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.     

盐酸丁咯地尔在人血浆和肝微粒体中代谢产物鉴定

目的研究盐酸丁咯地尔在人体的代谢情况。方法采用UPLC-QTOF/MS法发现并鉴定盐酸丁咯地尔在人血浆和肝微粒体孵化体系中的代谢产物。采用HSS T3 C18色谱柱(100 mm×2.1 mm,1.8μm),以5 mmol·L~(-1)醋酸铵水溶液(含体积分数0.05%的甲酸)(A)-甲醇(B)为流动相梯度洗脱,流速为0.4 m L·min~(-1),采用ESI离子源,正离子模式下检测。结果通过与空白生物样品进行比较,在人血浆和肝微粒体中共发现了丁咯地尔及其24种代谢产物,其中18种为首次发现的代谢产物。根据代谢产物的色谱保留时间、准分子离子及碎片离子等信息,确定并推断了丁咯地尔及其代谢产物的结构,阐明了丁咯地尔在人体的代谢途径,包括去甲基化、氧化、羟基化、硫酸和葡萄糖醛酸结合途径。结论建立了UPLC-QTOF/MS方法研究了丁咯地尔在人体的代谢情况,为其化学结构类似药物和候选化合物的代谢研究提供依据。     

Identification of trimetazidine metabolites in human urine and plasma

1. The objective was to use modern mass spectrometric techniques to update current information on the metabolism of trimetazidine in human subjects found by previous studies.

2. Urine and plasma samples were taken from four healthy human volunteers taking part in a larger kinetic study. Each subject received an oral dose of 80-mg trimetazidine daily for 4 days.

3. Identification and quantitation of trimetazidine and its metabolites in urine and plasma were achieved using modern liquid chromatography-mass spectrometric methods.

4. The major drug-related component observed in urine and plasma was unchanged trimetazidine. In addition to the parent drug, 10 metabolites were detected in urine in concentrations ranging from 0.008 (0.01% dose) to 1.094 μg.ml^-1 (1.4% dose). Metabolic profiles following acute and chronic doses of trimetazidine were qualitatively similar.     

Metabolism of (2S)-pterosin A: identification of the phase I and phase II metabolites in rat urine

The metabolic profile of the potent hypoglycemic agent, (2S)-pterosin A (1), in rat urine via intragastrical oral administration was investigated. In total, 19 metabolites (M1-M19) were identified. Among these, 16 metabolites were characterized by high-performance liquid chromatography solid-phase extraction-tube transfer-NMR, and seven metabolites were further isolated from the treated urine to enable further structural determination. Twelve of these are new compounds. The phase I metabolites of 1 were formed via various oxidations at positions C-3, C-10, C-12, C-13, or C-1 followed by decarboxylation of C-10 or C-14, and lactonization at C-12/C-14 or C-14/C-12. The phase II metabolites were glucuronide conjugates from the parent compound or phase I metabolites. The major metabolites were found to be (2S)-14-O-glucuronylpterosin A (M9), (2S)-2-hydroxymethylpterosin E (M14), and (±)-pterosin B (M19). Quantitative HPLC analysis of metabolites, based on similar UV absorption and use of the regression equation of 1, indicated that ～71% 1 was excreted as metabolites in rat urine.     

