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11.
Brandon EF van Ooijen RD Sparidans RW Lázaro LL Heck AJ Beijnen JH Schellens JH 《Journal of mass spectrometry : JMS》2005,40(6):821-831
The cyclic depsipeptide aplidine is a new anti-cancer drug of marine origin. Four metabolites of this compound were found after incubation with pooled human microsomes using gradient high-performance liquid chromatography with ultraviolet detection. After chromatographic isolation, the metabolites have been identified using nano-electrospray triple quadrupole mass spectrometry. A highly specific sodium-ion interaction with the cyclic structure opens the depsipeptide ring, and cleavage of the amino acid residues gives sequence information when activated by collision-induced dissociation in the second quadrupole. The aplidine molecule could undergo the following metabolic reactions: hydroxylation at the isopropyl group (metabolites apli-h 1 and apli-h 2); C-dealkylation at the N(Me)-leucine group (metabolite apli-da); hydroxylation at the isopropyl group and C-dealkylation at the N(Me)-leucine group (metabolite apli-da/h), and C-demethylation at the threonine group (metabolite apli-dm). The identification of these metabolites formed in vitro may greatly aid the elucidation of the metabolic pathways of aplidine in humans. 相似文献
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In vitro metabolic stability experiments using microsomes or other liver preparations are important components in the discovery and lead-optimization stages of compound selection in the pharmaceutical industry. Currently, liquid chromatography-tandem mass spectrometric (LC-MS/MS) support of in vitro metabolic stability studies primarily involves the monitoring of disappearance of parent compounds, using selected reaction monitoring (SRM) on triple-quadrupole instruments. If moderate to high turnover is observed, separate metabolite identification experiments are then conducted to characterize the biotransformation products. In this paper, we present a novel method to simultaneously perform metabolite screening in addition to the quantitative stability measurements, both within the same chromatographic run. This is accomplished by combining SRM and SRM-triggered, information-dependent acquisition (IDA) of MS/MS spectra on a hybrid triple-quadrupole linear ion trap (QqQLIT) mass spectrometer. Microsomal stability experiments using model compounds, bufuralol, propranolol, imipramine, midazolam, verapamil and diclofenac, were used to demonstrate the applicability of our approach. This SRM + SRM-IDA approach generated metabolic stability results similar to those obtained by conventional SRM-only approach. In addition, MS/MS spectra from potential metabolites were obtained with the enhanced product ion (EPI) scan function of LIT during the same injection. These spectra were correlated to the spectra of parent compounds to confirm the postulated structures. The time-concentration profiles of identified metabolites were also estimated from the acquired data. This approach has been successfully used to support discovery programs. 相似文献
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Montoudis A Boileau S Simoneau L Mounier C Lafond J 《Biomedical chromatography : BMC》2004,18(9):706-713
Alternative HPLC and solid-phase extraction column methods were developed to separate metabolites of enzymes involved in cholesterol metabolism in rabbit liver microsomes: hydroxyl-methylglutaryl-CoA reductase, cholesterol-7alpha-hydroxylase and acyl-CoA:cholesterol acyltransferase. A comparison method of thin-layer chromatography and solid-phase extraction column were assayed to separate substrate and metabolite of hydroxy-methylglutaryl-CoA reductase, whereas for cholesterol-7alpha-hydroxylase and acyl-CoA:cholesterol acyltransferase, this comparison was done between thin layer chromatography and HPLC. The results obtained by the new analytical chromatographic methods are not significantly different than those observed in literature. Moreover a larger percentage recovery was obtained for analysed metabolites. Our results demonstrate the reliability of these alternative chromatographic techniques and showed that they are valuable tools to precisely and rapidly measure the activity of those enzymes. 相似文献
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Dehydroepiandrosterone (DHEA) is an important neurosteroid with neuronal protection and memory enhancement functions. 7α‐OH DHEA and 7β‐OH DHEA are the two important metabolites of DHEA in the brain. We have developed an LC/MS method to quantitatively analyze 7α‐OH DHEA and 7β‐OH DHEA. Chromatographic separation was carried out on a C18 column with gradient elution using mobile phases of formic acid in acetonitrile and in water formic acid. Mass spectral detection was performed with a ThermoFinnigan LCQ advantage quadruple ion trap mass spectrometer with electrospray ionization. Positive ion chromatograms were acquired using single ion monitoring. The protonated molecule was 305 m/z, but the most abundant ion (269 m/z) was used for quantification. This method was validated and applied to investigate the 7‐hydroxylation of DHEA. When incubating DHEA with rat brain microsomes, both 7α‐OH DHEA and 7β‐OH DHEA were observed, but 7α‐OH DHEA was the major metabolite. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献
15.
