounds (trans-anethole and estragole), which can alter positively or negatively different cellular processes, and necessity to a simple and efficient method for extraction and sensitive determination of these compounds in the mentioned samples. Under the optimum conditions (including extraction solvent: 120 µL of n-octanol; dispersive solvent: 600 µL of acetone; collecting solvent: 1000 µL of acetone, sample pH 3; with no salt), limits of detection (LODs), linear dynamic ranges (LDRs) and recoveries (R) were 79-81 ng mL(-1), 0.26-6.9 µg mL(-1) and 94.1-99.9%, respectively. The obtained results showed that the IS-R-DLLME was a simple, fast and sensitive method with low level consumption of extraction solvent which provides high recovery under the optimum conditions. The present method was applied to investigate the absorption amounts of the mentioned analytes through the determination of the analytes before (in the plant extracts) and after (in the human plasma and urine samples) the consumption which can determine the toxicity levels of the analytes (on the basis of their dosages) in the extracts.

A data-independent acquisition workflow for qualitative screening of new psychoactive substances in biological samples.

Identification of new psychoactive substances (NPS) is challenging. Developing targeted methods for their analysis can be difficult and costly due to their impermanence on the drug scene. Accurate-mass mass spectrometry (AMMS) using a quadrupole time-of-flight (QTOF) analyzer can be useful for wide-scope screening since it provides sensitive, full-spectrum MS data. Our article presents a qualitative screening workflow based on data-independent acquisition mode (all-ions MS/MS) on liquid chromatography (LC) coupled to QTOFMS for the detection and identification of NPS in biological matrices. The workflow combines and structures fundamentals of target and suspect screening data processing techniques in a structured algorithm. This allows the detection and tentative identification of NPS and their metabolites. We have applied the workflow to two actual case studies involving drug intoxications where we detected and confirmed the parent compounds ketamine, 25B-NBOMe, 25C-NBOMe, and several predicted phase I and II metabolites not previously reported in urine and serum samples. The screening workflow demonstrates the added value for the detection and identification of NPS in biological matrices.

Nontargeted SWATH acquisition for identifying 47 synthetic cannabinoid metabolites in human urine by liquid chromatography-high-resolution tandem mass spectrometry.

Clandestine laboratories constantly produce new synthetic cannabinoids to circumvent legislative scheduling efforts, challenging and complicating toxicological analysis. Sundstrom et al. (Anal Bioanal Chem 405(26):8463-8474, [9]) and Kronstrand et al. (Anal Bioanal Chem 406(15):3599-3609, [10]) published nontargeted liquid chromatography, high-resolution, quadrupole/time-of-flight mass spectrometric (LC-QTOF) assays with validated detection of 18 and 38 urinary synthetic cannabinoid metabolites, respectively. We developed and validated a LC-QTOF urine method for simultaneously identifying the most current 47 synthetic cannabinoid metabolites from 21 synthetic cannabinoid families (5-fluoro AB-PINACA, 5-fluoro-AKB48, 5-fluoro PB-22, AB-PINACA, ADB-PINACA, AKB48, AM2201, JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, JWH-398, MAM2201, PB-22, RCS-4, UR-144, and XLR11). ß-Glucuronidase-hydrolyzed urine was extracted with 1-mL Biotage SLE+ columns. Specimens were reconstituted in 150-µL mobile phase consisting of 80% A (0.1% formic acid in water) and 20% B (0.1% formic acid in acetonitrile). Fifty microliters was injected, and SWATH™ MS data were acquired in positive electrospray mode. The LC-QTOF instrument consisted of a Shimadzu UFLCxr system and an ABSciex 5600+ TripleTOF® mass spectrometer. Gradient chromatographic separation was achieved with a Restek Ultra Biphenyl column with a 0.5-mL/min flow rate and an overall run time of 15 min. Identification criteria included molecular ion mass error, isotopic profiles, retention time, and library fit criteria. Limits of detection were 0.25-5 µg/L (N = 10 unique fortified urine samples), except for two PB-22 metabolites with limits of 10 and 20 µg/L. Extraction efficiencies and matrix effects (N = 10) were 55-104 and -65-107%, respectively. We present a highly useful novel LC-QTOF method for simultaneously confirming 47 synthetic cannabinoid metabolites in human urine.

