Serum and urine concentrations of flunitrazepam and metabolites, after a single oral dose, by immunoassay and GC-MS.

A clinical study was conducted to assess the ability of commercially available immunoassays to detect flunitrazepam (FNP) in plasma and urine samples and to compare the results with those obtained by gas chromatography-mass spectrometry (GC-MS). The clinical study consisted of four individuals (two male and two female) who had taken a single 2-mg dose of FNP. Serum was collected over a 48-h period and urine was collected over a 72-h period. The serum and urine samples were analyzed by the COBAS INTEGRA Serum Benzodiazepines assay (SBENZ), the TDx serum and urine Benzodiazepines assay, and GC-MS. The GC-MS procedure was developed for analysis of FNP and metabolites in plasma and urine using an acid hydrolysis step resulting in the formation of specific benzophenones corresponding to FNP and its metabolites. The relative sensitivities of the assays for the detection of FNP and metabolites in serum and urine were GC-MS > SBENZ > TDx. The immunoassay results for serum samples showed peak concentrations of FNP metabolites at 8 h after FNP ingestion for three individuals and at about 1 h for the fourth individual. The GC-MS, SBENZ, and TDx urine immunoassays detected drug above the stated limit of detection (LOD) in 44, 41, and 35 serial FNP urine samples, respectively. FNP metabolites were detected in urine samples with all three assays for up to 72 h after a 2-mg dose. The improved detection rate with the SBENZ assay as compared to the TDx assay is likely explained by its higher cross-reactivity with the major metabolite, 7-amino-flunitrazepam (7-amino-FNP), and its lower LOD.     

Urinary excretion kinetics of 1-hydroxypyrene in rats subchronically exposed to pyrene or polycyclic aromatic hydrocarbon mixtures

The urinary excretion kinetics of 1-hydroxypyrene (1-OHP) were studied in male Sprague-Dawley rats exposed orally, on Tuesdays and Fridays for 10 consecutive weeks, to 0.046 mg/kg/d of pyrene or 0.3, 1 or 3 mg/kg/d of polycyclic aromatic hydrocarbon (PAH) mixtures containing pyrene (0.046, 0.15, and 0.46 mg/kg/d, respectively). During the subchronic exposure, 24-h urine samples were collected on Mondays (prior to exposure) and Tuesdays (after exposure) for all exposure groups. During a 14-d period following last exposure in rats treated with 3 mg/kg/d of PAH mixture, 24-h urine samples were collected at frequent time intervals (0-24, 48-72, 96-120, 144-168, 193-217, 313-338 h). Whatever the administered dose, repeated exposures to pyrene and PAH mixtures resulted in a progressive time-dependent increase in the daily urinary excretion of 1-OHP. After 10 wk of treatment, daily excretion rates were on average 5 times higher than those observed after the first administration. Following the subchronic exposure to 3 mg/kg/d of PAH mixture, the time profile of 1-OHP excretion in rat urine showed a multiphasic elimination. An average first-order apparent elimination half-life of 26.5 h could be estimated for the 48-168 h period following the end of the exposure. The observed time-dependent increase in 1-OHP excretion values upon repeated exposures to PAHs does not appear to result from a PAH enzyme induction effect of pyrene metabolism to 1-OHP. Rather, the slow release of residual pyrene accumulated in a long-term compartment and/or the enterohepatic recirculation of 1-OHP and other pyrene metabolites may play significant roles in the observed urinary excretion kinetics of 1-OHP. Furthermore, the absence of mixture effect on the urinary excretion of 1-OHP suggests that 1-OHP is a good bioindicator of exposure to complex PAH mixtures, in the dose range used in this study.     

