用液相质谱法鉴定盐酸关附甲素在大鼠胆汁中Ⅰ相和Ⅱ相代谢产物(英文)

目的:研究盐酸关附甲素在大鼠胆汁中的代谢产物.方法:建立了液相质谱和串联质谱法(LC-MS)对关附甲素及其代谢产物鉴定的方法.大鼠静脉注射盐酸关附甲素后采集胆汁,通过与对照化合物的色谱保留时间、分子离子峰、碎片离子峰和紫外图谱对照从而鉴定Ⅰ相代谢物.胆汁经过葡萄糖醛酸酶或硫酸酯酶水解,鉴定其水解产物(苷元),从而确定Ⅱ相结合物,再通过LC-MS分离和确定分子离子峰,最后利用MS-MS寻找特征子离子和母离子的方法进行验证.结果:大鼠胆汁中存在Ⅰ相代谢物关附壬素;Ⅱ相结合物经葡萄糖醛酸酶和硫酸酯酶酶解后,出现关附甲素和关附壬素;LC-MS检测发现胆汁中m/z 606和 510两个准分子离子峰,推测分别为关附甲素葡萄糖醛酸苷和关附甲素硫酸酯:经MS-MS鉴定出m/z 606特征子离子m/z 177和m/z 430,进一步确证大鼠胆汁中存在关附甲素葡萄糖醛酸苷.结论:大鼠胆汁中存在Ⅰ相代谢产物关附壬素,以及Ⅱ相结合物关附甲素葡萄糖醛酸和硫酸结合物、关附壬素葡萄糖醛酸和硫酸结合物.     

Identification of ginkgolic acid (15:1) metabolites in rats following oral administration by high-performance liquid chromatography coupled to tandem mass spectrometry

1.?In this article, metabolites of ginkgolic acid (GA) (15:1) in rats plasma, bile, urine and faeces after oral administration have been investigated for the first time by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) with the aid of on-line hydrogen/deuterium (H/D) exchange technique and β-glucuronidase hydrolysis experiments.

2.?After oral administration of GA (15:1, M0) to rats at a dose of 10?mg/kg, it was found that metabolites M1-M5 together with parent compound (M0) existed in rat plasma; parent compound (M0) and metabolites M2–M5 were observed in rat bile, and parent compound (M0) with metabolites M1 and M2 were discovered in rat faeces, and there was no parent compound and metabolite detectable in rat urine.

3.?Two oxidative metabolites of GA (15:1, M0) were identified as 2-hydroxy-6-(pentadec-8-enyl-10-hydroxy) benzoic acid (M1) and 2-hydroxy-6-(pentadec-8-enyl-11-hydroxy-13-carbonyl) benzoic acid (M2), respectively. Metabolites M3, M4 and M5 were identified as the mono-glucuronic acid conjugates of parent compound (M0), M1 and M2, respectively.

4.?The results indicated that M1 and M2 with parent compound (M0) were mainly eliminated in faeces and three glucuronide metabolites (M3, M4 and M5) excreted in bile as the predominant forms after oral administration of GA (15:1) to rats.     

Characterization of the metabolites of alosetron in experimental animals and human

The metabolism of radiolabelled alosetron was studied in rat, dog, rabbit, mouse and human. The metabolism in rat and dog was studied at a low and an elevated dose designed to generate sufficient quantities of metabolite for definitive identification. A strategy for the characterization of metabolites in cases of extensive metabolism was developed and demonstrated for alosetron. Semi-preparative high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR) and liquid chromatography-nuclear magnetic resonance (HPLC-NMR) enabled the isolation and characterization of 28 metabolites of alosetron. The characterization of the metabolites in animal excreta facilitated the identification of human systemic metabolites.     

大鼠肠内菌转化连翘苷的代谢产物

应用液相色谱-串联质谱法研究肠内菌转化连翘苷的代谢产物.将连翘苷与大鼠肠内菌于体外厌氧温孵培养,在温孵液中检测到连翘苷及其3种代谢产物.放大制备了转化产率高的代谢物,依据1H NMR和ESI-MS数据推测代谢物结构并假设了连翘苷可能的代谢途径.     

