基于UPLC—Q—TOF/MS的大鼠体内茵芋苷代谢特征分析

目的：应用超高效液相色谱-四级杆串联飞行时间质谱法（UPLC-Q-TOF/MS）鉴定并分析大鼠茵芋苷给药后的代谢途径及其在胆汁、血浆、尿液、粪便中的代谢产物。方法：通过UPLC-Q-TOF/MS分析大鼠给药前、灌胃和静脉注射给药后得到的不同时间段的生物样品,并在电喷雾正负离子模式下获取生物样品的质谱数据;使用MassLynx V4.1软件处理UPLC-Q-TOF/MS数据;鉴定茵芋苷在胆汁、血浆、尿液、粪便中的代谢产物。结果：在大鼠的血浆、胆汁、尿液、粪便样品中除原型外共鉴定得到10种代谢产物,其中有8种代谢产物均通过肾脏途径代谢。结论：水解、葡萄糖苷酸结合、氧化、羟基化等是茵芋苷在体内代谢的主要途径;根据尿液中广泛代谢的结果,应重视茵芋苷经肾途径代谢的临床用药指导。     

片叶苔素D大鼠体内代谢产物的结构鉴定

目的 研究片叶苔素D在大鼠体内的代谢,并对片叶苔素D在大鼠体内代谢产物进行分离与结构鉴定。方法 大鼠按40mg/kg剂量口服片叶苔素D后收集胆汁,采用高效液相色谱仪分离、纯化代谢产物,利用HPLC-MS/MS分析代谢产物,利用NMR鉴定结构。结果 大鼠口服片叶苔素D后,在胆汁中检测到2个Ⅱ相代谢产物,均为片叶苔素D与葡萄糖醛酸的结合产物(片叶苔素D-1’-O-β-D-吡喃葡萄糖醛酸),且两个代谢产物互为旋阻异构体,室温下可互相转化。结论 片叶苔素D在大鼠体内产生与葡萄糖醛酸结合的Ⅱ相代谢产物,且2个代谢产物互为旋阻异构体。     

甘草对雷公藤二萜内酯体内代谢产物的影响研究

目的：研究甘草对雷公藤甲素和雷公藤内酯酮体内代谢产物的影响,为阐明甘草对雷公藤的减毒增效作用机制奠定基础。方法采用LC-MS/MS法研究雷公藤甲素和雷公藤内酯酮单独给药以及与甘草联合给药后在大鼠尿液中的相应代谢成分,分析比较单独给药及联合给药后代谢产物种类和生成量的差异。结果在雷公藤甲素单独给药和与甘草联合给药的大鼠尿液中,均发现代谢产物5个：m/z（[M＋H]^＋）377,359,359,359,375;在雷公藤内酯酮单独给药和与甘草联合给药的大鼠尿液中,均发现代谢产物3个：m/z（[M＋H]^＋）413,397,375;各代谢产物生成量在单独给药组和联合给药组中有很大差异。结论雷公藤甲素和雷公藤内酯酮单独给药和与甘草联合给药后其部分代谢产物相同,但是代谢产物的生成量存在很大差异。     

Qualitative analysis of catechins from green tea GMB-4 clone using HPLC and LC-MS/MS

Objective:To identify the bioactive compounds in catechins isolation and its components from green tea GMB-4 clone.Methods:Green tea GMB-4 clones were extracted with distilled water at 90C.Samples were eluted into the column with 10%ethanol.Subsequently,the column was eluted with95%ethanol and evaporated separately.Green tea extract was identified by thin layer chromatography.Catechins were separated by the stationary phase in column chromatography using polyamide with 10%ethanol eluent and 95%ethanol.The results of isolations were analyzed by high performance liquid chromatographic(HPLC)and LCMS/MS.Analysis of catechins by HPLC was done by external standard.Results:Fraction from 10%ethanol showed that four major peaks at retention time of1.663,2.367,2.950 and 4.890,indicated the presence of four catechins components including catechin,epicatechins,gallocatechin and epigallocatechin.Whereas,fraction from 95%ethanol showed two main peaks at retention time of 5.167 and 9.82,which indicated the presence of epigallocatechin gallate(EGCG)and epicatechin gallate(ECG).EGCG(m/z 459),epigallocatechin(m/z 307),ECG(m/z 443),and epicatechin(m/z 291)were isolated and separated successfully using HPLC and LC-MS/MS.Conclusions:The HPLC and LC-MS/MS methods were successfully tuned for the qualitative analysis of green tea extract with EGCG and ECG.Four major catechins were separated and identified by LC-MS/MS,such as EGCG,epigallocatechin,ECG and epicatechin.The result of HPLC analysis showed that EGCG and ECG were main components from catechins isolation of green tea GMB-4 clone.     