Sweat testing of MDMA with the Drugwipe analytical device: a controlled study with two volunteers

Rapid on-site tests for the analysis of drugs of abuse in unconventional specimens (e.g., sweat) have recently been developed. Two healthy volunteers familiar with the effects of methylenedioxymethamphetamine (MDMA) were given 100 mg of the drug as a single oral dose. MDMA and its main metabolite 4-hydroxy-3-methoxymethamphetamine (HMMA) were determined in plasma and urine by gas chromatography-mass spectrometry (GC-MS). MDMA was also investigated in sweat with the Drugwipe (an immunochemical strip test). Subjects' armpits were swabbed for 10 s at 0 time (predose) and at 2, 6, 8, 12, and 24 h after MDMA administration. MDMA consumption could be detected using Drugwipe at 2 h and for as long as 12 h after drug administration. However, in one of the volunteers, a faint color change appeared at 0 time, when plasma and urine tested negative for MDMA and did not disappear even 48 h later. Plasma concentrations of MDMA and HMMA measured by GC-MS peaked at 2-4 h, and values greater than 20 ng/mL for MDMA and of 40 ng/mL for HMMA were still detected at 24 h. Urine tested positive by GC-MS for MDMA and HMMA in the 48-h collection period. These findings preliminarily support sweat testing with Drugwipe for monitoring MDMA use.     

Absolute bioavailability of tolvaptan and determination of minimally effective concentrations in healthy subjects

Tolvaptan is a selective vasopressin V2 receptor antagonist that can be given orally once daily for treatment of clinically significant hypervolemic and euvolemic hyponatremia (US) or cardiac edema (Japan). Tolvaptan absolute bioavailability was determined in a single-center, open-label, sequential administration trial in which intravenous (i.v.) placebo (Day -2), i.v. 1 mg tolvaptan (Day 1) and an oral 30 mg tablet (Day 8) were administered to 14 healthy subjects. Urine volume and osmolality were determined on Days -2, 1 and 8 at multiple intervals postdose; 24-h fluid balance was also assessed. On Days 1 and 8, blood samples for tolvaptan were collected for 48 h postdose. Mean absolute bioavailability was determined to be 56% (range 42 - 80). Mean peak tolvaptan concentration at 1 h (end-of-infusion) was 32.7 (range 18 - 45) ng/ml compared to 231 (range 87 - 410) ng/ml for the oral dose. In the 4-h period from start of the 1 mg tolvaptan i.v. infusion, 12 of 14 subjects experienced increased urine volume and decreased urine osmolality; both parameters were affected for 24 h postdose following the 30 mg oral dose. Minimally effective concentrations are rapidly achieved after oral dosing as all subjects had tolvaptan concentrations > 20 ng/ml at 1 h postdose.     

8-Oxo-7, 8-dihydro-2'-deoxyguanosine as a biomarker of DNA damage by mobile phone radiation

We examined the effect of exposure to mobile phone 1800?MHz radio frequency radiation (RFR) upon the urinary excretion of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG), one major form of oxidative DNA damage, in adult male Sprague-Dawley rats. Twenty-four rats were used in three independent experiments (RFR exposed and control, 12 rats, each). The animals were exposed to RFR for 2?h from Global System for Mobile Communications (GSM) signal generator with whole-body-specific absorption rate of 1.0?W/kg. Urine samples were collected from the rat while housed in a metabolic cage during the exposure period over a 4-h period at 0.5, 1.0, 2.0 and 4.0?h from the beginning of exposure. In the control group, the signal generator was left in the turn-off position. The creatinine-standardized concentrations of 8-oxodG were measured. With the exception of the urine collected in the last half an hour of exposure, significant elevations were noticed in the levels of 8-oxodG in urine samples from rats exposed to RFR when compared to control animals. Significant differences were seen overall across time points of urine collection with a maximum at 1?h after exposure, suggesting repair of the DNA lesions leading to 8-oxodG formation.     