Ultrasound-assisted temperature-controlled ionic-liquid dispersive liquid-phase microextraction method for simultaneous determination of anethole, estragole, and para-anisaldehyde in different plant extracts and human urine: a comparative study.

In this study, the performances of four ionic-liquid-based microextraction methods, ionic-liquid-based dispersive liquid-liquid microextraction (IL-DLLME), ionic-liquid-based ultrasound-assisted emulsification microextraction (IL-USA-ME), temperature-controlled ionic-liquid dispersive liquid-phase microextraction (TC-IL-DLME), and ultrasound-assisted temperature-controlled ionic-liquid dispersive liquid-phase microextraction (USA-TC-IL-DLME), were investigated for extraction of three bioactive compounds (anethole, estragole, and anisaldehyde) from different plant extracts and human urine. Anethole and estragole were chosen because they can alter cellular processes positively or negatively, and an efficient method is needed for their extraction and sensitive determination in the samples mentioned. Because there is no previous report on the separation of anethole and estragole (structural isomers), first, simultaneous gradient elution and flow programming were used. The microextraction methods were then applied and compared for analysis of these compounds in plant extracts and human urine by use of high-performance liquid chromatography (HPLC). The effect of conditions on extraction efficiency was studied and under the optimum conditions, the best enrichment factors (58-64), limits of detection (14-18 ng mL(-1)), limits of quantification (47-60 ng mL(-1)), and recovery (94.4-101.7 %) were obtained by use of USA-TC-IL-DLME. The optimized conditions were used to determine anethole, estragole, and para-anisaldehyde in fennel, anise, and tarragon extracts and in human urine.

Determination of 4-bromo-2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine (25B-NBOMe) in serum and urine by high performance liquid chromatography with tandem mass spectrometry in a case of severe intoxication.

We present a case of 4-bromo-2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine (25B-NBOMe), an N-benzyl phenethylamines derivative, intoxication and a high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) method for detection and quantification of 25B-NBOMe. A 19-year-old male was found unresponsive with generalized grand mal seizure activity. On the second day of hospitalization, a friend admitted that the patient used 'some unknown drug' called 25B. Serum and urine collected were sent to the Virginia Commonwealth University Medical Center Toxicology Laboratory for analysis. An HPLC-MS/MS method for the identification and quantification of 25B-NBOMe using 2-(2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25H-NBOMe) as the internal standard (ISTD) was developed. As this is a novel, single-case presentation, an assay validation was performed prior to testing to ensure the reliability of the analytical results. The serum and urine specimens were determined to contain 180 pg/ml and1900 pg/ml of 25B-NBOMe, respectively.

In vivo validation of DNA adduct formation by estragole in rats predicted by physiologically based biodynamic modelling.

Estragole is a naturally occurring food-borne genotoxic compound found in a variety of food sources, including spices and herbs. This results in human exposure to estragole via the regular diet. The objective of this study was to quantify the dose-dependent estragole-DNA adduct formation in rat liver and the urinary excretion of 1'-hydroxyestragole glucuronide in order to validate our recently developed physiologically based biodynamic (PBBD) model. Groups of male outbred Sprague Dawley rats (n = 10, per group) were administered estragole once by oral gavage at dose levels of 0 (vehicle control), 5, 30, 75, 150, and 300mg estragole/kg bw and sacrificed after 48h. Liver, kidney and lungs were analysed for DNA adducts by LC-MS/MS. Results obtained revealed a dose-dependent increase in DNA adduct formation in the liver. In lungs and kidneys DNA adducts were detected at lower levels than in the liver confirming the occurrence of DNA adducts preferably in the target organ, the liver. The results obtained showed that the PBBD model predictions for both urinary excretion of 1'-hydroxyestragole glucuronide and the guanosine adduct formation in the liver were comparable within less than an order of magnitude to the values actually observed in vivo. The PBBD model was refined using liver zonation to investigate whether its predictive potential could be further improved. The results obtained provide the first data set available on estragole-DNA adduct formation in rats and confirm their occurrence in metabolically active tissues, i.e. liver, lung and kidney, while the significantly higher levels found in liver are in accordance with the liver as the target organ for carcinogenicity. This opens the way towards future modelling of dose-dependent estragole liver DNA adduct formation in human.