HPLC-PDA-ESI-ITMS~n法鉴定氯唑沙宗在SD大鼠尿液中的代谢产物

目的通过高效液相色谱-光电二极管阵列检测器/离子阱质谱（HPLC-PDA-ESI-ITMSn）联用技术定性分析氯唑沙宗在SD大鼠尿液中的代谢产物。方法高效液相色谱条件：采用Benatnach C18（250 mm×4.6 mm,5μm）色谱柱,以甲醇-水（含10 mmol乙酸铵）进行梯度洗脱,流速为0.8 mL/min,检测波长为287 nm。ESI质谱条件：负离子扫描模式,喷针电压为5 000 V,毛细管电压为20 V,干燥气（N2）压力为137.896 kPa,质量扫描范围为m/z 50-500,雾化温度为300℃。结果通过HPLC-PDA-ESI-ITMSn联用技术,分离并鉴定出SD大鼠口服氯唑沙宗原料药后,尿液中的5种氯唑沙宗代谢产物,并揭示了这5种代谢产物的质谱裂解规律。结论本方法灵敏度高、专属性强,为氯唑沙宗在SD大鼠体内的代谢途径及代谢过程的研究提供了依据。     

UPLC-Q-TOF/MS based metabolomic profiling of serum and urine of hyperlipidemic rats induced by high fat diet

Hyperlipidemia is considered to be a high lipid level in blood, can induce metabolic disorders and dysfunctions of the body, and results in some severe complications. Therefore, hunting for some metabolite markers and clarifying the metabolic pathways in vivo will be an important strategy in the treatment and prevention of hyperlipidemia. In this study, a rat model of hyperlipidemia was constructed according to histopathological data and biochemical parameters, and the metabolites of serum and urine were analyzed by UPLC-Q-TOF/MS. Combining pattern recognition and statistical analysis, 19 candidate biomarkers were screened and identified. These changed metabolites indicated that during the development and progression of hyperlipidemia, energy metabolism, lipid metabolism, amino acid metabolism and nucleotide metabolism were mainly disturbed, which are reported to be closely related to diabetes, cardiovascular diseases, etc. This study demonstrated that a UPLC-Q-TOF/MS based metabolomic approach is useful to profile the alternation of endogenous metabolites of hyperlipidemia.     

HPLC-MS/MS法在骨髓移植患者全血CSA及AM1浓度测定中的应用

目的 建立同时测定骨髓移植患者全血环孢素A(CSA)及AM1浓度的HPLC-MS/MS法,探索CSA及其主要代谢产物AM1在个体内及个体间的关系,为临床合理选用CSA血药浓度监测方法提供可靠依据.方法 采用环孢素D(CSD)作内标,全血样品经含内标甲醇沉淀蛋白处理.色谱柱为Ultimate XB-C18,柱温65℃,流动相为含0.1％甲酸和2 mmol/L乙酸铵的水和含0.1％甲酸的甲醇,梯度洗脱.质谱检测方式为电喷雾离子阱正离子模式,质谱多反应监测(MRM)扫描,监测CSA1219.9～1203.1 m/z,AM1 1236.1～1219.1 m/z,CSD 1234.0～1217.0 m/z.结果 CSA浓度在16～1600 ng/mL范围内线性关系良好,Y=0.0143X+0.0213(r=0.9976);AM1浓度在10～1000 ng/mL范围内线性关系良好,Y=0.00363X-0.00528(r=0.9973).CSA与AM1个体间比率(AM1/CSA)范围为32％～356％;CSA与AM1个体内比率(AM1/CSA)范围为27％～147％.结论 建立了同时检测CSA及AM1的HPLC-MS/MS法;CSA在骨髓移植患者个体间及个体内代谢存在变异;HPLC-MS/MS法可用于临床骨髓移植患者全血CSA浓度的监测.     