盐酸关附甲素对大鼠心室肌细胞膜L型钙通道(ICa-L)的影响

目的研究盐酸关附甲素对正常大鼠心室肌细胞L型钙离子通道(ICa-L)的影响.方法应用膜片钳全细胞记录法,记录关附甲素对ICa-L的作用并对通道动力学进行初步评价.结果关附甲素呈浓度依赖性阻滞ICa-L,在25,125,250和1 000μm01·L-1时ICa-L峰值电流密度(去极化至0 mV时)分别减少为用药前的81.76%,77.54%,76.46%和64.77%(P＜0.05).关附甲素使ICa-L的Ⅰ-V曲线上移,但不改变其激活电位、峰值电位.关附甲素呈轻度使用依赖性阻滞ICa-L,对ICa-L静息态有阻滞作用.关附甲素对ICa-L激活曲线无明显影响,但使ICa-L失活后再激活的恢复时间常数(τ)延长,有作用于ICa-L的失活态的可能.结论因为关附甲素产生明显ICa-L阻滞的浓度远大于临床浓度,其对ICa-L的阻滞作用可能不是抗心律失常的主要作用机制.关附甲素呈浓度依赖性阻滞ICa-L,其作用于L型钙通道的静息态,也有可能作用于失活态.     

Identification of the n-heptane metabolites in rat and human urine

Numerous n-heptane metabolites have been identified and quantified by gas chromatography and mass spectrometry in some tissues and in the urine of Sprague Dawley rats exposed for 6 h to 1800 ppm n-heptane. 2-Heptanol and 3-heptanol were the main biotransformation products of the solvent. 2-Heptanone, 3-heptanone, 4-heptanol, 2,5-heptanedione, -valerolactone, 2-ethyl-5-methyl-2, 3-dihydrofuran and 2,6-dimethyl-2,5-dihydropyran were also found as metabolites of n-heptane. In five shoe factory workers and in three rubber factory workers the mean exposure to technical heptane was measured (n-heptane ranged between 5 and 196 mg/m³). In the urine collected at the end of their work shift some n-heptane biotransformation products were found: 2-heptanol, 3-heptanol, 2-heptanone, 4-heptanone and 2,5-heptanedione. 2-Heptanol was the main n-heptane metabolite and its urinary concentrations ranged between 0.1 and 1.9 mg/l. Urinary 2,5-heptanedione was detectable only in some samples and at very low concentration (0.1–0.4 mg/1).These data suggest that n-heptane can be considered as a neurotoxic product, since it gives rise to 2,5-heptanedione, but the small amount of the urinary metabolite is very unlikely to cause clinical damage to the peripheral nervous system.Part of this work was presented at the 48 Congr. Naz. Soc. Ital. Med. Lav. Igiene Ind. Pavia 18–21 September 1985     

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.     

Identification of thymol phase I metabolites in human urine by headspace sorptive extraction combined with thermal desorption and gas chromatography mass spectrometry

Development of a novel highly sensitive headspace sorptive extraction (HSSE) method in combination with thermal desorption gas chromatography coupled to a mass spectrometer (TD-GC/MS) allowed the identification of thymol and several phase I metabolites in human urine. Combined with an enzymatic hydrolysis of glucuronated or sulphated phase II metabolites of thymol and of the respective phase I metabolites prior to analysis, even trace quantities of hitherto not detected thymol phase I metabolites could be identified in urine samples of test persons after oral administration of 50 mg thymol. It was proven, that human metabolism leads to a hydroxylation of the aromatic ring as well as of the iso-propyl side chain. Hydroxylation of the iso-propyl group results in the formation of the rather unstable p-cymene-3,8-diol and the corresponding dehydration product p-cymene-3-ol-8-ene which could be clearly detected in human urine samples. Furthermore, the aromatic hydroxylation products p-cymene-2,5-diol, its oxidation product p-cymene-2,5-dione and p-cymene-2,3-diol were also unambiguously identified by comparison with synthesized reference compounds.     

Human moricizine metabolism. I. Isolation and identification of metabolites in human urine

