Metabolic studies of turinabol in horses

Turinabol (4-chloro-17alpha-methyl-17beta-hydroxy-1,4-androstadien-3-one) is a synthetic oral anabolic androgenic steroid. As in the case of other anabolic steroids, it is a prohibited substance in equine sports. The metabolism of turinabol in human has been reported previously; however, little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of turinabol in racehorses with an objective to identify the most appropriate target metabolites for detecting turinabol administration. For the in vitro studies, turinabol was incubated with fresh horse liver microsomes. Metabolites in the incubation mixture were isolated by liquid-liquid extraction and analysed by gas chromatography-mass spectrometry (GC-MS) after trimethylsilylation. The results showed that the major biotransformation of turinabol was hydroxylation at the C6, C16 and C20 sites to give metabolites 6beta-hydroxyturinabol (M1), 20-hydroxyturinabol (M2), two stereoisomers of 6beta,16-dihydroxyturinabol (M3a, M3b) and 6beta,20-dihydroxyturinabol (M4). The metabolite 6beta-hydroxyturinabol was confirmed using an authentic reference standard. The structures of all other turinabol metabolites were tentatively identified by mass spectral interpretation. For the in vivo studies, two horses were administered orally with turinabol. Pre- and post-administration urine samples were collected for analysis. Free and conjugated metabolites were isolated using solid-phase extraction and analysed by GC-MS as described for the in vitro studies. The results revealed that turinabol was extensively metabolised and the parent drug was not detected in urine. Two metabolites detected in the in vitro studies, namely 20-hydroxyturinabol and 6beta,20-dihydroxyturinabol, these were also detected in post-administration urine samples. In addition, 17-epi-turinabol (M5) and six other metabolites (M6a-M6c and M7a-M7c), derived from D-ring hydroxylation and A-ring reduction, were also detected. Except for 17-epi-turinabol, none of these metabolites has ever been reported in any species. All in vivo metabolites were detected within 48 h after administration.     

Extraction method and thin-layer chromatographic system for the determination of alpha-l-acetylmethadol and metabolites in biological fluids.

An extraction method and thin-layer chromatographic (TLC) system for the determination of alpha-l-acetylmethadol and its known metabolites (methadol, noracetylmethadol, dinoracetylmethadol, normethadol, 6-acetamide-4,4-diphenyl-3-heptanol, and N-methyl-6-acetamido-4,4-diphenyl-3-heptanol) are described. The parent drug and metabolites are extracted from biological fluids with ethyl acetate and separated by TLC using silica gel plates and a developing system of ethyl acetate-methanol-water-ammonia (85:10:1:1). This system may be used to quantitatively determine levels of radiolabeled drug and metabolites by scraping the TLC plates into 3-mm zonal fractions and measuring the amount of radioactivity by scintillation counting. A representative radiochromatogram obtained from an extract of monkey urine is shown.     

Structure elucidation by synthesis of four metabolites of the antitumor drug ENMD-1198 detected in human plasma samples

ENMD-1198 is a biologically active analogue of the antitumor drug 2-methoxyestradiol. Four human metabolites of ENMD-1198 were identified through synthesis and liquid chromatography/mass spectrometry comparisons of the metabolites with the synthetic standards. Two metabolites (3 and 4) are epimers resulting from benzylic hydroxylation at C-6. Two additional metabolites (5 and 6) are formed by epimeric hydroxylation at C-6 and α-epoxidation of the 16,17-alkene. The syntheses provided sufficient quantities of the metabolites for cytotoxicity studies to proceed. The 6-β-ol 4 was moderately less cytotoxic than the parent drug, while the remaining three metabolites (3, 5, and 6) were significantly less cytotoxic.     