LC-MS／MS法与 HPLC 法测定青娥丸中药效成分比较

目的：通过液质联用（ LC-MS/MS）与高效液相色谱（ HPLC）测定青娥丸中药效成分-松脂醇二葡萄糖苷、补骨脂素和异补骨脂素的含量比对,为其质量控制提供方法参考。方法：采用Kromasil C18色谱柱,以甲醇-0．1％甲酸为流动相进行梯度洗脱,使用多反应监测（ MRM）模式,监测离子对为m/z 700．5→m/z 235．4（松脂醇二葡萄糖苷）、m/z 187．1→m/z 131．1（补骨脂素和异补骨脂素）进行LC-MS/MS法同时测定,与药典HPLC法分别测定的结果进行对比分析,比较二者的差异性。结果：3种成分LC-MS/MS法测定的方法回收率分别为99．08％、98．95％和98．56％,RSD分别为1．33％、1．43％和1．27％。结论：LC-MS/MS法同时测定青娥丸中的药效成分,与药典HPLC法相比具有简便、灵敏、准确等特点,适用于青娥丸的含量测定。     

基于UHPLC-Q-TOF/MS的地榆化学成分分析及大鼠体内代谢研究

目的 采用超高效液相色谱和-四级杆串联飞行时间质谱(UHPLC-Q-TOF/MS)法分析地榆水提物在大鼠血清、尿液及粪便中的原型成分及代谢产物,探讨地榆中活性成分在大鼠体内的代谢途径.方法 采用Thermo AcclaimTM RSLC 120 C18色谱柱(3.0 mm×100 mm,2.2 μm),以0.1％甲酸水...     

LC-MS/MS定性分析大鼠尿液中益智提取物的成分及其代谢产物

目的 通过LC-MS/MS法检测大鼠尿液中益智提取物中10种活性成分的存在形式.方法 采用LC-MS/MS法检测灌胃给药后0~4 h、4~8 h、8~24 h大鼠尿液中的益智代谢产物,与给药前大鼠空白尿液MRM图进行比对.结果 实验组大鼠尿液中9种主要活性成分均以原形化合物的形式存在,而未发现山柰酚及其葡萄糖酸代谢产物.结论 大鼠灌胃益智提取物后,益智活性成分在大鼠尿液中的物质形式以原形为主,为理解这些物质在大鼠体内的消除途径提供部分参考依据.     

Software-aided detection and structural characterization of cyclic peptide metabolites in biological matrix by high-resolution mass spectrometry

Compared to their linear counterparts, cyclic peptides show better biological activities, such as antibacterial, immunosuppressive, and anti-tumor activities, and pharmaceutical properties due to their conformational rigidity. However, cyclic peptides could form numerous putative metabolites from potential hydrolytic cleavages and their fragments are very difficult to interpret. These characteristics pose a great challenge when analyzing metabolites of cyclic peptides by mass spectrometry. This study was to assess and apply a software-aided analytical workflow for the detection and structural characterization of cyclic peptide metabolites. Insulin and atrial natriuretic peptide (ANP) as model cyclic peptides were incubated with trypsin/chymotrypsin and/or rat liver S9, followed by data acquisition using TripleTOF® 5600. Resultant full-scan MS and MS/MS datasets were automatically processed through a combination of targeted and untargeted peak finding strategies. MS/MS spectra of predicted metabolites were interrogated against putative metabolite sequences, in light of a, b, y and internal fragment series. The resulting fragment assignments led to the confirmation and ranking of the metabolite sequences and identification of metabolic modification. As a result, 29 metabolites with linear or cyclic structures were detected in the insulin incubation with the hydrolytic enzymes. Sequences of twenty insulin metabolites were further determined, which were consistent with the hydrolytic sites of these enzymes. In the same manner, multiple metabolites of insulin and ANP formed in rat liver S9 incubation were detected and structurally characterized, some of which have not been previously reported. The results demonstrated the utility of software-aided data processing tool in detection and identification of cyclic peptide metabolites.     