大鼠肠内菌转化间尼索地平的代谢产物

应用LC-MS/MS法研究肠内菌转化间尼索地平的代谢产物.将间尼索地平与大鼠肠内菌于体外厌氧温孵培养,放大制备转化产率最高的代谢物.依据1H NMR和ESI-MS/MS数据确认代谢物结构,推断可能的代谢途径.结果在温孵液中发现了间尼索地平及其2种代谢产物.     

Metabolism of the A(1)1 adenosine receptor positron emission tomography ligand [18F]8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine ([18F]CPFPX) in rodents and humans.

Studies of plasma from mice, rats, and human volunteers evaluated methods for the extraction and quantification of the positron emission tomography ligand [(18)F]8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine ([(18)F]CPFPX) and identification of its metabolites in plasma by thin-layer chromatography and high-performance liquid chromatography (HPLC). Analysis of human, mouse, and rat plasma extracts by HPLC identified four identical radioactive metabolites in each species. The low mass of radioligand administered to humans (0.5 - 5 nmol) prevented direct identification of metabolites. However, incubating liver microsomes with CPFPX and analysis by means of liquid chromatography-mass spectrometry (LC-MS) identified seven compounds, four having the same retention times as the metabolites in human plasma. Analysis of microsomal metabolites by LC-MS identified five [M + H](+) ions of m/z equivalent to hydroxy derivatives, 339, one of m/z equivalent to an oxo derivative, m/z 337, and one of m/z equivalent to a difunctionalized oxo-desaturation species, m/z 335, which is prominent in rat and mouse plasma and is the main metabolite in human plasma. An [M + H](+) ion corresponding to a N-dealkylated derivative was not detected. Thus, like the natural methylxanthines, CPFPX seems to undergo oxidation by liver microsomes but, unlike those methylxanthines, dealkylation did not occur. LC-MS experiments with "in source" fragmentation identified the cyclopentyl moiety to be the most functionalized part of the molecule by liver microsomes and in vivo oxidations. Except for two metabolites, hydroxylated at the N1 propyl chain, all oxidative modifications found took place at the cyclopentyl ring.     

Sensitive and specific liquid chromatographic-tandem mass spectrometric assay for atropine and its eleven metabolites in rat urine

A sensitive and specific method is described for the simultaneous determination of atropine and its metabolites in rat urine by combining liquid chromatography and tandem mass spectrometry (LC-MS(n)). Various extraction techniques (free fraction, acid hydrolyses and enzyme hydrolyses) and their comparison were carried out for investigation of the metabolism of atropine. After extraction procedure the pretreated samples were separated on a reversed-phase C18 column using a mobile phase of methanol/ammonium acetate (2 mM, adjusted to pH 3.5 with formic acid) (70: 30,v/v) and detected by an on-line LC-MS(n) system. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular masses (DeltaM), retention-times and full scan MS(n) spectra with those of the parent drug. The results revealed that at least eleven metabolites (N-demethyltropine, tropine, N-demethylatropine, p-hydroxyatropine, p-hydroxyatropine N-oxide, glucuronide conjugates and sulfate conjugates of N-demethylatropine, p-hydroxyatropine and the parent drug) and the parent drug existed in rat urine after ingesting 25 mg/kg atropine. p-Hydroxyatropine and the parent drug were detected in rat urine for up 106 h after ingestion of atropine.     

Correlative analysis of metabolite profiling of Danggui Buxue Tang in rat biological fluids by rapid resolution LC-TOF/MS

In this work, the metabolite profiles of Danggui Buxue Tang (DBT) in rat bile and plasma were qualitatively described, and the possible metabolic pathways of DBT were subsequently proposed. Emphasis was put on correlative analysis of metabolite profiling in different biological fluids. After oral administration of DBT, bile and plasma samples were collected and pretreated by solid phase extraction. Rapid resolution liquid chromatography coupled to time-of-flight mass spectrometry (RRLC-TOFMS) was used for characterization of DBT-related compounds (parent compounds and metabolites) in biological matrices. A total of 142 metabolites were detected and tentatively identified from the drug-containing bile and plasma samples. Metabolite profiling shows that rat bile contained relatively more glutathione-derived conjugates, more saponins compounds and more diverse forms of metabolites than urine. The metabolite profile in plasma revealed that glucuronide conjugates of isoflavonoids, dimmers, acetylcysteine conjugates and parent form of phthalides, as well as saponin aglycones were the major circulating forms of DBT. Collectively, the metabolite profile analysis of DBT in different biological matrices provided a comprehensive understanding of the in vivo metabolic fates of constituents in DBT.     