Capillary electrophoresis contributions to the hydromorphone metabolism in man

CE-ESI multistage IT-MS (CE-MS(n), n < or = 4) and computer simulation of fragmentation are demonstrated to be effective tools to detect and identify phase I and phase II metabolites of hydromorphone (HMOR) in human urine. Using the same CE conditions as previously developed for the analysis of urinary oxycodone and its metabolites, HMOR and its phase I metabolites produced by N-demethylation, 6-keto-reduction and N-oxidation and phase II conjugates of HMOR and its metabolites formed with glucuronic acid, glucose, and sulfuric acid could be detected in urine samples of a patient that were collected during a pharmacotherapy episode with daily ingestion of 48 mg of HMOR chloride. The CE-MS(n) data obtained with the HMOR standard, synthesized hydromorphol and hydromorphone-N-oxide, and CYP3A4 in vitro produced norhydromorphone were employed to identify the metabolites. This approach led to the identification of previously unknown HMOR metabolites, including HMOR-3O-glucide and various N-oxides, structures for which no standard compounds or mass spectra library data were available. Furthermore, the separation of alpha- and beta-hydromorphol, the stereoisomers of 6-keto-reduced HMOR, was achieved by CE in the presence of the single isomer heptakis(2,3-diacetyl-6-sulfato)-beta-CD. The obtained data indicate that the urinary excretion of alpha-hydromorphol is larger than that of beta-hydromorphol.     

Biotransformation of phorbol by human intestinal bacteria

Anaerobic incubation of phorbol (1) from Croton tiglium with human intestinal bacteria afforded five metabolites: isophorbol (2), deoxyphorbol (3), 4beta,9alpha,20-trihydroxy-13,15-seco-1,6,15-tigliatriene-3,13-dione (4), 4beta,9alpha,20-trihydroxy-15,16,17-trinor-1,6-tigliadiene-3,13-dione (5) and 4beta,9a,20-trihydroxy-14(13-->12)-abeo-12alphaH-1,6-tigliadiene-3,13-dione (6). All these metabolites (2-6) were identified and characterized by spectroscopic means, including two-dimensional (2D)-NMR. Nine defined strains from the human intestine showed an ability to transform 1 to these metabolites.     

Identification of carbadox metabolites formed by liver microsomes from rats, pigs and chickens using high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry

Carbadox (methyl-3-(2-quinoxalinylmethylene)-carbazate-N(1),N(4)-dioxide) is a chemotherapeutic growth promoter added to feed for starter pigs. In this work, the metabolism of carbadox in rat, pig and chicken liver microsomes has been studied firstly. The incubation mixtures were then processed and analyzed for metabolites with a sensitive and reliable method based on high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry (LC/MS-IT-TOF). With the help of chromatographic behavior and accurate mass measurements, it is possible to rapidly and reliably characterize the metabolites of carbadox. The structural elucidations of these metabolites were performed by comparing the changes in the accurate molecular masses and fragment ions generated from precursor ions with those of parent drug. The present results showed that the metabolism of carbadox in liver microsomes had qualitative species-difference. A total of seven metabolites were identified in rat liver microsomes. Five metabolites (Cb1-Cb3, Cb5, Cb7) were observed in pig and chicken liver microsomes. In addition, metabolite Cb6 was also detected in chicken liver microsomes. The peak areas of the metabolites in the three species are different. For the formations of Cb1, Cb2, Cb5 and Cb6, the rank order was rat>chicken>pig; Cb3; pig~chicken>rat. Cb1, Cb2 and Cb3 have been previously reported, whereas the other four metabolites were novel. The N→O group reduction and hydroxylation followed by N→O group reduction were the main metabolic pathways for carbadox in the three species.     

