Structure Elucidation Of Metabolites Research Articles

Comparison of LC-NMR and conventional NMR for structure elucidation in drug metabolism studies

Liquid chromatography-nuclear magnetic resonance (LC-NMR) has proven to be a useful technique for the structure elucidation of novel metabolites from pharmaceutical compounds. Proponents of LC-NMR tout the advantage of eliminating the step of a separate chromatographic isolation. However, the advantages of directly coupling NMR and HPLC instrumentation must be weighed against compromises in performance made to each technique to achieve a hyphenated system. While significant advances have been made in LC-NMR technology, a strong case can be made that HPLC purification of metabolites followed by conventional tube NMR is equally useful. It is relatively rare that one approach will be successful and the other not. The fundamental consideration is whether there is sufficient chromatographic expertise in the NMR laboratory to adequately design and execute appropriate experiments such that a pure chromatographic peak will be produced in the hyphenated system. Due to speed and sensitivity differences between NMR spectroscopy and mass spectrometry, liquid chromatography/mass spectrometry (LC/MS) continues to be the front-line approach for the structure elucidation of metabolites.

Metabolism of olaquindox in rat liver microsomes: structural elucidation of metabolites by high‐performance liquid chromatography combined with ion trap/time‐of‐flight mass spectrometry

Olaquindox (N-(2-hydroxyethyl)-3-methyl-2-quinoxalincarboxamide-1,4-dioxide) is a growth-promoting feed additive for food-producing animals. Its toxicity is closely related to the metabolism. The complete metabolic pathways of olaquindox are not revealed. To improve studies of the metabolism and toxicity of olaquindox, its biotransformation in rat liver microsomes and the structure of its metabolites using high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry (LC/MS-ITTOF) were investigated. When olaquindox was incubated with an NADPH-generating system and rat liver microsomes, ten metabolites (M1-M10) were detected. The structures of these metabolites were identified from mass spectra and comparison of their changes in their accurate molecular masses and fragment ions with those of the parent drug. With the high resolution and good mass accuracy achieved by this technique, the elemental compositions of the metabolites and their fragment ions were exactly determined. The results indicate that the N --> O group reduction is the main metabolic pathway of olaquindox metabolism in rat liver microsomes, because abundant 1-desolaquindox (M2), 4-desolaquindox (M1) and bisdesoxyolaquindox (M9) were produced during the incubation step. Seven other minor metabolites were revealed which were considered to be hydroxylation metabolites, based on the position of the quinoxaline ring or 3-methyl group and a carboxylic acid derivative on the side chain at position 2 of the quinoxaline ring. Among the identified metabolites, five new hydroxylated metabolites (M3-M7) were found for the first time in rat liver microsomes. This work will conduce to complete clarification of olaquindox metabolism, and improve the in vivo metabolism of olaquindox in food animals.

Identification of ginkgolide B metabolites in urine and rat liver cytochrome P450 enzymes responsible for their formation in vitro

To identify metabolites of ginkgolide B in rat urine, the predominant metabolism of ginkgolide B and the major cytochrome (CYP) P450 enzymes responsible for the metabolism of ginkgolide B in rat liver microsomes. A liquid chromatography quadrupole mass spectrometer and liquid chromatography ion-trap-time-of-flight mass spectrometer with electrospray ionization in negative-ion mode were used for the structure elucidation of metabolites in rat urine and liver microsome incubation. Various selective CYP450 inhibitors were applied to investigate their effects on the metabolism of ginkgolide B and the formation of the major metabolite in rat liver microsomes. Three metabolites were identified in rat urine. One hydroxyl metabolite of ginkgolide B were identified in rat liver microsomes, and quinidine uncompetitively inhibited the formation of the metabolite; its inhibitor constant (Ki) value for the inhibition of hydroxyl metabolite was estimated to be 8 micromol/L, while alpha-naphthoflavone, ketoconazole, sulfaphenazole, and diethyldithiocarbamate had no inhibitory effects. Ginkgolide B was metabolized to its hydroxyl metabolite in rats, and CYP2D6 was the major rat CYP isoform responsible for the ginkgolide B metabolism in rat liver microsomes.