Characterization of ornidazole metabolites in human bile after intraveneous doses by ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry

Ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) was used to characterize ornidazole metabolites in human bile after intravenous doses. A liquid chromatography tandem mass spectrometry (LC–MS/MS) assay was developed for the determination of the bile level of ornidazole. Bile samples, collected from four patients with T-tube drainage after biliary tract surgery, were prepared by protein precipitation with acetonitrile before analysis. A total of 12 metabolites, including 10 novel metabolites, were detected and characterized. The metabolites of ornidazole in human bile were the products of hydrochloride (HCl) elimination, oxidative dechlorination, hydroxylation, sulfation, diastereoisomeric glucuronation, and substitution of NO₂ or Cl atom by cysteine or N-acetylcysteine, and oxidative dechlorination followed by further carboxylation. The bile levels of ornidazole at 12 h after multiple intravenous infusions were well above its minimal inhibitory concentration for common strains of anaerobic bacteria.     

Metabolism and disposition of the flame retardant tris(2,3-dibromopropyl)phosphate in the rat

The metabolism and disposition of the flame retardant, tris(2,3-dibromopropyl)phosphate (Tris-BP), were studied after po and iv administration of the 14C-labeled compound to the male rat. Tris-BP was readily absorbed from the gastrointestinal tract and rapidly distributed throughout the body. The distribution and excretion of Tris-BP derived radioactivity were similar after either po or iv administration. The only effects of route of administration on tissue distribution were slightly higher concentrations in liver after po administration and in lung after iv administration. The initial elimination of Tris-BP derived radioactivity in urine, feces, and as CO2 accounted for approximately 50% of the dose in 24 hr. An analysis of Tris-BP derived radioactivity remaining in the tissues one day after administration indicated that most of the radioactivity in all tissues was in the form of various metabolites rather than the parent compound. The terminal clearance of Tris-BP derived radioactivity from most of the tissues studied was best described by a single component exponential decay with a half-life of approximately 2.5 days. Clearance from liver and kidney was somewhat slower having a half-life of approximately 3.8 days. Approximately 33% of the radioactivity excreted in urine and approximately 50% of the radioactivity excreted in bile were identified by cochromatography with synthesized standards on high performance liquid chromatography (HPLC). Six metabolites and a trace of the parent compound were identified in urine and bile by this method. The six metabolites products of dealkylation and dehydrobromination of the parent compound. The metabolites of Tris-BP isolated from urine and bile were also formed in vitro by NADPH-dependent microsomal enzymes from rat liver. The soluble enzymes from liver metabolized Tris-BP to at least three unidentified polar metabolites.     

Toxicokinetics and metabolism of 1,2-diethylbenzene in male Sprague Dawley rats--part 2: evidence for in vitro and in vivo stereoselectivity of 1,2-diethylbenzene metabolism.

In a previous study, it was shown that the neurotoxic compound 1,2-diethylbenzene (1,2-DEB) is mainly hydroxylated in the alkyl chain to give 1-(2'-ethylphenyl)ethanol (1,2-EPE) and excreted in urine of rats as two glucuronide compounds (GA1 and GA2). Some findings have suggested that the two enantiomers of 1,2-EPE are formed in vivo. In the present study, a chiral high-performance liquid chromatography method was developed to separate the two enantiomers of 1,2-EPE from a synthesized racemic mixture. Absolute configuration of both enantiomers was determined after esterification with (R)-(+)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetic acid and analysis of their (1)H NMR spectra in CCl(4) added with Eu (fod)(3). The two main urinary metabolites, GA1 and GA2, from [(14)C]1,2-DEB-treated Sprague-Dawley rats (80 mg/kg, i.p.) were identified, after hydrolysis with beta-glucuronidase from Escherichia coli, as (R) and (S) glucuronide conjugates of 1,2-EPE, respectively. In vitro hydroxylation of 1,2-DEB and glucuroconjugation of 1,2-EPE were under stereoselective control in S9 fraction or microsomes from male Sprague-Dawley rat liver. The V(max) and K(m) constants for (R)1,2-EPE enantiomer formation determined in S9 fraction were greater than those for the (S) enantiomer. In the plasma of bile duct-cannulated rats, the ratio was 1.2 +/- 0.02 over the 1- to 4-h period after oral administration of [(14)C]1,2-DEB (100 mg/kg). In contrast, the glucuroconjugation rate of (S)1,2-DEB enantiomer was 4 times that of (R)1,2-EPE glucuroconjugation. A similar ratio of (R) to (S)1,2-EPE glucuronide conjugates was obtained in the plasma of bile duct-cannulated rats.     