Bioanalytical investigation of asarone in connection with Acorus calamus oil intoxications.

Preparations of the plant Acorus calamus (calamus or sweet flag) (A. calamus) are available via internet trade and marketed as being hallucinogenic. In 2003-2006, the Swedish Poisons Information Centre received inquiries about 30 clinical cases of intentional intoxication with A. calamus products. The present investigation aimed to identify alpha- and beta-asarone, considered active components of A. calamus, and metabolites thereof in urine samples collected in seven of these cases. To further aid the identification of asarone biotransformation products, a calamus oil preparation was incubated with the fungus Cunninghamella elegans, which is used as a microbial model of mammalian drug metabolism. Using gas chromatography-mass spectrometry (GC-MS) analysis in selected ion monitoring mode, alpha-asarone was detected in five urine samples at concentrations ranging between approximately 11 and 1150 microg/L and beta-asarone in four of those at approximately 22-220 microg/L. A previously identified asarone metabolite, trans-2,4,5-trimethoxycinnamic acid (trans-TMC), was detected in the fungus broth by liquid chromatography-tandem mass spectrometry whereas cis-TMC was tentatively identified in the human urine samples. Using GC-MS, a hydroxylated asarone metabolite was identified both in fungus broth and urine samples. However, this study demonstrated no evidence for the presence of 2,4,5-trimethoxyamphetamine, claimed as a hallucinogenic component of A. calamus. The main clinical symptom reported by the patients was prolonged vomiting that sometimes lasted more than 15 h.

Study of the metabolism of estragole in humans consuming fennel tea.

The metabolism of the potent carcinogen estragole was investigated in humans after consumption of fennel tea by analyses of its metabolites in blood plasma and urine. Stable isotope dilution assays based on LC-MS/MS detection revealed that 1'-hydroxylation of estragole happened very fast as the concentration of conjugated 1'-hydroxyestragole in urine peaked after 1.5 h, whereas it was no longer detectable after 10 h. Besides the formation of less than 0.41% conjugated 1'-hydroxyestragole of the estragole dose administered, the further metabolite p-allylphenol was generated from estragole in a higher percentage (17%). Both metabolites were also detected in blood plasma in less than 0.75-2.5 h after consumption of fennel tea. In contrast to this, no estragole was present in these samples above its detection limit. From the results, it can be concluded that an excess of the major fennel odorant trans-anethole principally does not interfere with estragole metabolism, whereas influences on the quantitative composition of metabolites cannot be excluded. The presence of a sulfuric acid conjugate of estragole could not be confirmed, possibly due to its high reactivity and lability.

The bioactivity of 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2) and its detection in rat urine by capillary electrophoresis combined with an on-line sample concentration technique.

The bioactivity of 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2) on nitric oxide (NO) production and the proliferation of spleen and thymus lymphocytes to mitogen stimulation in mice are reported for the first time. NO production by T and B lymphocytes in spleen and T cells in the thymus of mice decreased after the oral administration of 2C-T-2. This indicates that 2C-T-2 intake may perturb both neural and immune activity since a decrease in NO production is indicative of a weakened defense function. 2C-T-2 (the parent drug) in rat urine samples was detected by means of capillary electrophoresis/UV absorbance combined with an on-line sample concentration technique. When the CZE and MEKC modes were employed, the detection limit was found to be 4.5 and 5.0 microg/mL (at a 92.1% confidence level); whereas when on-line sample concentration methods, including stacking and sweeping-micellar electrokinetic chromatography were used, the detection limits were improved to 19.2 and 9.1 ng/mL, respectively. In an analysis of some actual samples from animal experiments, three male rats were administered 20 microg/g of body weight of 2C-T-2 by intra-peritoneal injection. The first- and second-day urine fractions were collected after the administration, for use in the analysis. As a result, 2.9 microg/mL and 0.25 microg/mL of 2C-T-2, respectively, were detected after ingestion of the doses.