Characterization of in vitro metabolites of irisflorentin by rat liver microsomes using high‐performance liquid chromatography coupled with tandem mass spectrometry
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Yan‐Wei Jia Zhong‐Qiu Zeng Hai‐Li Shi Jian Liang Yi‐Ming Liu Ya‐Xiong Tang Xun Liao 《Biomedical chromatography : BMC》2016,30(9):1363-1370
Belamcanda chinensis has been extensively used as antibechic, expectorant and anti‐inflammatory agent in traditional medicine. Irisflorentin is one of the major active ingredients. However, little is known about the metabolism of irisflorentin so far. In this work, rat liver microsomes (RLMs) were used to investigate the metabolism of this compound for the first time. Seven metabolites were detected. Five of them were identified as 6,7‐dihydroxy‐5,3′,4′,5′‐tetramethoxy isoflavone (M1), irigenin (M2), 5,7,4′‐trihydroxy‐6,3′,5′‐trimethoxy isoflavone (M3), 6,7,4′‐trihydroxy‐5,3′,5′‐trimethoxy isoflavone (M4) and 6,7,5′‐trihydroxy‐5,3′,4′‐trimethoxy isoflavone (M5) by means of NMR and/or HPLC‐ESI‐MS. The structures of M6 and M7 were not elucidated because they produced no MS signals. The predominant metabolite M1 was noted to be a new compound. Interestingly, it was found to possess anticancer activity much higher than the parent compound. The enzymatic kinetic parameters of M1 revealed a sigmoidal profile, with Vmax = 12.02 μm /mg protein/min, Km = 37.24 μm , CLint = 0.32 μL/mg protein/min and h = 1.48, indicating the positive cooperation. For the first time in this work, a new metabolite of irisflorentin was found to demonstrate a much higher biological activity than its parent compound, suggesting a new avenue for the development of drugs from B. chinensis, which was also applicable for other herbal plants. Copyright © 2016 John Wiley & Sons, Ltd. 相似文献
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Stanisaw Przestalski Halina Kleszczyska Zenon Trela Zofia Spiak Maria Zamarajeva Natalia Glazyrina Andrea Gagelgans 《应用有机金属化学》2000,14(8):432-437
We have studied the effect of triphenyl‐lead chloride on the lipid phase of erythrocyte membranes, on lipid monomolecular layers and Na+/K+‐ATPase of the microsomal fraction of rat brain. It was found that the haemolytic effect induced by this compound occurs when its concentration exceeds 30 µM . The minimal lead concentration inducing measurable effects in monomolecular lecithin layers is about 1 µM . Inhibition of Na+/K+‐ATPase activity begins at a concentration exceeding 0.5 µM . Maximum inhibition is observed at around 40 µM —a concentration at which haemolysis also occurs. It can thus be thought that at very low lead concentrations the main (or exclusive) role in modifying membrane function is played by direct interaction between lead and the sulphydryl groups of ATPase, whereas at higher concentrations two effects seem to overlap: direct interaction between lead and enzymic proteins via their sulphydryl groups and as indirect influence on the proteins via changes in the organization of the lipid phase of the membrane. Copyright © 2000 John Wiley & Sons, Ltd. 相似文献
18.