Structure elucidation of metabolites of gambogic acid in vivo in rat bile by high-performance liquid chromatography–mass spectrometry and high-performance liquid chromatography–nuclear magnetic resonance

Gambogic acid (GBA), the main component of Gamboge, possesses significant anti-tumour activity. Due to its structural complexity, little is known about GBA metabolism. Here, we investigate the metabolism of GBA in vivo in rat bile. Identification of the metabolites formed was elucidated using high-performance liquid chromatography (HPLC) with UV–vis detection, HPLC/ion trap electrospray ionization-mass spectrometry, as well as HPLC/nuclear magnetic resonance. Four main metabolites were determined. Two phase I metabolites, 10-hydroxygambogic acid and 9,10-epoxygambogic acid, were oxides on the 9,10-olefinic bond of GBA. The others phase II metabolites, were 9,10-epoxygambogic acid-30- O-glucuronide and 10-hydroxylgambogic acid-30- O-glucuronide.

Structure elucidation of metabolites of swertiamarin produced by Aspergillus niger

The in vitro metabolism of swertiamarin was carried out in preparative scale using the fungus Aspergillus niger and the metabolites were isolated by semi-preparative HPLC combined with liquid-liquid extraction. Two metabolites, erythrocentaurin and one new compound were obtained and identified by 1H, 13C and 2D NMR and high resolution MS. The anti-inflammatory activity of the novel metabolite was tested and compared with that of swertiamarin in a mice model.

Discovery, Biosynthesis, and Structure Elucidation of Metabolites of a Doping Agent and a Direct Analogue, Tetrahydrogestrinone and Gestrinone, Using Human Hepatocytes

Tetrahydrogestrinone (18a-homo-pregna-4,9,11-trien-17beta-ol-3-one, THG) is an anabolic androgenic steroid sold to athletes as an undetectable performance enhancer. Being an unapproved substance, no legitimate in vivo human excretion studies could be performed to identify urinary markers of this doping agent. In vitro systems were used as an alternative approach to study the human metabolism of THG and the gestrinone analogue, which is a marketed drug. Incubations of both compounds in the presence of human hepatocytes led to formation of oxidative and glucuroconjugated metabolites. Microgram quantities of the major in vitro metabolites were biosynthesized using human hepatocytes, characterized by HPLC/MS/MS, and their structures elucidated by NMR. Due to high structure similarity, both THG and gestrinone had an analogous in vitro metabolic pathway leading to successive addition of a hydroxyl and a beta-glucuronic acid at C-18. This in vitro metabolite of gestrinone was consistent with a previously reported major but unknown human urinary metabolite. The structure of another metabolite of THG was proposed to be a glucuroconjugate of an oxidative product with a hydroxyl group most likely at C-16epsilon. In vitro information reported therein could significantly impact the identification of new urinary markers of THG for doping control purposes.

Metabolism Profiling, and Cytochrome P450 Inhibition & Induction in Drug Discovery

To reduce the high attrition rates of NCEs in preclinical and clinical development uncovering pharmacokinetics, toxicokinetics, drug metabolism, and drug-drug interactions early in drug discovery would be highly valuable. There have been many in vitro screens developed for these areas that have higher sample throughput, which is consistent with the iterative cycle of a typical drug discovery research project. We have presented the present status and given detailed descriptions of biotransformation, metabolic stability assays, identification of drug metabolizing P450 enzymes, prediction of pharmacokinetic parameters from in vitro metabolism data, structure elucidation of metabolites, CYP450 inhibition assays and CYP450 induction assays from a drug discovery perspective. Strategies for the proper sequencing of primary and secondary assays employedfor drug metabolism and CYP450 inhibition & induction is discussed.