1. Using synthetic standards and or spectral data, seven moricizine metabolites were structurally identified in human urine. Two novel metabolites were identified as phenothiazine-2-carbamic acid and ethyl [10-(3-aminopropionyl) phenothiazin-2-yl] carbamate. Two novel human moricizine metabolites, 2-amino-10-(3-morpholinopropionyl) phenothiazine, a previously identified dog metabolite, and 2-aminophenothiazine, a previously identified rat metabolite, were also identified. Three additional human metabolites, phenothiazine-2-carbamic acid ethyl ester sulphoxide (P2CAEES), moricizine sulphoxide, and ethyl {10-[N-(2′-hydroxyethyl)3-aminopropionyl] phenothiazin-2-yl} carbamate, all previously described in the literature, were observed. 2. Both 2-amino-10-(3-morpholinopropionyl) phenothiazine and ethyl [10-(3- aminopropionyl) phenothiazin-2-yl] carbamate, and possibly ethyl {10-[N-(2′-hydroxyethyl)3-aminopropionyl]phenothiazin-2-yl} carbamate, possess the structural characteristics thought to be necessary for class 1 antiarrhythmic activity.     

l-bunolol metabolites in human urine.

Nine radiolabeled compounds were identified in human urine after administering a single oral dose of 3H-l-bunolol (3 mg) to 5 male volunteers. These compounds represented 54.7% of the dose and 71.4% of the isotope excreted in 3 days. Intact bunolol accounted for 14.7% of the dose and its conjugates totaled an additional 5.0%. The major drug metabolite (28.2% of dose) was dihydrobunolol, a reduction product known to have the same pharmacological activity and potency as bunolol. Dihydrobunolol conjugates amounted to 3.9% of the dose. Two minor acidic metabolites were produced by oxidative cleavage of the bunolol side chain, and another minor metabolite (hydroxydihydrobunolol) resulted from both reductive and oxidative biotransformation. Bunolol metabolism in man showed qualitative and quantitative differences from patterns observed in the rat and dog.     

Microbial production of phase I and phase II metabolites of propranolol

1. The production in multimilligram amounts of 4- and 5-hydroxylated metabolites of (R)- or (S)-propranolol by biotransformation with two fungal strains, an Absidia sp. M50002 and a Cunninghamella sp. M50036, was carried out, starting from either the racemic drug or pure enantiomers.

2. While both enantiomers of propranolol were hydroxylated in the 5-position by incubation with strain M50002, the strain M50036 operated a chiral discrimination, resulting in the exclusive formation of the 4-hydroxy-(R)-enantiomer.

3. In addition, a Streptomyces sp. strain M52104, isolated from a soil sample, was selected for the high-yield regioselective β-glucuronidation of propranolol and its 4- and 5-hydroxylated derivatives.

4. NMR and mass spectroscopic data have been extensively used for the unambiguous characterization of 4- and 5-hydroxylated and glucuronidated derivatives, all of them corresponding to the major animal and human metabolites of propranolol, a typical substrate of CYP2D6.

     

Microbial production of phase I and phase II metabolites of midazolam

Midazolam, a potent benzodiazepine derivative and a typical substrate of CYP3A4/3A5, is essentially metabolized in human into 1'-hydroxymidazolam, then eliminated as the corresponding phase II metabolite, the 1'-O-β-D-glucuronide derivative. A high yield alternative to the current multistep synthesis of 1'-hydroxymidazolam is described, using a biotransformation of midazolam by a fungal microorganism, Beauveria bassiana. The corresponding phase II metabolite, 1'-hydroxymidazolam-1'-O-β-D-glucuronide, has been then prepared by chemical synthesis (3 steps, 20% yield), or by microbial glucuronidation (one step, 20% yield) using a Streptomyces sp. strain. The use of the same Streptomyces strain allows a direct and expeditive synthesis of the same glucuronide conjugate from midazolam itself in an advantageous 17% yield.     

Microbial production of phase I and phase II metabolites of propranolol

1. The production in multimilligram amounts of 4- and 5-hydroxylated metabolites of (R)- or (S)-propranolol by biotransformation with two fungal strains, an Absidia sp. M50002 and a Cunninghamella sp. M50036, was carried out, starting from either the racemic drug or pure enantiomers. 2. While both enantiomers of propranolol were hydroxylated in the 5-position by incubation with strain M50002, the strain M50036 operated a chiral discrimination, resulting in the exclusive formation of the 4-hydroxy-(R)-enantiomer. 3. In addition, a Streptomyces sp. strain M52104, isolated from a soil sample, was selected for the high-yield regioselective β-glucuronidation of propranolol and its 4- and 5-hydroxylated derivatives. 4. NMR and mass spectroscopic data have been extensively used for the unambiguous characterization of 4- and 5-hydroxylated and glucuronidated derivatives, all of them corresponding to the major animal and human metabolites of propranolol, a typical substrate of CYP2D6.     