以睾酮为探针采用高效液相色谱法测定细胞色素CYP450 3A4的酶活性 总被引:6,自引:0,他引:6
建立了一种快速、高效的以睾酮作为探针药物评价细胞色素P450 3A4(CYP3A4)酶活性的高效液相色谱-紫外检测方法。采用的色谱柱为Phenomenex C18柱(4.6 mm×150 mm,5 μm),梯度洗脱,流速1.0 mL/min,紫外检测波长245 nm,柱温30 ℃。睾酮与大鼠肝微粒体温孵后,过已活化好的C18固相萃取小柱,收集甲醇洗脱液,于37 ℃水浴中通N2吹干,用50%甲醇复溶后进样分析测定。研究结果表明,6β-羟基睾酮的 保留时间为11.60 min,线性范围为0.5~32 μg/mL,最低检出质量浓度为0.02 μg/mL,提取率为88.41%~92.73%,方法的回收率为99.07%~101.30%;睾酮的保留时间为19.27 min,线性范围为0.5~40 μg/mL,最低检出质量浓度为0.01 μg/mL,提取率为89.59%~92.66%,方法的回收率为96.50%~98.03%。两者的日内、日间相对标准偏差均小于10%,温孵体系中的其他内源性物质不干扰测定。该方法快速、稳定、灵敏度高,适合体外睾酮及其代谢物6β-羟基睾酮的测定,可应用于体外CYP3A4酶活性的评价及酶动力学的研究。 相似文献
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S. Hamm G. E. Von Unruh W. D. Paar H. J. Dengler 《Isotopes in environmental and health studies》2013,49(2-3):99-110
Tramadol (T), racemic 1(e)-(m-methoxyphenyl)-2(e)-(dimethylaminomethyl)-cyclohexane-1(a)-ol is an effective analgesic drug. Metabolites were formed by O- and N-demethylation. Six deuterated tramadol isotopomers have been synthesized; their kinetic isotope effects in oxidative demethylation reactions were investigated in vitro using human liver microsomes. In comparison to unlabelled (±)-tramadol, (±)-T-OCD3 and (±)-T-D9 displayed an unequivocal (< 3), (±)-T-ND6 and (±)-T-ND3 a noticeable, and (±)-T-cyclohexyl-D3 as well as (±)-T-15ND2 no measurable isotope effect. Metabolic switching (favoring the N-demethylation) was observed only after incubation of a tramadol with a trideuterated methoxy group. Additional N-CD3-groups prevented this metabolic switching. Metabolic switching favoring the O-demethylation was not observed. The isoenzyme responsible for the O-demethylation was always saturated under the experimental conditions required to detect the metabolites. The two tramadol isotopomers containing deuterium in metabolic inert positions (other than the methyl groups, i.e., (±)-T-15ND2 and (±)-T-cyclohexyl-D3) were expected to display no isotope effects. This expectation could be verified in this study. 相似文献
20.
K. D. Mooiman R. F. Maas‐Bakker H. Rosing J. H. Beijnen J. H. M. Schellens I. Meijerman 《Biomedical chromatography : BMC》2013,27(9):1107-1116
Complementary and alternative medicines (CAM) can affect the pharmacokinetics of anticancer drugs by interacting with the metabolizing enzyme cytochrome P450 (CYP) 3A4. To evaluate changes in the activity of CYP3A4 in patients, levels of 1‐hydroxymidazolam in plasma are often determined with liquid chromatography–quadrupole mass spectrometry (LC‐MS/MS). However, validated LC‐MS/MS methods to determine in vitro CYP3A4 inhibition in human liver microsomes are scarce and not optimized for evaluating CYP3A4 inhibition by CAM. The latter is necessary because CAM are often complex mixtures of numerous compounds that can interfere with the selective measurement of 1‐hydroxymidazolam. Therefore, the aim was to validate and optimize an LC‐MS/MS method for the adequate determination of CYP3A4 inhibition by CAM in human liver microsomes. After incubation of human liver microsomes with midazolam, liquid–liquid extraction with tert‐butyl methyl ether was applied and dried samples were reconstituted in 50% methanol. These samples were injected onto a reversed‐phase chromatography consisting of a Zorbax Extend‐C18 column (2.1 × 150 mm, 5.0 µm particle size), connected to a triple quadrupole mass spectrometer with electrospray ionization. The described LC‐MS/MS method was validated over linear range of 1.0–500 nm for 1‐hydroxymidazolam. The results revealed good inter‐assay accuracy (≥85% and ≤115%) and within‐day and between‐day precisions (coefficient of variation ≤ 4.43%). Furthermore, the applicability of this assay for the determination of CYP3A4 inhibition in complex matrix mixtures was successfully demonstrated in an in vitro experiment in which CYP3A4 inhibition by known CAM (β‐carotene, green tea, milk thistle and St. John's wort) was determined. Copyright © 2013 John Wiley & Sons, Ltd. 相似文献