Identification of phase-I and phase-II metabolites of fluphenazine in rat bile. Intact glucuronide and sulfate conjugates.

In rat bile following ip administration of fluphenazine (FLU) dihydrochloride (20 mg/kg body weight), phase-I metabolites 7-hydroxyfluphenazine (7-HOFLU) and FLU sulfoxide (FLUSO), together with unmetabolized FLU, were isolated and identified by HPLC and fast atom bombardment spectrometry (FAB/MS) and also by comparison with authentic compounds. Two intact glucuronide conjugates of FLU were isolated and identified as phase-II metabolites: 7-hydroxyfluphenazine ring glucuronide (glucuronic acid linked to the aromatic hydroxyl group of 7-hydroxyfluphenazine), and FLU glucuronide (glucuronide linked to the aliphatic group of the side chain of FLU) by HPLC and FAB/MS in comparison with authentic compounds. Further confirmed by FAB/MS were several sulfate conjugates of FLU that were isolated and identified indirectly as phase-II sulfate metabolites: FLU sulfate, 7-hydroxyfluphenazine sulfate and/or 7-hydroxyfluphenazine ring sulfate, and FLU sulfoxide sulfate by HPLC and FAB/MS; their aglycones were identified after sulfatase hydrolysis as FLU, 7-HOFLU and FLUSO. A further phase-II metabolite, for which no authentic standard was available, was tentatively identified as a monoglucuronide of dihydroxy derivative of FLU. To our knowledge, this report provides the first direct evidence of the presence of intact phase-II metabolites of FLU in rat bile.     

Identification of major metabolites of the catechol-O-methyltransferase-inhibitor nitecapone in human urine.

Metabolites of nitecapone [3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione], a potent new catechol-O-methytransferase-inhibitor, were isolated from human urine both after hydrolysis with beta-glucuronidase and as intact conjugates. Seven phase-I metabolites and corresponding glucuronides were identified using electron ionization and fast atom bombardment mass spectrometry, IR spectroscopy, and proton NMR spectrometry. The most abundant metabolite in urine was the glucuronide of unchanged nitecapone, representing 60-65% of the metabolites found. The main phase-I metabolic reaction was reduction of the side chain double bond and carbonyl groups. One of the major metabolites was formed by cleavage of the side chain by retro aldol condensation. All phase-I metabolites were present mainly as their glucuronic acid conjugates. The 3-nitrocatechol-structure of nitecapone seems to hinder nitro-reduction, catechol-O-methylation, and sulfation reactions.     

Studies on the metabolism of propafenone. 3rd Comm.: Isolation of the conjugated metabolites in the dog and identification using fast atom bombardment mass spectrometry

The metabolism and urinary and biliary excretion of propafenone (2-(2'-hydroxy-3'-propylamino-propoxy)-omega-phenyl-propiophenone hydrochloride) were studied in the dog. Approximately 20% of the dose was excreted in 24 h with the urine and about 65% with the bile. Propafenone was extensively metabolized. Less than 4% of the dose was recovered unchanged in urine and bile. The metabolites were mainly excreted as conjugates. Free metabolites accounted for less than 20% of the dose. Separation methods were developed to isolate and purify the conjugated metabolites. Fractionation on an Al2O3-column yielded a sulphate and a glucuronide fraction, further separation and purification was done by TLC and HPLC. Positive and negative ion fast atom bombardment mass spectra (FAB/MS) were obtained of the purified glucuronides. The structures of two hydroxylated propafenone derivatives and two O-methylated catechol-like derivatives were definitely proven by FAB/MS, the glucuronic acid moiety being conjugated to the hydroxyl function in the different aromatic rings. Two isomeric propafenone glucuronides were found in the bile, probably diastereomeric forms of the O-glucuronide. Thus FAB/MS proved to be a complementary method to electron impact ionization mass spectrometry (EI/MS) for studying drug metabolism. The structures of free and conjugated metabolites can be defined from the combination of both mass spectrometric techniques.     