Hepatic uptake, disposition, and metabolism of rubratoxin B in isolated perfused rat liver

Isolated perfused rat liver preparations were utilized to measure the hepatic uptake, biliary excretion and metabolism of rubratoxin B. Livers were perfused with 30% rat blood perfusate containing 0.24 μmol labeled rubratoxin B, and a series of timed blood and bile samples were analyzed by high-pressure liquid chromatography, and treated enzymatically for the determination of glucuronide and sulfate conjugates. Blood, bile and liver samples were also radioassayed. Rubratoxin B was removed from the perfusate by a first-order process (monophasically) with a half-life of 207.5 ± 23.7 min (mean ± SE). By 3.5 hr of perfusion, 30% of the total rubratoxin B-derived radioactivity was excreted into the bile. More than 8% of the total dose of rubratoxin B was excreted unchanged into the bile by 3.5 hr. The rates of biliary excretion of rubratoxin B- derived radioactivity and parent compound reached a maximum at 30 min, after which time the rates of excretion decreased monophasically with half-lives of 35.5 and 72 min, respectively. Two major metabolites detected in the bile were the glucuronide and sulfate conjugates, together accounting for 22% of the radioactivity excreted into the bile by 3.5 hr. In addition, at least one major unidentified organosoluble metabolite was detected in the bile.     

LC-MS/MS法快速分析大鼠尿液中他喷他多的代谢物

目的建立快速分析大鼠尿液中他喷他多代谢物的方法。方法大鼠灌胃给予他喷他多,收集空白和给药后0～12 h尿液,以液相色谱-串联质谱法,采用多离子反应监测(multiple reactionmonitoring,MRM)及二级全扫描质谱(full scan MS2)方式,分析尿液中他喷他多的代谢物。结果在大鼠尿液中发现了他喷他多原形药物及其15种代谢物,首次发现了他喷他多脱氢化合物、他喷他多脱氢葡萄糖醛酸结合物。结论本方法简便、快速,适用于大鼠尿液中他喷他多代谢物结构及代谢路径分析。     

Identification of novel metabolites of pioglitazone in rat and dog

1. Four new metabolites of pioglitazone were identified by liquid chromatography-mass spectrometry (LC-MS/MS) as being formed by hydroxylation (M-VII and M-VIII), opening of the thiazolidinedione ring (M-X) and by desaturation of the terminal ethyl side chain or tether ethoxy moiety (M-IX), respectively. The structure of one of the hydroxylated metabolites (M-VII) was confirmed by chemical modification using the Jones reaction. 2. Oxidative cleavage of the thiazolidinedione ring is a novel pathway not previously reported for pioglitazone. 3. The hydroxylated M-VII was detected in incubations with rat, dog and human liver and kidney microsomes, and in plasma from rats and dogs dosed orally with [(3)H]pioglitazone. 4. The carboxylic acid derivative of M-VII (M-V) and its taurine conjugate were the major radioactive components in dog bile.     

Characterization by liquid chromatography-nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry of two coupled oxidative-conjugative metabolic pathways for 7-ethoxycoumarin in human liver microsomes treated with alamethicin.

The microsomal metabolism of 7-ethoxycoumarin (7-EC) was investigated using liquid chromatography (LC)-NMR and liquid chromatography-mass spectrometry (LC-MS) to characterize the coupling of oxidative-conjugative metabolism events. Within microsomes, cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs) are spatially disparate, each having surface and luminal localization, respectively. To optimize cofactor and substrate transit to UGT without compromising P450 activity, the pore-forming peptide alamethicin was used for microsomal perforation. Aqueous extracts of microsomal incubations containing NADPH and UDP-glucuronic acid were injected for LC-NMR and LC-MS analysis. The analytical complementarity of LC-NMR and LC-MS permitted the identification of four metabolites (M1 to M4). The metabolites M1 and M2 are novel microsomal metabolites for 7-EC, consistent with 3-hydroxylation and subsequent glucuronidation, respectively. Metabolites M3 and M4 were 7-hydroxycoumarin (7-HC) and 7-HC glucuronide, respectively. Viewed collectively, these results illustrate the utility of alamethicin in the examination of coupled oxidative-conjugative metabolism and the synergy of LC-NMR and LC-MS in metabolite identification.     