The microbial hydroxylation of levonorgestrel

The microbial transformation of levonorgestrel (1) by Cunningham elegans resulted in the formation of five hydroxylated metabolites, 13-ethyl-10beta, 17beta-dihydroxy-18,19-dinor-17alpha-pregn-4-en-20-yn-3-one(2), 13-ethyl-6beta,17beta-dihydroxy-18,19-dinor-17alpha-pregn-4-en-20-yn-3-one (3) 13-ethyl 6beta, 10beta, 17beta-trihydroxy-18,19-dinor-17alpha-pregn-4-en-20-yn-3-one (4) 13-ethyl-15alpha-17beta-dihydroxy-18,19-dinor-17alpha-pregn-4-en-20-yn-3-one (5) and 13-ethyl-11alpha, 17beta-dihydroxy-18,19-dinor-17alpha-pregn-4en-20-yn-3-one. The fermentation of one with Rhizopus stolonifer, Fusarium lini and Curvularia lunata afforded compound 2 as a major metabolise. These metabolites were structurally characterized on the basis of spectroScopic techniques. Metabolite 6 was identified as a new compound. Compounds 2 2 ad 5 displayed inhibitory activity against the acetylcholinesterase ( AChE, EC. 3.1.1.7) with IC50 values of 79.2 and 24.5 microM, respectively. The metabolites 2 and 5 also showed inhibitory activity against the butyryLcholinesterase ( BChE, E.C 3.1.1.8) with IC50 values ranging between 9.4 and 309.8 microM.     

Biliary excretion of metabolites of baicalin and baicalein in rats

Biliary excretion of metabolites of baicalin, present in Scutellariae Radix, was investigated using rats. The bile of rats administered baicalin orally was shown to contain five major metabolites, which were identified as baicalein 6-O-beta-glucopyranuronoside (M1), 6-O-methyl-baicalein 7-O-beta-glucopyranuronoside (oroxylin A 7-O-beta-glucuronide (M2], baicalein 7-O-beta-glucopyranuronoside (M3), 6-O-beta-glucopyranuronosyl- baicalein 7-O-sulfate (M4), and baicalein 6,7-di-O-beta-glucopyranuronoside (M5) on the basis of chemical and spectroscopic evidence. The bile of rats treated with baicalein also contained the above metabolites. Slower biliary excretion of the metabolites after baicalin administration suggested that it was absorbed as baicalein after hydrolysis in the gastrointestinal tract. The total cumulative amounts of the five metabolites excreted in the bile during 30 h after oral administration of baicalin and that of baicalein were approximately 54% and 40% of the doses, respectively. In addition the bilary metabolites of both drugs were shown to be mainly composed of M5 and M4, which have high polarity and large molecular weight.     

Metabolism of eupatilin in rats using liquid chromatography/electrospray mass spectrometry

Eupatilin (5,7-dihydroxy-3',4',6-trimethoxy flavone) is an active ingredient of an ethanol extract of Artemisia asiatica (DA-9601) that is used in the treatment of gastritis. In vitro and in vivo metabolism of eupatilin in the rats has been studied by LC-electrospray mass spectrometry. Rat liver microsomal incubation of eupatilin in the presence of NADPH and UDPGA resulted in the formation of four metabolites (M1-M4). M1, M2, M3 and M4 were tentatively identified as 3'- or 4'-O-demethyl-eupatilin glucuronide, eupatilin glucuronide, 6-O-demethyleupatilin and 3'- or 4'-O-demethyl-eupatilin, respectively. Those metabolites from in vitro study were also characterized in bile, plasma or urine samples after an intravenous administration of eupatilin to rats. In rat bile, plasma and urine samples, eupatilin glucuronide (M2) was a major metabolite, whereas M3, M4 and M4 glucuronide (M1) were the minor metabolites.     