Taurine conjugates as metabolites of arylacetic acids in the ferret.

1. The pattern of conjugation in the ferret of 8 arylacetic acids and, for comparison, benzoic acid and 4-nitrobenzoic acid was examined. 2. The arylacetic acids, phenylacetic, 4-chloro- and 4-nitro phenylacetic, alpha-methylphenylacetic (hydratropic), 1- and 2-naphthylacetic and indol-3-ylacetic acids, were excreted in the urine as taurine and glycine conjugates. Diphenylacetic acid did not form an amino acid conjugate and was excreted as a glucuronide. 3. The taurine conjugate was the major metabolite of 4-nitrophenylacetic, alpha-methylphenylacetic, 1- and 2-naphthylacetic and indol-3-ylacetic acids, whereas the glycine conjugate was the major metabolite of phenylacetic and 4-chlorophenylacetic acids. Taurine conjugation did not occur with benzoic and 4-nitrobenzoic acids which were excreted as glycine and glucuronic acid conjugates. 4. Phenacetylglutamine and 4-hydroxyphenylacetic acid were minor urinary metabolites of phenylacetic in the ferret. 5. A number of taurine conjugates of aliphatic and aromatic acids were synthesized and their characterization and properties were studied. The role of taurine as an alternative to glycine in the metabolic conjugation of arylacetic acids is discussed.     

Stereoselective ring oxidation of propranolol in man.

The objective of this study was to elucidate stereoselective mechanisms of propranolol metabolism in man. Five normal subjects were given single 80 mg oral doses of deuterium-labeled pseudoracemates of propranolol, and the stereochemical composition of propranolol and its major metabolites in urine was determined by GC/MS. The (-)/(+)-enantiomer ratios for unchanged propranolol, 1.50 +/- 0.10 (mean +/- s.e. mean), and propranolol glucuronide, 1.76 +/- 0.10, were similar to previous findings in plasma. All products of side-chain oxidation also consisted mainly of the (-)-enantiomer, with an overall (-)/(+) ratio of 1.61 +/- 0.11. A (-)/(+) ratio of 1.04 +/- 0.17 for 4-hydroxypropranolol did not indicate stereoselectivity in ring oxidation. However, the ratio for its glucuronic acid conjugate of 1.78 +/- 0.19 and for its sulphate conjugate of 0.27 +/- 0.03 suggested stereoselectivity in either the glucuronidation or sulphation of 4-hydroxypropranolol, or both. When the stereoselectivity in these secondary pathways was taken into consideration, the overall ring oxidation strongly favoured (+)-propranolol with a (-)/(+)-enantiomer ratio of 0.59 +/- 0.09. The composite observations of the stereochemistry of propranolol metabolism in man are consistent with stereoselective ring oxidation of (+)-propranolol, leading to a greater bioavailability of the pharmacologically more active (-)-propranolol and subsequent preferential side-chain oxidation and glucuronidation of this enantiomer.     

Quantitative account of propranolol metabolism in urine of normal man

A recent study, identifying several sulfate conjugates, appears to have led to a full qualitative account of propranolol metabolism in man. The objective of the present investigation was to determine the quantitative fate of propranolol, including the relationship between the primary metabolic pathways, i.e. glucuronidation, side-chain oxidation and ring oxidation. Single 80-mg oral doses of propranolol together with [3H]propranolol were administered to seven normal subjects. Urinary metabolites were determined by HPLC with radiometric detection after hydrolysis of glucuronic acid conjugates and fractionation by solvent extraction. About 90% of the dose was recovered in urine. Twelve metabolites accounted for 91% of the recovered dose. When examining the metabolites based on the primary metabolic pathways, 17% of the dose (range, 10-25%) was going through glucuronidation, 41% (range, 32-50%) through side-chain oxidation, and 42% (range, 27-59%) through ring oxidation. These data show that the net elimination of propranolol is largely due to oxidative metabolism. The relative contribution of the primary pathways is well reflected by the four major propranolol metabolites, i.e. propranolol glucuronide, naphthoxylactic acid, and the glucuronic acid and sulfate conjugates of 4'-hydroxypropranolol. These observations should greatly facilitate future studies of the biochemical mechanisms of propranolol disposition.     

