Identification of etamiphylline and metabolites in equine plasma and urine by accurate mass and liquid chromatography/tandem mass spectrometry

Etamiphylline camsylate (Millophylline V) was administered intravenously to two horses at a dose of 2.8 mg/kg. Urine and blood samples were taken up to 32 h post administration. Unhydrolyzed plasma and urine was extracted using solid phase extraction (SPE). The identity of the parent drug and metabolites was confirmed using a linear ion trap mass spectrometer and accurate mass analysis on an orbitrap mass spectrometer. Desethyletamiphylline (molecular weight 251) was the main metabolite observed in the urine and plasma samples and resulted from the N‐deethylation of etamiphylline. The second metabolite detected in urine and plasma resulted from the demethylation of etamiphylline (molecular weight 265). The third minor metabolite detected in urine was proposed to have resulted from a simultaneous N‐deethylation and demethylation of etamiphylline (molecular weight 238). Copyright © 2010 John Wiley & Sons, Ltd.     

Comprehensive characterization of the in vitro and in vivo metabolites of geniposide in rats using ultra-high-performance liquid chromatography coupled with linear ion trap–Orbitrap mass spectrometer

1.?Geniposide (genipin 1-O-glucose), one of the major bioactive constituents isolated from Fructus Gardeniae, possesses many biological activities. In this study, an efficient strategy was developed using ultra-high-performance liquid chromatography coupled with linear ion trap–Orbitrap mass spectrometer (UPLC–LTQ–Orbitrap) to profile the in vitro and in vivo metabolic patterns of geniposide in rat liver microsomes (RLMs), plasma, urine, and various tissues. And post-acquisition data-mining methods including extracted ion chromatogram (EIC), multiple mass defect filters (MMDF), fragment ion searching (FISh), and isotope pattern filtering (IPF) were adopted to characterize the known and unknown metabolites.

2.?A total of 33 metabolites were detected and interpreted according to accurate mass measurement, diagnostic fragment ions, relevant drug biotransformation knowledge, and bibliography data. Among them, 17 metabolites were detected in the plasma, 31 metabolites were identified in the urine, six metabolites could be found in rat heart, 12 in liver, three in spleen, six in lung, 12 in kidney, six in brain, and four in RLMs.

3.?A series of corresponding reactions such as hydrolysis, hydroxylation, taurine conjugation, hydrogenation, decarboxylation, demethylation, sulfate conjugation, cysteine S-conjugation, glucosylation, and their composite reactions were all discovered.

4.?The results could provide comprehensive insights and guidance for elucidation of side effect mechanism and safety monitoring as well as for rational formulation design in drug delivery system. The newly discovered geniposide metabolites could be targets for future metabolism studies on the important chemical constituents from herbal medicines.     

Metabolism of aildenafil in vivo in rats and in vitro in mouse,rat, dog,and human liver microsomes

Aildenafil, 1‐{[3‐(6, 7‐dihydro‐1‐methyl‐7‐oxo‐3‐propyl‐1H‐pyrazolo [4, 3‐d] primidin‐5‐yl)‐4‐ethoxyphenyl] sulfonyl}‐cis‐3, 5‐dimethylpiperazine, a phosphodiesterase type V enzyme inhibitor (PDE5I), is under development for treatment of erectile dysfunction (ED). The purpose of this study was to elucidate metabolism of aildenafil in vivo in rats and in vitro in mouse, rat, dog, and human liver microsomes. Thirty‐one phase I metabolites have been found by LTQ/Orbitrap hybrid mass spectrometry in rat urine, faeces, and bile after oral administration. Major biotransformation pathways of aildenafil included N‐dealkylation of the piperazine ring, hydroxylation and dehydrogenation, aliphatic hydroxylation and loss of alkyl group of piperazine ring. Minor pathways involved hydroxylation on the phenyl ring, pyrazole N‐demethylation, O‐deethylation, loss of piperazine ring (cleavage of N‐S bond) and dehydrogenation on the piperazine ring. Similar metabolic pathways of aildenafil were observed in the incubations of liver microsomes from mouse, rat, and dog as well as from human. The depletion rate of parent drug in mouse and rat liver microsomes was significantly different from that in human liver microsomes. The cytochrome P450 reaction phenotyping analysis was conducted using isozyme‐specific inhibitors. The results indicated that CYP3A was the main isoenzyme involved in oxidative metabolism of aildenafil. Overall, these in vitro and in vivo findings should provide valuable information on possible metabolic behaviours of aildenafil in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