In vivo metabolism of lidocaine in the rat. Isolation of urinary metabolites as pentafluorobenzoyl derivatives and their identification by combined gas chromatography-mass spectrometry

The metabolism of a large dose (40 mg/kg intraperitoneally) of lidocaine (LIDO) in mature male Sprague-Dawley rats is described. Pentafluorobenzoyl chloride was used to derivatize the hydrolyzed urinary metabolites prior to extraction and analysis as pentafluorobenzoyl-derivatives by combined gas chromatography-mass spectrometry. Total ion and selected ion current (m/z 195; C6F5CO+) traces were recorded and metabolites of LIDO were readily identified. Only one major metabolite, 3-hydroxy-N-(N-ethylglycyl)-2,6-xylidine, was excreted in urine. A new metabolite, 3-hydroxy-N-glycyl-2,6-xylidine was also present in significant amounts, as well as minor quantities of four oxygenated metabolites of N-(N-ethylglycyl)-2,6-xylidine. Other known metabolites of LIDO, including 3-hydroxylidocaine, were excreted in trace quantities. The results suggest that metabolism of LIDO in rats may be age- and/or dose-dependent.     

Isolation and identification of metabolites from dihydromyricetin

Dihydromyricetin (DHM) is the major bioactive constituent of Rattan Tea, which is the tender stems and leaves of Ampelopsis grossendentata. Seven metabolites (2-8) of DHM (1) were obtained by the chromatographic method. The metabolites 2-5 were obtained from the urine of rats administered orally with DHM; and metabolites 6-8 were detected in the fecal specimens of rats, which were also produced by human intestinal bacteria (HIB) in vitro, and were separated from the cultured media of HIB containing DHM. Their structures were elucidated as 5,7,3',5'-tetrahydroxyflavanonol (2); 5,7,3',5'-tetrahydroxy-4'-methoxyflavanonol (3); 5,7,4',5'-tetrahydroxy-3'-methoxyflavanonol (4); and dihydromyricetin-O-5-beta-D-glucuronide (5); (2R,3S)-5,7,3',4',5'-pentahydroxyflavanonol (6); 3,4,5,7,3',4',5'-hepthydroxyflavan (7) and 5,7,3',4',5'-pentahydroxyflavanone (8) on the basis of UV, NMR and LC-MS/MS data. These seven metabolites were formed through familiar metabolic reactions. Dihydromyricetin-O-5-beta-D-glucuronide (5) is a new compound. The (13)C-NMR data of (2) and (4) are reported for the first time.     

Human urinary metabolite profile of tea polyphenols analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry with data-dependent acquisition

Tea is rich in polyphenols and has a variety of biological activities. In order to better understand the biological effects of tea constituents on human health, markers for their exposure and their metabolic fates are needed. Previously, we have characterized several catechin metabolites in the blood and urine, but more information on the metabolite profile of tea polyphenols is needed. In the present study, the human urinary metabolite profile of tea polyphenols was investigated using liquid chromatography/electrospray ionization tandem mass spectrometry with data-dependent acquisition. With data-dependent MS/MS analysis by collecting the MS2 and MS3 spectra of the most intense ions in the sample, we identified more than twenty metabolites of tea polyphenols from human urine samples. (-)-Epigallocatechin (EGC) glucuronide, methylated EGC glucuronide, methylated EGC sulfate, (-)-epicatechin (EC) glucruronide, EC sulfate, methylated EC sulfate, as well as the glucuronide and sulfate metabolites of the ring-fission metabolites of tea catechins, 5-(3',4',5'-trihydroxyphenyl)-gamma-valerolactone (M4), 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone (M6) and 5-(3',5'-dihydroxyphenyl)-gamma-valerolactone (M6'), were the major human urinary metabolites of tea polyphenols. To our knowledge, this is the first report of the direct simultaneous analysis of the human urinary metabolite profile of tea polyphenols using single sample analysis. This method can also be used for thorough investigations of the metabolite profiles of many other dietary constituents.     