Glucuronides of hydroxylated metabolites of amitriptyline and nortriptyline isolated from rat bile

Polar conjugates were isolated from the bile of rats given amitriptyline (AT, unlabeled or labeled with 14C), nortriptyline (NT), or 10-hydroxy (10-OH) derivatives of the drugs. The procedure involved extraction on a column of polystyrene resin, elution with methanol, and separation by preparative TLC followed by reversed phase HPLC. Individual metabolites were characterized by NMR spectroscopy and fast atom bombardment mass spectrometry and by enzymatic or acid deconjugation with subsequent identification of aglycones and glucuronic acid. Conversely, they were compared with conjugates obtained from hydroxy compounds by incubation with rat liver microsomes and UDP-glucuronic acid. Glucuronides isolated from the bile of rats given AT were derived from 2-OH-AT, (E)- and (Z)-10-OH-AT, 2-hydroxy-3-methoxy- (or 3-hydroxy-2-methoxy) AT, 10, 11-(OH)2-AT, and some of the N-demethylated analogues of these compounds. In most cases, 10-OH compounds form two diastereoisomeric glucuronides produced from the enantiomeric alcohols; 10, 11-(OH)2 metabolites occur as cis- and trans-isomers that are conjugated with glucuronic acid. Administration of synthetic (E)- and (Z)-10-OH-AT and -NT leads to the excretion of their glucuronides along with conjugates formed after demethylation and/or introduction of a second OH group. NT gives rise to 2-OH-NT glucuronide besides those conjugates derived from (E)-10-OH-NT. No glutathione conjugates could be detected.     

Pharmacokinetics and metabolism of the antiinflammatory agents S-H 766 in man]

Blood and plasma levels as well as urinary and fecal excretion were measured in humans after oral administration of radioactively labelled 4-[j-(2'-fluorobiphenylyl)]-4-hydroxycrotonic acid (S-H 766 MO). The radioactivity in the plasma reaches maximum values of about 10 mug eq./ml 1 to 2 h after application with either form. After repeated administration good agreement is found between the plasma levels measured and those simulated according to the pharmacokinetic parameters obtained after single application. The S-H 766 metabolites were investigated in blood and urine. The substance was found to undergo considerable metabolism, only approximately 2% being excreted in the urine unchanged. The conjugates, which constitute over 60% of the radioactivity of the urine, consist mainly of glucuronides and sulfates. The structure of the aglycones shows that the metabolism occurs along two pathways, by beta-oxidation of the aliphatic side chain into aryl acetic acids and by hydroxylation of the aromatic nucleus to phenolic compounds. It must be assumed that these biotransformations take place both simultaneously and successively.     

Direct gradient reversed-phase HPLC analysis and preliminary pharmacokinetics of nalidixic acid, 7-hydroxymethylnalidixic acid, 7-carboxynalidixic acid,and their corresponding glucuronide conjugates in humans

A gradient reversed-phase high pressure liquid chromatographic analysis was developed for the direct measurement of nalidixic acid with its acyl glucuronide, 7-hydroxymethylnalidixic acid with its acyl and ether glucuronides, and 7-carboxynalidixic acid in human plasma and urine. The glucuronides and 7-carboxynalidixic acid were not present in plasma after an oral dose of 1,000 mg nalidixic acid. The acyl glucuronides of 7-carboxynalidixic acid were not present in plasma and urine. The acyl glucuronides are stable in urine at pH 5.0–5.5. The subject's urine must therefore be acidified by the oral intake of 4×1 g of ammonium chloride per day. With acidic urine, hardly any nalidixic acid was excreted unchanged (0.2%). It was excreted as acyl glucuronide (53.4% of dose), 7-hydroxymethylnalidixic acid (10.0%), the latter's acyl glucuronide (30.9%), and 7-carboxynalidixic acid (4.2%).     