GHB urine concentrations after single-dose administration in humans

Gamma-hydroxybutyric acid (GHB) is used as an illicit drug and is implicated in drug-facilitated sexual assault, but it also has some therapeutic uses. Detection of GHB in urine is important for forensic testing and could be of clinical benefit in overdose management. Urine GHB concentration-time profiles have not been well-characterized or correlated with doses used therapeutically. GHB levels were measured by gas chromatography-mass spectrometry in urine collected over 24 h from 16 adults administered single doses of 50 mg/kg GHB (Xyrem) alone and combined with 0.6 g/kg ethanol. Peak GHB urine concentrations averaged 150-200 mg/L and occurred in the 0-3 h urine collection. Significant variability in GHB urine levels between individuals was observed. Caucasians had lower urine concentrations than other races/ethnicities (p = 0.03). Men had lower GHB levels than women in the first 3 h after dosing (p = 0.038). Coingestion of ethanol did not significantly affect renal clearance of GHB, but urine GHB concentrations were lower in the first 3 h when ethanol and GHB were coingested (p = 0.039). At a proposed cut-off of 10 mg/L to distinguish endogenous versus exogenous GHB levels, 12.5% of the samples collected from 3 to 6 h, 81.3% of samples collected from 6 to 12 h, and 100% of urine specimens collected from 12 to 24 h were below this level. We conclude that the detection time for GHB in urine may be shorter than the previously reported 12-h window in some people taking therapeutic doses of GHB.     

Contamination of rat urine with gut flora using all-glass metabolism cages for collection of urine and faeces.

In rat urine collected in all-glass metabolism cages, at least four strains of intestinal microflora were found: two types of E. coli, Enterobacter cloaceae and Proteus vulgaris. The number of bacteria of each strain increased with time. 2. The pH of the urine increased from 6-9 after 24 h to 8-95 after 120 h. The pH of the urine of neomycin-treated rats remained nearly constant over a period of two days. 3. Nitroreductase activity was present in the rat urine. Added p-nitrobenzoic acid was reduced within the first 24 h. Nitroreductase activity in the urine of neomycin-treated rats was significantly lower than in the urine of normal rats, during the second 24-h period only. 4. Collection of urine at -10 degrees prevented the consequences of contamination.     

Disposition of 2-(2-quinolyl)-1,3-indandione (D. C. yellow #11) in rats dosed orally or intravenously

The disposition of 2-(2-quinolyl)-1,3-indandione (D. C. yellow #11, DCY) in male Fischer rats dosed intravenously or by feeding was determined. For rats given [14C]DCY in the feed (0.00044-0.41% of the diet), recovery of radioactivity during the 24-h dosing period and the 72-h period thereafter ranged from 89.1 to 93.9% for feces and from 4.98 to 6.25 for urine. Tissues contained only trace amounts. Following intravenous dosing with [14C]DCY (0.93 mg/kg), radioactivity distributed readily into most tissues; maximum amounts were present at 5 min, the earliest time of assay. Maximum amounts of radioactivity in fat, skin, and gut tissue, however, were present at 30 min after dosing. These three tissues also had relatively long alpha phases for the elimination of radioactivity. In 24 h after intravenous dosing, rats excreted 81.1% of the dose in the feces and 16.0% of the dose in the urine. For rats fitted with biliary cannulas, 54.5% of the dose, all of which was metabolites of [14C]DCY, was recovered in the bile in 4 h. Associated with the rapid and extensive biliary excretion of metabolites of intravenously administered [14C]DCY was the appearance of large amounts of radioactivity in the feces and also, at intermediate time points, in the liver, gut contents, and gut tissue. In conclusion, rats rapidly distribute, metabolize, and excrete [14C]DCY.     

LC-MS/MS及离子簇技术分析鉴定盐酸戊乙奎醚外消旋体在大鼠体内的代谢产物

目的研究盐酸戊乙奎醚外消旋体在大鼠体内的代谢产物。方法健康大鼠同时等量im盐酸戊乙奎醚外消旋体及其氘标盐酸戊乙奎醚,收集尿样并处理。用LC/MS/MS,GC-MS,FAB-MS及氘标离子簇示踪技术分析鉴定盐酸戊乙奎醚在大鼠体内的代谢产物。结果共检测到8个代谢产物,分别为原形环戊基上的单氧化产物(M1与M1^*)、原形环戊基上的单羟基化产物(M2与M2^*)、原形环戊基间位上的氧化羟基化产物(M3与M3^*)及原形环戊基与奎宁环上的羟基化产物(M4与M4^*)。其中,M1与M1^*,M2与M2^*,M3与M3^*及M4与M4^*互为异构体。结论 此法为临床合理用药及新抗胆碱能手性药物研究提供有价值的信息。     