卡德沙星代谢物的合成、结构确证及其体外抗菌活性

目的:合成并确证卡德沙星两种代谢产物,初步研究其抗菌活性。方法:大鼠灌胃卡德沙星(9mg/kg)后,通过LC-MS分析,在胆汁和尿液中发现有m/z385.90的成分(M-1),在尿液中发现有m/z329.1的成分(M-2)。参考类似物的代谢方式,推测其结构分别为1-环丙基-6-氟-8-二氟甲氧基-1,4-二氢-7-(2-氨基-丙胺基)-4-氧代-3-喹啉羧酸和1-环丙基-6-氟-8-二氟甲氧基-1,4-二氢-7-氨基-4-氧代-3-喹啉羧酸。化学合成这两种物质,与卡德沙星的代谢产物进行对比,并研究体外抗菌活性。结果与结论:本文对卡德沙星代谢物的结构推断正确,它们的体外抗菌活性很弱。M-1和M-2均未见文献报道。     

川芎嗪的体内代谢

本研究使用氘代川芎嗪(TMPz)为示踪剂,结合稳定同位素和GC/MS分析的离子簇技术,发现接受灌胃给予TMPz后大鼠的24h尿中存在有TMPz本身及其三种代谢产物.     

人体尿中川芎嗪代谢产物的研究

使用气相色谱／质谱研究了人体尿中的川芎嗪代谢物，证实主要代谢物为３，５，６－三甲基吡嗪甲酸。     

A sensitive HPLC technique for the quantitation of dauricine

trahydroisoquinoline alkaloid, which is extractedfrom the root of Dauricum D. C.. Both clinical trials and experiments have proved its good antiarrhythmic effect, especially its use--dependence inprolonging APD in guinea myocardial[" Zj, whichmakes it very promising to work dauriclne into anew type of antiarrhythmlc agent. Therefore, It isof great significance to study the Dan's pharmacokinetics. Some determination techniques of Danhave been previously reported, such as TLC fluo. [3].resc…     

白首乌中抗肿瘤活性成分告达庭-3-O-β-D-磁麻糖苷在大鼠体内的代谢产物分析

目的 研究白首乌中甾苷成分告达庭-3-O-β-D-磁麻糖苷在大鼠体内的代谢情况.方法 大鼠单剂量灌胃给予20 mg/kg的告达庭-3-O-β-D-磁麻糖苷,分别收集24 h内的血浆、尿液、粪便和胆汁,样品经处理后,采用UPLC-Q-TOF分析,结合MetabolynxTM软件初步鉴定告达庭-3-O-β-D-磁麻糖苷在大...     

LC-MS/MS法同时测定Beagle犬血浆中柚皮苷和新橙皮苷的含量

目的:建立同时测定Beagle犬血浆中柚皮苷和新橙皮苷的LC-MS/MS分析方法。方法:以芦丁为内标,血浆样品经乙腈沉淀蛋白处理,采用Diamonsil C18(150 mm×4.6 mm,5μm)色谱柱,以乙腈-0.1%甲酸水(25:75)为流动相,流速为0.4 ml/min,柱温20℃;采用ESI(+)离子源,MRM扫描检测离子对为柚皮苷m/z 581→273、新橙皮苷m/z 611→303、芦丁(内标)m/z611→303。结果:血浆中柚皮苷在0.025～2.5μg/ml、新橙皮苷在0.015～1.5μg/ml浓度范围内线性关系良好,二者的方法回收率均近100%,分析方法的日内日间精密度RSD值均小于10%,内源性物质不干扰样品测定。结论:本方法简便、准确、灵敏度高、专属性好,适用于Beagle犬血浆中柚皮苷和新橙皮苷测定及其体内药代动力学的研究。     