Stereoselective formation of glucuronides in metabolism of hexobarbital enantiomers in vivo: isolation and quantitation of glucuronides in rabbit urine

An improved column-chromatographic method was described for isolation and purification on preparative scale of a glucuronide from urine of rabbits administered (RS)-hexobarbital. The analytically pure preparation obtained was found to be a mixture of two glucuronides of diastereomeric 3'-hydroxyhexobarbitals. Rates of hydrolysis of the glucuronides were dependent on the enzyme preparations used as well as on the configuration of the substrate. For quantitative determination, the glucuronides were hydrolyzed completely by beta-glucuronidases from either Escherichia coli or abalone entrails under the conditions used. In vivo studies on the metabolism of (R)-(-)- and (S)-(+)-hexobarbital in the rabbit showed that the glucuronides excreted in 24-hr urine accounted for about 30% of the dose of each enantiomer, and that conjugation of the hydroxy isomers with glucuronic acid was so stereoselective that the isomers with S-configuration at the 3'-position were preferentially conjugated. There were almost no differences in the urinary metabolite profile between normal an PB-treated rabbits; however, a noticeable change was observed in recovery of unchanged (S)-hexobarbital after phenobarbital treatment.     

Pharmacokinetics,metabolism and excretion of the glycine antagonist GV150526A in rat and dog

1. The pharmacokinetics, metabolic fate and excretion of 3-[-2(phenylcarbamoyl) ethenyl-4,6-dichloroindole-2-carboxylic acid (GV150526), a novel glycine antagonist for stroke, in rat and dog following intravenous administration of [C14]-GV150526A were investigated. 2. Studies were also performed in bile duct-cannulated animals to confirm the route of elimination and to obtain more information on metabolite identity. 3. Metabolites in plasma, urine and bile were identified by HPLC-MS/MS and NMR spectroscopy. 4. GV150526A was predominantly excreted in the faeces via the bile, with only trace metabolites of radioactivity in urine (< 5%). Radioactivity in rat bile was predominantly due to metabolites, whereas approximately 50% of the radioactivity in dog bile was due to parent GV150526. 5. The principal metabolites in bile were identified as glucuronide conjugates of the carboxylic acid, whereas in rat urine the main metabolite was a sulphate conjugate of an aromatic oxidation metabolite. Multiple glucuronide peaks were observed and identified as isomeric glucuronides and their anomers arising from acyl migration and muta-rotation.     

Pharmacokinetics,metabolism and excretion of the glycine antagonist GV150526A in rat and dog

1. The pharmacokinetics, metabolic fate and excretion of 3-[-2(phenylcarbamoyl) ethenyl-4,6-dichloroindole-2-carboxylic acid (GV150526), a novel glycine antagonist for stroke, in rat and dog following intravenous administration of [C14]-GV150526A were investigated. 2. Studies were also performed in bile duct-cannulated animals to confirm the route of elimination and to obtain more information on metabolite identity. 3. Metabolites in plasma, urine and bile were identified by HPLC-MS/MS and NMR spectroscopy. 4. GV150526A was predominantly excreted in the faeces via the bile, with only trace metabolites of radioactivity in urine (< 5%). Radioactivity in rat bile was predominantly due to metabolites, whereas approximately 50% of the radioactivity in dog bile was due to parent GV150526. 5. The principal metabolites in bile were identified as glucuronide conjugates of the carboxylic acid, whereas in rat urine the main metabolite was a sulphate conjugate of an aromatic oxidation metabolite. Multiple glucuronide peaks were observed and identified as isomeric glucuronides and their anomers arising from acyl migration and muta-rotation.     