Metabolic study of new psychoactive substance methoxpropamine in mice by UHPLC-QTOF-HRMS

Methoxpropamine (MXPr) is an arylcyclohexylamine dissociative drug structurally similar to 3-methoxyeticyclidine, ketamine, and deschloroketamine, recently appeared in the European illegal market, and was classified within the new psychoactive substances (NPS). Our study investigated the metabolism of MXPr to elucidate the distribution of the parent drug and its metabolites in body fluids and fur of 16 mice. After the intraperitoneal administration of MXPr (1, 3, and 10 mg/kg), urine samples from eight male and eight female mice were collected every hour for six consecutive hours and then at 12- to 24-h intervals. Additionally, plasma samples were collected 24 h after MXPr (1 and 3 mg/kg) administration. Urine and plasma were diluted 1:3 with acetonitrile/methanol (95:5) and directly injected into the UHPLC-QTOF-HRMS system. The phase-I and phase-II metabolites were preliminarily identified by means of the fragmentation patterns and the exact masses of both their precursor and fragment ions. Lastly, the mice fur was analyzed following an extraction procedure specific for the keratin matrix. Desmethyl-MXPr-glucoronide was identified in urine as the main metabolite, detected up to 24 h after administration. The presence of norMXPr in urine, plasma, and fur was also relevant, following a N-dealkylation process of the parent drug. Other metabolites that were identified in fur and plasma included desmethyl-MXPr and dihydro-MXPr. Knowledge of the MXPr metabolites evolution is likely to support their introduction as target compounds in NPS toxicological screening analysis on real samples, both to confirm intake and extend the detection window of the dissociative drug MXPr in the biological matrices.     

Effects of age on serum tryptophan and urine indican in adults given a tryptophan load test

An oral load of L-tryptophan (490 mumol/kg) was administered to 25 men and 25 women between the ages of 30 and 80 years. Blood samples were drawn before the load and at 2-h intervals for 6 h after the load. Urine samples were collected for 5 days. Fasting serum tryptophan levels averaged 79.5 mumol/l (+/- 12.9 s.d.). Peak serum tryptophan levels (911-1002 mumol/l) occurred 2 h after the load. Urinary indican excretion on the day of the load averaged 6.4 mumol/24h/kg of body weight (+/- 2.8 s.d.). The concentration of tryptophan in serum, the amount of indican excreted in urine, and the indican:creatinine ratio in urine depended on the age of the subjects. The findings are discussed in relation to previous reports on effects of age on tryptophan pharmacokinetics.     

三聚氰胺污染饲料致大鼠肾毒性的代谢组学研究

目的探讨三聚氰胺污染饲料致大鼠尿液和肾脏组织内源性代谢产物的变化,寻找三聚氰胺污染饲料致大鼠肾毒性的差异代谢物。方法大鼠喂食三聚氰胺污染饲料20 d后,通过光学显微镜观察大鼠肾脏组织病理变化,并采用气相色谱-质谱联用(GC-MS)分析手段对正常对照组和三聚氰胺组大鼠尿液和肾脏组织的代谢物进行分析。利用代谢组学分析软件SIMCA-P+11对大鼠尿液和肾脏组织采集的GC-MS数据进行多元统计分析。结果与正常对照组相比,三聚氰胺组大鼠肾脏组织部分纤维化,有大量尿酸盐结晶,可见颗粒管形病变。正常对照组和三聚氰胺组大鼠尿液和肾脏组织样品在主成分分析(PCA)中得到了很好的分离,并通过偏最小二乘判别分析(PLS-DA)指认引起差异的特征代谢物。与正常对照组相比,三聚氰胺组大鼠肾脏组织中丙氨酸显著上升;缬氨酸和乙醇胺等明显下降;尿液中乳酸显著上升,而半胱氨酸和尿嘧啶等明显下降。结论三聚氰胺污染饲料引起大鼠明显的肾毒性,尿液中有17种物质,肾脏组织中有11种物质被判定为可能特征代谢物。     