Metabolism of pamaqueside, a cholesterol absorption inhibitor, in Long-Evans rat: effect of bile duct cannulation on absorption

1. The metabolism of pamaqueside, a cholesterol absorption inhibitor, was studied in the bile duct cannulated and non-cannulated rat after an oral dose (100 mg/kg) and an i.v. dose (6 mg/kg) of [14C]pamaqueside. 2. Faeces was the major route of excretion in all rat. Only 0.1% of the radioactivity was recovered in the urine of the non-cannulated rat. In contrast, ∼ 17% of the total dose was recovered in the bile and urine in the bile duct cannulated rat. Following an i.v. dose, an almost equal percentage of radioactivity was excreted in the bile and urine of the bile duct cannulated rat. 3. The aglycone (M1) was the major metabolite in rat and was present in greater amounts in the faeces of the bile duct cannulated rat. 4. The structural elucidation of metabolites in the bile and urine indicated that M1 was metabolized oxidatively via a novel ring opening, and the oxidative metabolites further underwent sulphate conjugation. 5. The oxidative ring opening of pamaqueside (the cellobioside ring intact) was also observed following an i.v. dose to rat suggesting that oxidative ring opening was the major route of metabolism of saponins, at least in rat. 6. The study demonstrated that the absorption and metabolism of pamaqueside was altered by surgical cannulation of rat.

薬物代謝におけるＭＳを用いた代謝物の構造解析とその代謝経路の究明

The aim of drug metabolism study is to elucidate the phenomena associated with the efficacy or the toxicity of a drug by tracing its biotransformation after administration to an organism. The structure determination of metabolites in plasma, urine and bile and the clarification of metabolic pathways have been considered as the important tasks indispensable to the development of a new drug. However the structure determination is not a final object, but the investigation of pharmacological activity of determined metabolites and species differences in metabolism is important for application of the drug candidate to human. In particular, mass spectrometry (MS) has played a major role in this field.This study explained two examples of such elucidation of chemical structure of metabolites and metabolic pathways using MS. In the structural elucidation of metabolites, the most suitable ionization method should be selected for each drug and metabolite from various ionization modes of EI, FAB, and ESI.

On-line liquid chromatography coupled with high field NMR and mass spectrometry (LC-NMR-MS): a new technique for drug metabolite structure elucidation

High performance liquid chromatography has been coupled simultaneously to high field NMR and MS detectors, giving UV, NMR and mass spectra for each component in a mixture, after on-line separation. This powerful new tool for the structure elucidation of components in mixtures without isolation has been successfully applied to the analysis of the metabolites of paracetamol in human urine.

Meloxicam: metabolic profile and biotransformation products in the rat.

1. The metabolic fate of 14C-labelled meloxicam was investigated in the urine and bile of rat following oral and intraduodenal administration. Structural elucidation of metabolites was performed by nuclear magnetic resonance, mass spectrometry (electron impact and fast atom bombardment). 2. A mean total of 76.3% 14C-radioactivity was recovered in urine over 96 h, with the remainder in the faeces. The metabolic pattern in the excreta was independent of dose (1 versus 10 mg/kg) and collection period (0-8 versus 24-48 h). In bile one of the main metabolites was absent. 3. Meloxicam underwent extensive metabolism with only small amounts of unchanged drug recovered in the urine (< 0.5%) or bile (4.5%). Principal routes of biotransformation were: oxidation of the 5-methyl group of the N-heteroaryl-carbamoyl side chain to yield the 5'-hydroxymethyl derivative (33% of metabolites in urine, 22% in bile) and the 5'-carboxy derivative (16% in urine, 49% in bile). Oxidative cleavage of the benzothiazine-ring yielded an oxamic acid metabolite in urine (23.5%), which was not present in bile. 4. The introduction of a methyl-group into the N-heteroaryl-carbamoyl side chain increased lipophilicity and facilitated metabolic excretion compared with structurally related compounds.

Drug metabolite identification: stable isotope methods.

This paper focuses on stable isotope labeling of drugs, in combination with mass spectrometry (MS)-based methods, to facilitate the recognition and identification of metabolites and the employment of stable isotope-labeled derivatization reagents (e.g., bis-trimethylsilylacetamide-d18) in the structure elucidation of metabolites from unlabeled drugs via gas-liquid chromatography-MS techniques. In both cases, it is the so-called isotope peak shift that permits generation of data useful for metabolite identification. Furthermore, judicious labeling of a drug permits characterization of drug-related species (metabolites) by MS-based recognition of isotope cluster signatures. Studies using stable isotope-labeled drugs are exemplified by work on aminopyrine and isopropylantipyrine metabolism; examples of the derivatization peak shift approach include those from studies of timolol and cyproheptadine.