Decreased absorption as a possible cause for the lower bioavailability of a sustained-release propranolol

The influence of sustained absorption on the oral availability of propranolol (P) and the metabolic disposition of P were investigated by obtaining the partial metabolic clearances (CLm) following long-acting P (LA) dosing in comparison with the conventional propranolol tablet (CP). Ten healthy volunteers were given a single oral dose of an LA capsule (60 mg) and CP (20 mg x 3) using a crossover design. Blood and urine samples were collected over 24- and 48-h postdose periods, respectively. Concentrations of P, propranolol glucuronide (PG), 4-hydroxypropranolol (4P), 4-hydroxypropranolol glucuronide (4PG), 4-hydroxypropranolol sulfate (4PS), and naphthoxylactic acid (NLA) were determined by HPLC with fluorescence and UV detection. Significant differences were observed between LA and CP in the area under the plasma concentration-time curves (AUCs) for P, PG, and NLA and in the amounts excreted into urine (Ae) for all measured metabolites (i.e., PG, 4P, 4PG, 4PS, and NLA). The parallel decrease of the AUC for P and the excreted amounts of all measured metabolites following LA dosing resulted in partial metabolic clearances (CLm) and renal clearances (CL) for P and its metabolites that were similar to those observed for CP. Therefore, the hepatic metabolism of P would not be affected by the slower absorption at a single oral dose of 60 mg. These results indicate that the poor absorption of P from the gastrointestinal tract might be one of the factors causing the low bioavailability of P observed after administration of the sustained-release formulation.     

Identification and distribution in the rat of acidic metabolites of tamoxifen

Chromatographic analysis of acidic fractions of urinary radioactivity from immature female rats which had received the triarylethylene antiestrogen tamoxifen (TAM), labeled with 14C, resulted in the identification of two new metabolites. These were tamoxifen acid (TA), in which the side chain of TAM was changed to an oxyacetic acid moiety, and 4-hydroxytamoxifen acid (4-HTA), which had a similarly altered side chain plus a phenolic hydroxy group in its structure. In contrast to other TAM metabolites and other arylacetic acids, neither TA nor 4-HTA were eliminated as glucuronic acid or glycine conjugates in urine. Only small amounts of acidic radioactivity were found in liver tissue 24 and 48 hr after dosing, and none was detected in uterine tissue. However, TA plus 4-HTA accounted for 2.8% and 2.9% of the administered dose eliminated within 24 hr in urine and feces, respectively. These results suggest that TA and 4-HTA are important final products of TAM metabolism and that these, unlike other hydroxylated and/or dealkylated metabolites of this drug, may not contribute to the antiuterotrophic effects of TAM.     

Deconjugation of N-glucuronide conjugated metabolites with hydrazine hydrate--biomarkers for exposure to the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

The metabolism of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) has been investigated in rabbit liver S9. Two phase I metabolites, N(2)-OH-PhIP and 4'-OH-PhIP were identified based on UV and mass spectra and co-elution with reference standards. Fortification of the incubation with UDGPA resulted in a complete glucuronidation of PhIP and N(2)-OH-PhIP, while 4'-OH-PhIP was only partly glucuronidated. Also, the PhIP metabolite 5-OH-PhIP was completely glucuronidated by rabbit liver S9, while 5-OH-PhIP was a poor substrate for CYP mediated hydroxylation. The glucuronic acid conjugates of PhIP metabolites were unsusceptible to treatment with beta-glucuronidase indicating that these are N-glucuronides. Treatment of the conjugates with hydrazine hydrate, however, resulted in complete hydrolysis of the glucuronic acid conjugates as well as in reduction to the parent amine of metabolites hydroxylated in the exocyclic amino group. Urine was collected from a male volunteer following consumption of fried chicken. Treatment of the urine with beta-glucuronidase/sulfatase resulted in release of 4'-OH-PhIP, while treatment with hydrazine hydrate in addition resulted in release of substantial amounts of PhIP and 5-OH-PhIP. The data show that hydrazine hydrate can hydrolyse N-glucuronides of metabolites of PhIP, glucuronides that are unsusceptible to enzymatic hydrolysis. In addition the data indicate that humans metabolise a large fraction of ingested PhIP to genotoxic metabolites. The chemical hydrolysis of glucuronide conjugates of PhIP metabolites with hydrazine hydrate observed in this study may also be a useful approach in the development of biomarkers for exposure and effect of other xenobiotics.     