Identification of 2,5-dimethoxy-4-ethylthiophenethylamine and its metabolites in the urine of rats by gas chromatography-mass spectrometry.

A simple and specific method based on gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS) for the analysis of in vivo metabolism of 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2) in rats is described. Three male rats were administered 20 mg/kg of 2C-T-2 by intra-peritoneal injection, and 24 h urine fractions were collected before and after the administration for analysis. After acidic hydrolysis of the urine samples, the metabolites were liquid-liquid extraction and analyzed by a quadruple mass spectrometer in the selected ion monitoring mode. The findings show that four metabolites of 2-(4-ethylthio-2,5-dimethoxyphenyl)-ethanol (Mw: 242), 4-ethylthio-2,5-dimethoxyphenyl acetic acid (Mw: 256), 1-acetoamino-2-(2-hydroxy-4-ethylthio-5-methoxyphenyl)-ethane (Mw: 269) and 1-acetoamino-2-(2-methoxy-4-ethylthio-5-hydroxyphenyl)-ethane (Mw: 269) are present and the metabolic pathway for 2C-T-2 in the rat is proposed.

Automated quantitative determination of the new renin inhibitor CGP 60536 by high-performance liquid chromatography.

A fully automated high-performance liquid chromatography method with fluorescence detection for the determination of the renin inhibitor CGP 60536 in animal and human plasma and urine has been developed and validated. After addition of an internal standard, the compounds were automatically extracted from 400 microl of plasma or urine with methyl alcohol-acetic acid (99:1, v/v) on 100-mg Bond-Elut CN cartridges using the Gilson ASPEC system. The on-line chromatographic separation was performed on a LiChrospher 100 RP8 5-microm particle size packed analytical column (25x0.4 cm I.D.). The mobile phase consisted of acetonitrile-0.01 M potassium dihydrogenphosphate (65:35, v/v) at a flow-rate of 0.8 ml/min. The analytes were detected using a fluorescence detector at excitation and emission wavelengths of 280 and 330 nm, respectively. The limit of quantitation was established at 4.5 ng/ml in plasma (accuracy 106% and precision 1%), and 9.0 ng/ml in urine (accuracy 101% and precision 13%). The method was applied to the investigation of the pharmacokinetics of CGP 60536.

Pathways of metabolism of [1'-14C]-trans-anethole in the rat and mouse.

This study describes the metabolic fate of trans-4'-methoxyprop-[1-14C]enylbenzene, the natural flavor compound trans-anethole, in rats and mice given single doses of 250 mg/kg body weight. In both rats and mice, an essentially quantitative (> 95% of dose) recovery of 14C was obtained with the majority in the 0-24 hr urine. Separation and identification of 18 urinary anethole metabolites were achieved by radio-HPLC, chemical derivatization, and GC/ MS. Anethole undergoes three primary oxidation pathways-O-demethylation, omega-side chain oxidation, and side chain epoxidation-followed by a variety of secondary pathways of oxidation and hydration, the products of which are extensively conjugated with sulfate, glucuronic acid, glycine, and glutathione. A novel major metabolite has been characterized in the rat, apparently originating from conjugation of the epoxide with glutathione, namely S-[1-(4'-methoxyphenyl)-2-hydroxypropane]-N-acetylcysteine. These metabolites are discussed in terms of the pathways responsible for and the toxicological consequences of their formation.

Distribution, metabolism and excretion of pentachloroanisole in the beagle dog and miniature pig.