Analysis of MDMA and its metabolites in urine and plasma following a neurotoxic dose of MDMA

3,4-Methylenedioxymethamphetamine (MDMA), a commonly encountered drug of abuse, has been shown in a variety of studies to cause neurotoxic effects. Because MDMA itself is not neurotoxic, identifying the potential neurotoxic metabolite(s) was of significant importance. Evaluation of urine and plasma concentrations of MDMA and three of its main metabolites, 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxyamphetamine (HMA), and 4-hydroxy-3-methoxymethamphetamine (HMMA), following administration of a neurotoxic dose (20 mg/kg) to male Dark Agouti rats was accomplished. Currently there are no data available describing urine and plasma concentrations of MDMA and these metabolites over a period of 7 days. The rats received a single 20 mg/kg i.p. dose of MDMA. Blood and urine samples were collected prior to administration and at 2, 4, 8, 12, 16, 20, 24, 48, 96, and 168 h following drug administration. Plasma and urine samples were extracted using solid-phase extraction, derivatized with N-methyl-bis(trifluoroacetamide), then analyzed using gas chromatography-mass spectrometry. Urine samples showed peak concentrations of MDMA at 4 h, MDA at 8 h, HMMA at 12 h, and HMA at 16 h post dose. MDMA and its metabolites were detectable (limit of detection 25 ng/mL) in the urine for up to 168 h post dose. Plasma samples showed mean peak concentrations of MDMA and MDA at 2 h post dose and HMMA at 4 h. Although the highest mean concentration of HMA was seen at 24 h post dose, variability between sample results for this time point was significant. No detectable levels of MDMA, MDA, HMA, and HMMA (LOD 10 ng/mL) were found in plasma at 96 and 168 h post dose.     

Contamination of rat urine with gut flora using all-glass metabolism cages for collection of urine and faeces

1. In rat urine collected in all-glass metabolism cages, at least four strains of intestinal microflora were found: two types of E. coli, Enterobacter cloaceae and Proteus vulgaris. The number of bacteria of each strain increased with time.

2. The pH of the urine increased from 6·9 after 24?h to 8·95 after 120 h. The pH of the urine of neomycin-treated rats remained nearly constant over a period of two days.

3. Nitroreductase activity was present in the rat urine. Added p-nitro-benzoic acid was reduced within the first 24 h. Nitroreductase activity in the urine of neomycin-treated rats was significantly lower than in the urine of normal rats, during the second 24-h period only.

4. Collection of urine at -10° prevented the consequences of contamination.     

The effect of the use of mouthwash on ethylglucuronide concentrations in urine

Two studies were performed to evaluate the effect of alcohol containing mouthwash on the appearance of ethyl glucuronide (EtG) in urine. In the first study, 9 volunteers were given a 4-oz bottle of mouthwash, which contained 12% ethanol. They gargled with all 4 oz. of the mouthwash at intervals over a 15-min period. All urine samples were collected over the next 24 h. Of 39 provided urine samples, there were 20 > 50 ng/mL, 12 > 100 ng/mL, 5 > 200 ng/mL, 3 > 250 ng/mL, and 1 > 300 ng/mL. The peak concentrations were all within 12 h after the exposure. In the second study, 11 participants gargled 3 times daily for 5 days. The first morning void was collected. Sixteen of the 55 submitted samples contained EtG concentrations of greater than 50 ng/mL. All of them were less than 120 ng/mL. These studies show that incidental exposure to mouthwash containing 12% ethanol, when gargling according to the manufacturer's instructions, can result in urinary EtG values greater than 50 ng/mL. All specimens were negative for ethanol. The limits of detection and quantitation for the EtG testing were 50 ng/mL.     