Unusual glucuronides

Glucuronidation reaction is catalyzed by mammalian uridine diphosphoglucuronosyl transferases by using uridine diphosphoglucuronic acid as a cosubstrate. Conjugation of glucuronic acid to nucleophilic functional groups in chemical entities results in formation of glucuronides. As anticipated, a number of nucleophilic functional groups such as hydroxyl, phenolic, acyl, primary secondary and tertiary amino, etc. in a diverse set of chemical compounds are known to form the corresponding glucuronides. Glucuronides have been reported to be formed at carbon atoms, selenium atoms, and upon N-carbamoylation of primary and secondary amino groups. Glucuronides are also believed to be the end products of metabolism. However, there are examples where glucuronidation results in further oxidative or conjugative biotransformation reactions. The objective of this review is to highlight unusual glucuronide conjugates. Diglucuronide conjugates reported in the literature fall under two distinct categories. Use of prefixes such as "bis" versus "di" has been previously proposed for separating the two types of diglucuronides. In spite of this, literature reports for diconjugative glucuronide metabolites reflect interchangeable use of "bisglucuronides" and "diglucuronides." Furthermore, the application of such prefixes does not adhere to recommendations of International Union of Pure and Applied Chemistry nomenclature for substituent groups. Therefore, an effort is made in this review to document the historic reports for diglucuronides into two distinct types for sake of clarity and to allow differentiation between the two types of diconjugative metabolites. Overall, this commentary centers on unusual glucuronide metabolites that result from conjugation at uncommon functional groups, glucuronides undergoing ensuing oxidative or conjugative metabolic transformations. Structural and mechanistic aspects are also discussed.     

Nonenzymic glycation of albumin by acyl glucuronides in vitro. Comparison of reactions with reducing sugars.

Acyl glucuronides are ubiquitous metabolites formed from acidic xenobiotics and endogenous compounds, such as bilirubin. Previous studies indicated that the covalent binding of acyl glucuronides to proteins occurs via an imine intermediate in a manner analogous to the glycation of proteins via reducing sugars. When glucuronic acid was incubated in solution with albumin, it formed 10 and 4 times more fluorescent, Maillard reaction products with albumin after 25 days than did glucose or fructose, respectively. However, radiolabeled glucuronic acid exhibited less covalent binding to albumin than either glucose or fructose. Circular dichroism measurements indicate that glucuronic acid is about 0.02% open chain form with exposure of the reactive aldehyde, whereas fructose and glucose have 2 and 0.0026% present in solution as the open chain; thus, differences in reactivity of the reducing sugars were not correlated with exposure of the free aldehyde. Methyl glucuronate formed little fluorescent product with albumin, suggesting that the C-6 carboxylate of glucuronic acid may facilitate the reactions after covalent binding that lead to the formation of fluorescent products. When acyl glucuronide metabolites of two previously marketed acidic drugs, zomepirac and suprofen, were incubated with albumin at a concentration of 2.5 mM, more fluorescent product was formed than by 500 mM glucose. Reversible binding of the acyl glucuronides to albumin was 60-90%, but almost zero for the free reducing sugars, which indicates that reversible binding may explain the enhanced reactivity of the acyl glucuronides in forming fluorescent products with albumin. These results indicate that acyl glucuronides are reactive metabolites that may cause significant glycation of proteins with glucuronic acid in vivo.     

Studies on the metabolism of propafenone. 2nd comm.: studies on the biotransformation in the dog