Identification of novel metabolites of pioglitazone in rat and dog

1. Four new metabolites of pioglitazone were identified by liquid chromatography-mass spectrometry (LC-MS/MS) as being formed by hydroxylation (M-VII and M-VIII), opening of the thiazolidinedione ring (M-X) and by desaturation of the terminal ethyl side chain or tether ethoxy moiety (M-IX), respectively. The structure of one of the hydroxylated metabolites (M-VII) was confirmed by chemical modification using the Jones reaction. 2. Oxidative cleavage of the thiazolidinedione ring is a novel pathway not previously reported for pioglitazone. 3. The hydroxylated M-VII was detected in incubations with rat, dog and human liver and kidney microsomes, and in plasma from rats and dogs dosed orally with [3 H]pioglitazone. 4. The carboxylic acid derivative of M-VII (M-V) and its taurine conjugate were the major radioactive components in dog bile.     

液相色谱-串联质谱法分析抗肿瘤新药乙烷硒啉在大鼠体内的代谢产物

本研究对抗肿瘤新药1,2-[二(1,2-苯并异硒唑-3(2H)-酮)]乙烷(乙烷硒啉,BBSKE)在大鼠体内的代谢产物进行鉴定。在灌胃给予大鼠单剂量乙烷硒啉200mg·kg-1后,采用液相色谱-串联质谱法(LC-MSn)对大鼠尿液、粪样、胆汁和血浆中的代谢产物进行检测,通过全扫描和选择离子扫描,以及根据多级质谱裂解规律对代谢物的结构进行分析。研究发现在大鼠尿样、粪样、胆汁和血浆中检测到3种Ⅰ相代谢产物和1种Ⅱ相代谢产物,其代谢途径分别为氧化、甲基化、硫甲基化和葡萄糖醛酸化反应,提示乙烷硒啉在大鼠体内的代谢方式可能是通过氧化、甲基化及葡萄糖醛酸化反应形成代谢产物。     

Identification of new metabolites of morroniside produced by rat intestinal bacteria and HPLC-PDA analysis of metabolites in vivo

Morroniside, the most abundant iridoid glycoside of traditional Chinese medicines Fructus Corni, was shown to prevent diabetic angiopathies. During the course of our studies on its metabolism by intestinal bacteria, two metabolites (mor-1 and mor-2) were isolated and purified by thin layer chromatography (TLC) and preparative high performance liquid chromatography (HPLC), and then identified as nitrogen-containing compounds along with the known aglycones on the basis of mass spectrometry (MS), and by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Mor-1 and mor-2 were proved to be new compounds. The structures of the metabolites of morroniside detected in rat urine, bile, feces and contents of intestine after oral administration of morroniside proved to be identical with those of the microbial metabolites mor-1 and mor-2.     

Simultaneous determination of glycyrrhizin, glycyrrhetic acid and glycyrrhetic acid mono-glucuronide in Shakuyaku-kanzo-to incubated with rat feces by semi-micro high-performance liquid chromatography

A method for semi-micro high-performance liquid chromatography (HPLC) has been established for the simultaneous determination of glycyrrhizin (GL), glycyrrhetic acid (GA) and glycyrrhetic acid mono-glucuronide (GAMG) in incubation mixtures of rat feces with Shakuyaku-kanzo-to decoction (combination of licorice root and peony root). The analysis could be accomplished within 20 min with a TSKgel ODS-80TsQA (150 x 2.0 mm i.d.) column by linear gradient elution using a mobile phase containing aqueous phosphoric acid and acetonitrile at a flow rate of 0.2 ml x min(-1), a thermostatic oven at 25 degrees C, and detection at 254 nm. The detection limits of these compounds were 0.1-0.85 pmol per injection (5 microl). The concentrations of GL and its metabolites in the incubation mixture after continuous consumption of Shakuyaku-kanzo-to were significantly different compared with those of untreated control. GL-hydrolysis of rat feces was enhanced by pre-consumption of Shakuyaku-kanzo-to.