Metabolite profile of sibutramine in human urine: a liquid chromatography-electrospray ionization mass spectrometric study

We present a detailed experimental approach to detection and subsequent structural characterization of unknown metabolites of sibutramine, using liquid chromatography-mass spectrometric techniques. The full-, precursor ion, and constant neutral loss scan modes of a triple quadrupole mass spectrometer were used for screening sibutramine metabolites in human urine. The structural assessment of unknown metabolites was based on MSn ion trap mass spectrometric analysis and comparison of MSn spectra between the standards and compounds detected. Two phase-I (M1 and M2) and eight phase-II (M3-M6) metabolites of sibutramine were found in human urine. Metabolites M1 and M2, which were found as minor metabolites, originated from N-demethylation of sibutramine. Carbamoyl glucuronides formed from metabolites M1, M2, and their hydroxylated analogs were the main metabolites of sibutramine and were characterized by tandem mass spectrometric analysis and by the chemical modification of their structure. We demonstrate the usefulness of the chemical derivatization approach for estimation of the site of glucuronidation and propose the formation of hydroxylated regioisomers of metabolites M4 and M6.     

Metabolism of 14C-miloxacin in rats--metabolites in urine, bile and feces (author's transl)

Isolation and characterization of metabolites of miloxacin, a new antimicrobial agent, were undertaken with rats. 14C-Miloxacin was orally administered to Sprague-Dawley rats at a dose of 50 mg/kg, and urine, bile and feces were collected. The metabolites extracted from the biological samples were isolated by column and thin-layer chromatographies. Characterization of the isolated metabolites was carried out by comparison with the authentic materials in various physicochemical analyses. Eight metabolites together with intact miloxacin were identified; containing the metabolites of N-demethoxy (M-1), catechol (M-3) and 6-methoxy (M-2 and M-4) types and their conjugates with glucuronic acid.     

Microbial metabolism. Part 6. Metabolites of 3- and 7-hydroxyflavones

Fermentation of 3-hydroxyflavone (1) with Beauveria bassiana (ATCC 13144) yielded 3,4'-dihdroxyflavone (3), flavone 3-O-beta-D-4-O-methylglucopyranoside (4) and two minor metabolites. 7-Hydroxyflavone (2) was transformed by Nocardia species (NRRL 5646) to 7-methoxyflavone (5) whilst Aspergillus alliaceus (ATCC 10060) converted it to 4',7-dihydroxyflavone (6). Flavone 7-O-beta-D-4-O-metylglucopyranoside (7) and 4'-hydroxyflavone 7-O-beta-D-4-O-methylglucopyranoside (8) were the metabolic products of 7-hydroxyflavone (2) when fermented with Beauveria bassiana (ATCC 7159). One of the minor metabolites of 3-hydroxyflavone (1) was tentatively assigned a beta'-chalcanol structure (9). Compounds 4, 7 and 8 are reported as new compounds. Structure elucidation of the metabolites was based on spectroscopic data.     

Microbial metabolism part 9. Structure and antioxidant significance of the metabolites of 5,7-dihydroxyflavone (chrysin), and 5- and 6-hydroxyflavones

5,7-Dihydroxyflavone (chrysin) (1) when fermented with fungal cultures, Aspergillus alliaceous (ATCC 10060), Beauveria bassiana (ATCC 13144) and Absidia glauco (ATCC 22752) gave mainly 4'-hydroxychrysin (4), chrysin 7-O-beta-D-4-O-methylglucopyranoside (5) and chrysin 7-sulfate (6), respectively. Mucore ramannianus (ATCC 9628), however, transformed chrysin into six metabolites: 4'-hydroxy-3'-methoxychrysin (chrysoeriol) (7), 4'-hydroxychrysin (apigenin) (4) 3',4'-dihydroxychrysin (luteolin) (8), 3'-methoxychrysin 4'-O-alpha-D-6-deoxyallopyranoside (9), chrysin 4'-O-alpha-D-6-deoxyallopyranoside (10), and luteolin 3'-sulfate (11). Cultures of A. alliaceous (ATCC 10060) and B. bassiana (ATCC 13144) metabolized 5-hydroxyflavone (2) into 5,4'-dihydroxyflavone (12) and 4'-hydroxyflavone 5-O-beta-D-4-O-methylglucopyranoside (13), respectively. 6-Hydroxyflavone (3) was transformed into 6-hydroxyflavanone (14), flavone 3-O-beta-D-4-O-methylglucopyranoside (15) and (+/-)-flavanone 6-O-beta-D-4-O-methylglucopyranoside (16) by cultures of Beauveria bassiana (ATCC 13144). The structures of the metabolic products were elucidated by means of spectroscopic data. The significance of the metabolites as antioxidants in relation to their structure is briefly discussed.     