PLC-LTQ-Orbitrap MS结合分子网络技术鉴别铁线莲属植物中三萜皂苷类成分

目的 构建6种铁线莲属植物中皂苷类成分的分子网络与快速分析。方法 超高效液相色谱-线性离子阱-静电场轨道阱质谱联用技术（UHPLC-LTQ Orbitrap MS）采集质谱信息，将信息上传至GNPS数据平台计算分析，借助Cytoscape软件生成可视化分子网络图，根据对照品的裂解规律和文献分析鉴定铁线莲植物中皂苷类成分。结果 从6种植物中鉴别出常春藤和齐墩果酸型皂苷共25个，其中16个为常春藤型皂苷、9个为齐墩果酸型皂苷，根据代表各成分节点的颜色及比例，得到各成分在6种铁线莲属植物中的分布情况。鉴定的25个三萜皂苷中，20个为2种或2种以上铁线莲共有成分，其中化合物clematichinenoside A和oleanolic acid 3-O-ribopyranosyl-（1→3）-α-L-rhamnopyranosyl-（1→2）-α-L-arabinopyranoside为5种铁线莲共有成分；6个三萜皂苷只在单种铁线莲中存在。结论 基于UPLC-LTQ-Orbitrap MS的分子网络技术与传统分析方法比较，其可快速、可视化的区分6种植物中三萜皂苷的差异；6种植物中三萜皂苷类成分相似之中又有不同，为药材替代提供参考依据。     

连翘酯苷A和连翘苷LTQ Orbitrap MS裂解途径

目的:对连翘酯苷A和连翘苷的电喷雾质谱裂解途径进行研究。方法:使用LTQ-Orbitrap高分辨质谱仪,采用蠕动注射泵直接进样的方法,在正离子扫描模式下,分析连翘酯苷A和连翘苷的一级和多级质谱图。结果:连翘酯苷A的[M+Na]+准分子离子峰容易失去碎片C6H10O4(鼠李糖)或C9H8O4(咖啡酸),并在MS3谱中发现同时有两种不同来源的m/z321的离子,推断是由m/z 501与m/z 467离子分别丢失咖啡酸或鼠李糖的裂解而来。连翘苷的[M+Na]+准分子离子峰容易失去碎片C6H10O5(葡萄糖);同时在多级碎裂时,容易失去H2O,推断与分子结构中的酯键和糖苷键以及羟基有关。结论:本文首次使用LTQ-Orbitrap高分辨质谱仪报道连翘酯苷A和连翘苷的质谱裂解途径,根据高分辨特征碎片离子可对其进行快速在线鉴别,同时也为进一步研究连翘酯苷A和连翘苷的体内代谢过程与结构修饰提供数据支撑。     

内皮细胞提取和UHPLC-LTQ-Orbitrap法分析筛选苦碟子注射液中的活性成分

应用细胞生物色谱法及UHPLC-LTQ-Orbitrap质谱联用技术快速筛选鉴定苦碟子注射液中可能的活性成分。选择人脐静脉内皮细胞(huma umbilical vein endothelid cells,HUVEC)作为靶细胞,先使苦碟子注射液中的活性成分与靶细胞特异性结合,经细胞靶点脱敏失活后,应用LC-MS快速鉴定与细胞靶向亲和的化学成分,从而筛选得到苦碟子注射液中可能的活性成分。根据获得的精确相对分子质量数据、保留时间以及结合苦碟子注射液中的化学成分进行鉴定归属。最终,从细胞裂解液中共筛选鉴定出9个化学成分,其中包括倍半萜内酯类成分4个、有机酸类成分3个以及黄酮类成分2个。应用细胞生物色谱法与UHPLC-LTQ-Orbitrap质谱联用技术快速筛选鉴定苦碟子注射液中可能的活性成分,可为苦碟子注射液药效物质基础研究提供一种快捷、有效的方法学借鉴。     

基于UPLC/LTQ Orbitrap Velos MS的创伤性脑损伤代谢组学分析*

摘要:目的联合非靶向和靶向代谢组学技术检测创伤性脑损伤(traumaticbraininjury,TBI)差异代谢物,并评估其与病情严重程度及预后的关系。方法选取徐州 医科大学附属医院2022年8月至12月就诊的TBI患者(TBI组)和体检健康者(健康 人对照组)各40例,应用超高效液相色谱-线性离子阱/静电场轨道阱组合式高分辨质谱( UPLC/LTQ Orbitrap Velos MS)筛选并验证差异代谢物。记录40例TBI患者入院12 h内格拉斯哥昏迷评分( glasgow coma scale, GCS)和伤后6个月格拉斯哥预后评分( glasgow outcome scale , GOS) ,结合差异代谢物评估疾病严重程度和预后。结果验证的4 个差异代谢物(谷氨酸、尿酸、肌酐和乳酸)存在显著分离趋势,TBI组谷氨酸和乳酸水平显著高于健康人对照组,尿酸和肌酐水平显著低于健康人对照组;随着伤情加重,TBI患者谷氨酸和乳酸水平逐渐升高,尿酸和肌酐水平逐渐降低;影响预后不良的4种代谢物组ROC曲线下面积( AUCROC)为0.818 ,敏感性为76.9% ,特异性为81.5%(P<0.05)。结论谷氨酸 ,乳酸、尿酸和肌酐与TBI患者病情密切相关,四者联合或可用于预测TBI的预后。