普罗帕酮在中国健康受试者体内的羟基化代谢产物研究

目的:阐明普罗帕酮羟基化代谢过程的种属及种族差异。方法:选择10 名中国健康受试者单剂量po300 mg 盐酸普罗帕酮片,收集0 ～12 h 的尿样,经液 液萃取后,采用LC/ MSⁿ 技术,对羟基化代谢产物进行选择性离子监测(m/z 358) 和多级全扫描质谱分析。结果:在服药后的尿样中检测到两种羟基化代谢产物,根据质谱数据,推测这两种代谢产物分别为4′-羟基普罗帕酮和5-羟基普罗帕酮。采用微生物转化法结合半制备HPLC制备并分离了4′-羟基普罗帕酮的对照品,通过NMR 证实了其结构。结论:在10 名受试者服药后的尿样中均能检测到代谢物4′ 羟基普罗帕酮和5 羟基普罗帕酮,与文献报道的白人受试者代谢结果相比,中国受试者有较宽的羟基化代谢谱。     

Novel device for quantitatively collecting small volumes of urine from laboratory rats

A reusable urinary collection device suitable for quantitative collection of uncontaminated urinary samples from rats without using a metabolism cage has been designed. The device can be attached to the pelvic skin within 5 min with minimum handling and discomfort to the unanesthetized rat using an adhesive. The suitability of this device was investigated by collecting and analyzing urine over a 48-h period following the intraperitoneal injection of [14C]inulin. A two-way crossover study was done with samples from one of the two experiments being collected while the animal was housed in a commercially available metabolism cage equipped to separate urine and feces. The percent of the dose excreted, food and water consumption, and the urinary output were not statistically different for rats housed in the metabolism cages or with the collection device attached. Histopathological examination of the rats revealed no pathology after a 24-h period except minor skin inflammation which occurred both in controls and animals to which the device was attached. These studies demonstrate the comparability of the new urine collection device to a commercially available metabolism cage in the quantitative collection of small (0-7 mL) urine samples with less contamination of the samples from the environment.     

Metabolism and disposition of ortho-benzyl-para-chlorophenol in male rats

The metabolism and disposition of ortho-benzyl-para-chlorophenol (BCP) has been investigated in the male rat following an oral dose of 69 mg/kg or 206 mg/kg. BCP was rapidly eliminated at both dose levels with 45-49% of the dose appearing in the urine and 44-49% in the feces during the 5-d period after dosing. After 5 d only 0.28-0.3% of the dose remained in the body, with almost half this value accounted for in the liver and kidney. The dynamics for the overall elimination of radioactivity from the body was biphasic at both dose levels. The initial rapid alpha phase had a well defined half-life of 8-9 h, and the slower beta phase had an estimated half-life of approximately 52-140 h. Analysis of the 12-24-h urine indicated that a majority of the radioactivity (41-61%) was present as sulfate and/or glucuronide conjugates. Treatment with purified aryl sulfatase suggested that sulfate esters were the predominate conjugate. Gas chromatography-mass spectroscopy (GC/MS) of the products isolated after enzymatic hydrolysis of the conjugates and purification by thin-layer chromatography (TLC) identified BCP, as well as two metabolites in which the benzyl ring was modified. One metabolite contained a hydroxyl substituent on the benzyl ring, and the other contained a hydroxyl and a methoxyl substituent. Preliminary analysis of the 12-24-h feces demonstrated the presence of BCP and two other components with chromatographic properties identical to the metabolites identified in the urine. A metabolic pathway for BCP has been proposed to account for the observed metabolites.     

An animal model for the study of chronopharmacokinetics of drugs and application to methotrexate and vinorelbine