Stereoselective convergent synthesis of 24,25-dihydroxyvitamin D3 metabolites: a practical approach

Vitamin D3 active metabolites 24R,25-(OH)2-D3, 24S,25-(OH)2-D3, and 1 alpha, 24R,25-(OH)3-D3 were synthesized by a convergent and stereoselective approach. In the synthetic route, the stereogenic center at C-24 was generated through ultrasonically induced aqueous conjugate addition of iodide 6 to Seebach's dioxolanone 5, and the vitamin D triene system was constructed using the Lythgoe approach. The synthesis, which is both short (seven steps from iodide 6) and efficient (32-40% overall yield), allows the preparation of large quantities of the metabolites and provides a novel example of a highly stereoselective reaction promoted by the zinc-copper couple in aqueous media.     

Studies on the metabolism of gomisin A (TJN-101). II. Structure determination of biliary and urinary metabolites in rat

After oral administration of gomisin A (1) to rats, the bile and urine were collected and treated with beta-glucuronidase and arylsulfatase. Seven metabolites, met B (2), met A-III (3), met E (4), met D (5), met F (6), met G (7), and met H (8) were isolated from the bile treated with the enzymes. Eight metabolites 2-8, and met A-II (9) were isolated from the urine treated with the enzymes. A major metabolite 2, and two minor metabolites 3 and 9 were identified as met B, met A-III, and met A-II, respectively, which are oxidative products of 1 formed by rat liver S9 mix. The structures of five new metabolites 4-7, and 8 were determined on the basis of chemical and spectral studies.     

Six immunosuppressive features from an ascomycete, Zopfiella longicaudata, found in a screening study monitored by immunomodulatory activity

In a screening study on immunomodulatory fungal metabolites, three known anthraquinones, carviolin (roseo-purpurin) (1), 1-O-methylemodin (2), omega-hydroxyemodin (citreorosein) (4), and a new anthraquinone, omega-acetylcarviolin (3), together with a known steroid, ergosta-4,6,8(14),22-tetraen-3-one (5) and a new steroid, 25-hydroxyergosta-4,6,8(14),22-tetraen-3-one (6) were isolated from an Ascomycete, Zopfiella longicaudata, and found to have moderate immunosuppressive activities. The structure-activity relationships of these metabolites are discussed.     

Lupane triterpenoids from Acacia mellifera with cytotoxic activity

Three new pentacyclic triterpenoids: (20R)-3-oxolupan-30-al (1), (20S)-3-oxolupan-30-al (2) and (20R)-28-hydroxylupen-30-al-3-one (3), along with (20S)-3beta-hydroxylupan-30-al (4), the latter previously described as a constituent of an epimeric mixture, were isolated from Acacia mellifera. In addition, the known metabolites 30-hydroxylup-20-(29)-en-3-one (5), 30-hydroxylup-20-(29)-en-3beta-ol (6), atranorin, methyl 2,4-dihydroxy-3,6 dimethyl benzoate, sitosterol-3beta-O-glucoside and linoleic acid were found in the analyzed plant species for the first time. The structures of the new metabolites were elucidated by extensive spectroscopic analyses and their relative stereochemistry was determined by NOESY experiments. The new metabolite 3 exhibited significant cytotoxic activity against the NSCLC-N6 cell line, derived from a human non-small-cell bronchopulmonary carcinoma.