Identification of In Vitro (Rat Liver Postmitochondrial S9 Fraction) Metabolites of the Antiprotozoal Agent 3a,4,5,6,7,7a-Hexahydro-3-(l-methyl-5-nitro-1H-imidazol-2-yl)-l,2-benzisoxazole

Twelve in vitro oxygenated metabolites of 3a,4,5,6,7,7a- hexahydro-3-(l-methyl-5-nitro-lH-imidazol-2-yl) -1,2-benzisoxazole (MK-0436) were produced by incubation of this antiprotozoal agent with the postmitochondriai supernatant (S9) fraction isolated from the livers of rats treated with phenobarbital. Metabolite structure elucidation was achieved using NMR and mass spectrometry. Seven monohydroxy and two dihydroxy metabolites were fully characterized; two other metabolites were partially characterized as dihydroxy derivatives of the drug. The major in vitro metabolite is the 5 axial hydroxy compound, and a minor metabolite is the corresponding ketone. In all cases metabolite formation involved biotransformation on the hexahydrobenzisoxazole ring.

Contributions to ecological chemistry CXLII: Separation and identification of metabolites excreted by rats after long term oral administration of imugan- 14C

Rapid conversion and high rate of excretion of the fungicide, Imugan- 14C (N-formyl-N′ - (3,4-dichlorophenyl)-2, 2, 2-trichloroacetaldehydeaminal, Fig. 1) has been observed in rats upon long term oral administration 2. In simulated waste composting experiments 3 this fungicide is metabolized to 3,4-dichloroaniline (3,4-DCA). 3,4-DCA and 3,3′, 4,4′-tetrachloroazobenzene (3,3′, 4,4′-TCAB) have been isolated from soil treated with it 4. Algae have been found to convert it to a ring hydroxylated monohydroxy 3,3′,4,4′-TCAB 5. In this work, the isolation and structure elucidation of metabolites of Imugan excreted with urine and faeces of rats are reported.

Use of ethyl ethers, deuteriomethyl ethers and cyclic n-butylboronates of hydroxychlorobiphenyls in identification of metabolites of polychlorinated biphenyls

The mass spectra and gas chromatographic properties of a number of chlorobiphenylols, chlorobiphenyldiols, chlorophenols and naphthols, as well as their methyl, deuteriomethyl and ethyl ethers and some cyclic n-butylboronates have been investigated. Metabolism experiments with 4′-chloro-4-biphenylol showed that the selective use of ethylation and methylation is most effective in both detection and structure elucidation of metabolites partly methylated by metabolic processes. The usefulness of deuteriomethylation in such studies seems to be limited. The formation of cyclic n-butylboronates provides specific information on o-dihydroxy derivatives of chlorobiphenyls.

Preparation, Gas Chromatographic Behavior, and Spectroscopic Properties of Hydroxylated Chlorobiphenyls

Abstract Some useful methods for the preparation of chlorobiphenylols have been investigated. Twenty monohydroxy and dihydroxy derivatives of chlorobiphenyls are described; 10 are new compounds and several are known metabolic products from animals or microorganisms. The relationship between structure of the chlorobiphenylols and gas chromatographic retention time as well as electron capture detector response, including those of derivatives, has been examined. The ultraviolet, fluorescence, and mass spectra of these hydroxylated derivatives have also been examined with a view towards analysis and structure elucidation of metabolites.

Pharmacology of pyrazoles I: Structure elucidation of metabolites of 4-methylpyrazole

The metabolism in mice of 4-methylpyrazole (4-MP), a potent inhibitor of alcohol dehydrogenase, has been investigated using gas chromatography and gas chromatography-mass spectrometry techniques. Radioactive 4-MP was synthesized to aid in the isolation of the metabolites. Of the administered radioactivity, 84% was recovered in the urine 24 hours after treatment. Analysis of the urine revealed the presence of several metabolites including 4-hydroxymethylpyrazole, 4-carboxypyrazole and 4-methylpyrazole-N-glucosiduronic acid, along with the parent compound, 4-methylpyrazole.