Following administration of 14C- and 2H-labelled propafenone hydrochloride (2-(2'-hydroxy-3'-propylaminopropoxy)-omega-phenyl-propiophenone hydrochloride) to beagle dogs, the metabolites were isolated from urine and bile and analysed by mass spectrometry. Reference substances were synthesized on the basis of the structures postulated from the mass spectra and compared with the substances isolated from the biological material. Propafenone was absorbed completely following i.d. administration and eliminated mainly with the bile. Within 28 h 10% of the dose was excreted with the urine and 87% with the bile. Propafenone was extensively metabolized. Less than 1% of the dose was recovered as unchanged substance in urine and bile. The urinary and biliary metabolites were almost exclusively conjugated. The main metabolite, accounting for more than 30% of the dose, was propafenone glucuronide, followed by conjugates of hydroxylated propafenone derivatives with glucuronic acid and sulphuric acid. 5-Hydroxy-propafenone, a propafenone derivative hydroxylated in the middle aromatic ring, and a derivative hydroxylated in the omega-phenyl ring each accounted for about 15% of the dose. Besides these monohydroxy metabolites, two other O-methylated catechol-like derivatives, substituted in the different aromatic rings were recovered. The remainder of the metabolic products identified were mainly substances resulting from degradation of the propoxyamine side chain.     

Isolation and characterization of carbinolamide and phenolic glucuronide conjugates of (+-)-N-methyl-N-(1-methyl-3,3-diphenylpropyl) formamide and N-formylmethamphetamine by FAB/MS, LC/MS/MS, and NMR.

The metabolic disposition of (+-)-N-methyl-N-(1-methyl-3,3- diphenyl-propyl)formamide, especially with regard to the formation of water soluble glucuronides, is described. The glucuronide conjugates, (+-)-N-hydroxymethyl-N-(1-methyl-3,3-diphenylpropyl)formamide glucuronide, (+-)-N-methyl-N-[1-methyl-3-(4'-hydroxyphenyl)-3-phenylpropyl]formamide glucuronide, and (+-)-N-methyl-N-[1-methyl-3-(4'-hydroxy-3'-methoxyphenyl)-3- phenylpropyl]formamide glucuronide were isolated from the bile of rats dosed with the parent compound. These conjugates were characterized spectroscopically by 1H-NMR, FAB/MS, and LC/MS/MS. Because it is becoming more common to isolate the intact glucuronide conjugates of xenobiotics, we investigated some common mass spectral fragmentation patterns of these conjugates, especially by LC/MS/MS. The fragmentation patterns for each of the conjugates were obtained under MS/MS conditions and compared. Specifically, the fragmentation patterns of phenolic glucuronide and an aliphatic O-glucuronide, in particular a carbinolamide glucuronide, were investigated. The data obtained from these studies was used to predict the nature of glucuronide conjugates obtained from rats dosed with the formamide analog, N-formylmethamphetamine. This is the first spectroscopic characterization of an intact carbinolamide glucuronide conjugate isolated from the bile of rats.     

Metabolism of 3-phenoxybenzoic acid and the enterohepatorenal disposition of its metabolites in the rat

The absorption, metabolism and disposition of 3-phenoxybenzoic acid (3PBA) and its 4'-hydroxylated derivative (4'HO3PBA) have been investigated in intact and bile duct-cannulated rats. Both acids are rapidly and extensively eliminated in the urine as the O-sulfate ester of 4'HO3PBA (4'HOSO2O3PBA). Biliary excretion was 45% of the dose when 3PBA was administered at 100 mg/kg, compared with 16% at 10 mg/kg. The major biliary metabolites, identified by mass spectrometry, were 3PBA glucuronide and the ether and ester glucuronic acid conjugates of 4'HO3PBA. These biliary metabolites were of minor importance in the urine. At a dose of 100 mg/kg, an apparent excretion balance figure approximately 120%, as determined from the extent of elimination of radioactivity in the urine (72%) of intact animals and bile of cannulated rats (45%) (only 7% appeared in the feces), suggests that the biliary glucuronides decompose and/or are enzymically cleaved in the gastrointestinal tract to the respective benzoic acids. The latter are subsequently reabsorbed and undergo further metabolism, principally to the sulfate ester which is excreted in the urine. This enterohepatorenal disposition of 3PBA has been confirmed by suppression of the gut microflora with antibiotics in vivo, which dramatically reduces the extent of deconjugation of biliary glucuronides and hence reabsorption of the free benzoic acids. The deconjugation by gut bacteria has also been demonstrated in vitro.