An animal model was designed to study the chronopharmacokinetics of intravenous drugs and applied to anticancer agents vinorelbine (VNB) and methotrexate (MTX). Each experiment was performed on four pigs housed in a standardized light-dark cycle (12:12). Four pigs received a 0.16-mg bolus of VNB, followed by a 60-h continuous infusion at 0.48 mg/h. After hydration and urine alkalinization, four other pigs received a 2 mg/kg bolus of MTX, followed by two concomitant 60-h continuous infusions, one with MTX (8 mg/kg/h) and the other for hydration and folinic acid rescue (1.5 mg/kg/24 h). Serum cortisol was determined in each blood sample collected in these pigs. Blood samples were collected each hour for 60 h. The infusion flow rates and drug solution concentrations were controlled throughout the experiments. Analysis of VNB serum concentrations did not show any circadian rhythm of VNB serum concentrations. One pig administered MTX exhibited severe toxicity. Interestingly, no circadian rhythm of serum cortisol concentration was observed in this pig, whereas the three others exhibited a statistically significant cortisol circadian rhythm with a peak secretion in the morning. Two of these three pigs showed a significant 24-h rhythm of MTX with acrophase occurring at approximately 1:00 PM in both. The maximal concentration was found at 12:00 AM in the third pig. After the data were pooled, a highly significant (P < 0.01) circadian rhythm in MTX serum concentrations (57%) was found, with acrophase at midday. The pig represents a useful model for the study of chronopharmacokinetics of drugs given intravenously in human. The MTX chronokinetic variation found herein may be of interest for the improvement of chemotherapy in cancer patients.     

Effects of ellagic acid by oral administration on distribution and metabolism of 2-aminofluorene in Sprague-Dawley rats

The effects of ellagic acid on the in vivo N-acetylation and metabolism of 2-aminofluorene (2-AF) were investigated in bladder, blood, colon, kidney, liver, feces and urine samples from male Sprague-Dawley rats. Major metabolites such as 1-OH-2-AAF, 8-OH-2-AAF and 9-OH-2-AAF were found in bladder tissues, 1-OH-2-AAF, 5-OH-2-AAF and 8-OH-2-AAF were found in blood samples, 1-OH-2-AAF, 3-OH-2-AAF, 5-OH-2-AAF, 8-OH-2-AAF and 9-OH-2-AAF were found in colon tissues, 1-OH-2-AAF, 3-OH-2-AAF and 9-OH-2-AAF were found in kidney tissues, 1-OH-2-AAF, 3-OH-2-AAF and 8-OH-2-AAF were found in liver tissues, 1-OH-2-AAF, 3-OH-2-AAF, 5-OH-2-AAF and 8-OH-2-AAF were found in feces samples and 1-OH-2-AAF, 3-OH-2-AAF, 5-OH-2-AAF and 8-OH-2-AAF were also found in urine samples after rats had been orally treated with 2-AF (50 mg/kg) for 24 h. Pretreatment of male rats with ellagic acid (10 mg/kg) 24 h prior to the administration of 2-AF (50 mg/kg) resulted in absence of 8-OH-2-AAF in bladder tissues, and there were significant decreases of 8-OH-2-AAF in blood and urine samples. In blood samples, amounts of 2-AAF and 8-OH-2-AAF were significantly decreased; in colon tissues, amounts of 2-AF, 1-OH-2-AAF and 3-OH-2-AAF, in liver tissues, amounts of 2-AAF, 1-OH-2-AAF and 3-OH-2-AAF, and in urine samples, amounts of 2-AF and 8-OH-2-AAF were significantly decreased in 24-h ellagic acid (EA)-treated rats before 2-AF was added to the diet. However, significantly increased 1-OH-2-AAF concentrations were found in urine samples in 24-h EA-treated rats before 2-AF was administered. In the EA and 2-AF rats, in the same time treated groups, bladder, colon and liver tissues, and feces and urine samples showed significant differences when compared to the ones without EA co-treatment. We saw significant decreases of the amounts of 2-AF and 1-OH-2-AAF in colon tissues. The feces samples showed increased amounts of 2-AAF in EA- and in 2-AF- treated rats in the same time groups, but urine samples showed a decreased amount of 8-OH-2-AAF in both EA-treated groups. The total amounts of 2-AF metabolites in bladder, blood, kidney and liver tissues showed significant difference between control and the group which was EA-treated 24 h before 2-AF was added. The total amounts of 2-AF metabolites in the liver, feces and urine showed significant decreases between control and EA-treated at the same time with 2-AF groups. This is the first report of EA affecting the N-acetylation and metabolism of 2-AF in rat tissues in vivo.