Tissue distribution, excretion and metabolism studies of pentachloroanisole (PCA), an environmental metabolite of pentachlorophenol (PCP), were conducted in the beagle dog and miniature pig following single oral doses (25 mg/kg) of radiolabelled PCA. PCA was readily demethylated by both species, with a half-life of 5-8 min. The resultant PCP was the major metabolite in dogs and pigs. In the dog, an average of 21.9% of the administered radiolabel was excreted in the urine and 62.3% in the faeces during a 7-day period. Of the tissues analysed, an average of 3.2% of the radiolabel remained in the liver, and blood and muscle accounted for averages of 3.0 and 2.3%, respectively, of the dose. Free and conjugated PCP were found in the urine of dogs; no PCA or tetrachlorohydroquinone (TCH) were found. In dog faeces, PCP and a trace of polar material were observed; no PCA was excreted in dog faeces. In the miniature pig, an average of 25.8% of the administered radiolabel was excreted in the urine and 32.0% in the faeces during a 2-wk period. An average of 4.4% of the radiolabel was found in the liver, 8.8% in the blood, 7.1% in the muscle and 6.4% in the fat. In pig urine, PCP and conjugated PCP were the only metabolites observed; no PCA or TCH was found. Pig faeces contained a trace of unchanged PCA; PCP and polar metabolites were also found. Since pig tissues retained a sizeable residue 2 wk after a single dose of PCA, various agents were used in an attempt to decrease the tissue level of radiolabel in pigs; anion exchange resin was found to be the most effective.

Stereochemical aspects of the hydration of trans-anethole epoxide in the rat.

Racemic trans-anethole epoxide [1-(4'-methoxyphenyl)-propane-1,2-oxide] was incubated with water, buffers, and rat liver microsomes and cytosol and the stereochemistry of the diols produced was determined by HPLC as their dicamphanyl esters. The diol metabolites were isolated by HPLC from the urine of rats administered [1'-14C] trans-anethole and their stereochemistry determined after derivatization to their camphanyl esters. The stereochemical course of the metabolism of trans-anethole by rat liver microsomes and cytosol is discussed.

Identification of 4-methoxybenzoyl-N-glycine in urine by gas chromatography/mass spectrometry.

An unusual metabolite was detected in the urine of two children with neurological dysfunctions of unclear aetiology by using gas chromatography/mass spectrometry (GC/MS). On the basis of the analysis of its fragmentation pathways, synthesis of tentative compound and its GC/MS analysis it was stated that the unknown metabolite is 4-methoxybenzoyl-N-glycine.

Metabolism of 4-hydroxyanisole: identification of major urinary excretory products.

The human metabolism of 4-hydroxyanisole was investigated by the analysis of urine samples from melanoma patients treated with this substance. The samples were hydrolyzed with glucoronidase and/or arylsulphatase, extracted with ethyl acetate, and, after derivatization with pentafluoropropionylanhydride, analyzed by gas chromatography-mass spectrometry. We were able to identify peaks by their retention times and mass spectra corresponding to 4-hydroxyanisole, 3,4-dihydroxyanisole, and two of its o-methyl derivatives, namely, 3-hydroxy-4-methoxy- and 4-hydroxy-3-methoxyanisole. We also detected a peak of hydroquinone, which may have originated (at least partly) from 4-hydroxyanisole. All the above-mentioned compounds were excreted predominantly as sulphates and glucuronides. Only a small proportion of the substances was present in urine in an unconjugated form. Our results demonstrate that 3,4-dihydroxyanisole is the most important metabolite of 4-hydroxyanisole.

Influence of dose size on the disposition of trans-[methoxy-14C]anethole in human volunteers.

The influence of dose size on the metabolic fate of the naturally occurring food flavouring trans-anethole has been investigated in human volunteers, using the [methoxy-14C]-labelled compound. The doses chosen were: 1 mg; close to the daily intake in the diet from foods, 50 mg; approximating to the amount present in a normal measure of an anise-flavoured beverage, and 250 mg. The order of administration was randomized. The major routes of elimination of 14C were in the urine (54-69% of the administered dose) and as exhaled 14CO2 (13-17%). Dose size had no systematic effect on either rate or route of excretion. Urinary metabolites were separated by high-pressure liquid chromatography, before and after treatment with beta-glucuronidase, and identified by comparison of their chromatographic mobilities with those of authentic standards. The principal metabolite (greater than 90% of urinary 14C) was 4-methoxyhippuric acid, accompanied by much smaller amounts of 4-methoxybenzoic acid and up to three other compounds, which were not examined further. The pattern of urinary metabolites was unaffected by dose size. These data are discussed with reference to the comparative metabolic disposition of trans-anethole in rats and mice, the species commonly used in toxicity testing and in which its fate exhibits a very marked dose dependence.