过敏性鼻炎患者肺虚感寒证和肾阳亏虚证血清代谢组学研究

[目的] 基于气相色谱/质谱（GC/MS）技术的代谢组学方法,分析过敏性鼻炎肺虚感寒证和肾阳亏虚证患者血清中的内源性小分子代谢物变化,为两种中医证型的科学辨证提供依据。[方法] 采用GC/MS技术检测过敏性鼻炎患者肺虚感寒证、肾阳亏虚证以及正常人的血清中小分子代谢物,利用SIMCA-P软件进行主成分分析（PCA）及偏最小二乘判别法（PLSA-DA）分析,研究血清中小分子代谢物在各组间的差异。[结果] 过敏性鼻炎肺虚感寒证、肾阳亏虚证患者代谢谱具有良好的分离趋势,并与健康人对照组有明显区分。利用商业化的代谢物谱库（如Wiley和NIST质谱库）及本实验室建立的标准品代谢物谱库鉴定了23个具有统计学意义的差异代谢物。在过敏性鼻炎肺虚感寒证和肾阳亏虚证患者中筛选得到了丙氨酸、十四烷酸、十六烷酸、9,12-十八碳二烯酸、9-（Z）-O-十八碳烯酸、十八烷酸6种差异代谢产物。[结论] 过敏性鼻炎肺虚感寒证和肾阳亏虚证的代谢差异主要体现在氨基酸代谢和脂肪酸代谢方面,基于GC/MS的代谢组学技术可用于临床不同中医证型的分类研究。     

杠柳毒苷在大鼠体内排泄的初步研究*

[目的]研究杠柳毒苷在大鼠体内的排泄情况.[方法]建立了液-液萃取后用高效液相色谱测定大鼠尿液、粪便和胆汁中杠柳毒苷含量的方法,并对大鼠静注及灌胃杠柳毒苷后的排泄情况进行分析.[结果]大鼠按12mg/kg剂量灌胃给予杠柳毒苷后,72 h尿中杠柳毒苷和其代谢物的累积排泄量占给药剂量的0.44%,粪中杠柳毒苷和其水解代谢物的累积排泄量占给药剂量的12.39%;按0.74 mg/kg剂量尾静脉注射给予杠柳毒苷后,72 h尿中杠柳毒苷及其代谢物的累积排泄量占给药剂量的6.24%,粪中杠柳毒苷及其水解代谢物的累积排泄量占给药剂量的7.33%;按0.74 mg/kg剂量尾静脉注射给予杠柳毒苷后,21 h内累积经胆汁排泄的杠柳毒苷占给药总量的76.13%.[结论]杠柳毒苷原形药物主要经胆汁排泄,但从尿液和粪便排泄则很少,尿液中可能存在结合代谢物,粪便中主要以肠菌代谢物(杠柳次苷)的形式排泄.     

HPLC测定人尿液三苯双脒次级代谢物对苯二甲酸浓度

目的建立测定人尿液三苯双脒次级代谢物对苯二甲酸（TPA）浓度的高效液相色谱法（HPLC）。方法以阿魏酸为内标（internal standard, IS）,志愿者尿样稀释后经Ultimate XB0C18柱（4.6mm×200mm,5μm）、流动相甲醇－1%醋酸（35∶65,V/V）分离,在检测波长为240nm、流速为0.8mL/min、柱温为30℃条件下采用HPLC分析。结果在1～400μg/mL内,TPA与IS的峰面积比值与浓度线性关系良好（r=0.9954）,定量下限为1μg/mL,低、中、高浓度的相对回收率分别为（106.0±4.4）%、（107.4±4.2）%和（108.5±2.4）%,批内和批间相对标准偏差（RSD）小于8%。结论方法符合生物样品分析要求,可用于三苯双脒次级代谢物TPA尿液浓度的测定。