Identification of polyphenols and their metabolites in human urine after cranberry-syrup consumption

As the beneficial effects of American cranberry (Vaccinium macrocarpon) can be partly attributed to its phenolic composition, the evaluation of the physiological behaviour of this fraction is crucial. A rapid and sensitive method by ultra-performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) has been used to identify phenolic metabolites in human urine after a single dose of cranberry syrup. Prior to the analysis, metabolites were extracted using an optimised solid-phase extraction procedure. All possible metabolites were investigated based on retention time, accurate mass data and isotope and fragmentation patterns. Free coumaroyl hexose (isomer 1 and 2), dihydroxybenzoic acid, caffeoyl glucose, dihydroferulic acid 4-O-β-d-glucuronide, methoxyquercetin 3-O-galactoside, scopoletin, myricetin and quercetin, together with other 23 phase-I and phase-II metabolites, including various isomers, could be tentatively identified in the urine. Afterwards, the metabolites were simultaneously screened in the urine of different subjects at 0, 2, 4, and 6 h after the ingestion of cranberry syrup by Target Analysis^TM software.     

Excretion of mephedrone and its phase I metabolites in urine after a controlled intranasal administration to healthy human volunteers

Mephedrone is a stimulant drug structurally related to cathinone. At present, there are no data available on the excretion profile of mephedrone and its metabolites in urine after controlled intranasal administration to human volunteers. In this study, six healthy male volunteers nasally insufflated 100 mg of pure mephedrone hydrochloride (Day 1). Urine was collected at different timepoints on Day 1 and then on Days 2, 3 and 30. Samples were analysed for the presence of mephedrone and its metabolites, namely, dihydro-mephedrone, nor-mephedrone (NOR), hydroxytolyl-mephedrone, 4-carboxy-mephedrone (4-carboxy) and dihydro-nor-mephedrone (DHNM), by a validated liquid chromatography-tandem mass spectrometry method. All analytes were detected in urine, where 4-carboxy (C_max = 29.8 μg/ml) was the most abundant metabolite followed by NOR (C_max = 377 ng/ml). DHNM was found at the lowest concentrations (C_max = 93.1 ng/ml). Analytes exhibited a wide range of detection windows, but only 4-carboxy and DHNM were detectable in all samples on Day 3, extending the detection time of mephedrone use. Moreover, mephedrone had a mean renal clearance of 108 ± 140 ml/min, and 1.3 ± 1.7% of unchanged parent drug was recovered in urine in the first 6 h post administration. It is hoped that this novel information will be useful in future studies involving mephedrone and other stimulant drugs.     

Amitriptyline metabolites in human urine. Identification of phenols, dihydrodiols, glycols, and ketones

From the urine patients being treated with amitriptyline, drug metabolites were extracted by adsorption to polystyrene. Nonconjugated compounds and aglycones liberated by enzymic hydrolysis were purified separately by repeated TLC and characterized by physicochemical and chemical methods. Besides the known E- and Z-10-hydroxy derivatives of amitriptyline (AT), nortriptyline (NT), and their primary amine analogue, two isomeric 10,11-dihydroxy compounds could be identified in each series. Metabolites with an oxo function in position 10 occurred in minor quantities. The phenols 2-hydroxy-NT and 2,11-dihydroxy-NT, as well as the 1,2-dihydrodiol derived from NT, were regularly present, while the corresponding tertiary amines as well as 3-hydroxy-AT and -NT were detected occasionally in very small amounts.