LC/MSn鉴定咖啡酸在大鼠体内的代谢产物

目的 研究咖啡酸（CA）在大鼠体内的代谢产物。方法 大鼠灌胃（50 mg·kg-1）给予CA后采集0 ~ 4 h尿样,用电喷雾离子阱多级质谱法对CA在大鼠体内的代谢产物进行分析。结果 大鼠灌胃给予CA后,在体内可测到2个原形药的甲基化代谢物、2个原形药的单葡萄糖醛酸结合物、1个原形药的双葡萄糖醛酸结合物、2个原形药的单硫酸结合物、2个甲基化物的葡萄糖醛酸结合物和2个甲基化物的硫酸结合物。结论 CA在大鼠体内广泛代谢,其代谢物的结构有待于进一步分析后确证。     

高效液相色谱-电喷雾串联四极杆质谱法研究大鼠尿内毛果芸香碱代谢产物

首次应用高效液相色谱-电喷雾串联四极杆质谱法鉴定了大鼠灌胃给予毛果芸香碱0.2mg后0-8小时内尿中的代谢物。4只大鼠给药毛果芸香碱盐酸盐,收集给药后尿液,固相萃取柱富集、然后应用高效液相色谱-电喷雾串联四极杆质谱法在线进行多反应监测,进而对尿中微量的代谢物进行了质谱解析。分别定性为毛果芸香碱的葡萄糖醛酸结合物、毛果芸香酸和毛果芸香酸的葡萄糖醛酸结合物。而且发现了各种代谢产物及原形药物在大鼠体内均有构型变化。     

液-质联用法测定反式白藜芦醇在大鼠和比格犬体内的代谢产物

目的：液相色谱串联多级质谱法测定大鼠和比格犬体内反式白藜芦醇（trans-resveratrol，TR）的代谢产物。方法：大鼠按150mg·kg^-1灌胃给予TR，收集给药后0～12h尿、粪和胆汁样品，比格犬按50mg·kg^-1灌胃给予TR，收集给药后0～12h尿样和粪样。用C18柱固相萃取后进行液-质联用法（LC／MS^n）分析。结果：在服药后的大鼠体内可测到4个Ⅱ相代谢物（2个O-葡萄糖醛酸化和2个硫酸化物），在比格犬体内可测到2个Ⅱ相代谢物（O-硫酸化物）。结论：TR在大鼠和比格犬体内广泛代谢，并存在着种属差异。     

联苯双酯代谢产物的分离和鉴定

给大鼠灌胃联苯双酯后,用TLC法分离尿中代谢产物。发现尿中的主要代谢物为葡萄糖醛酸结合物,将此结合物水解后,用TLC法分离到一种极性较葡萄糖醛酸结合物为弱的代谢物,经光谱分析测定其结构为4-羟基-4′-甲氧基-5,6,5′,6′-二次甲二氧基-2,2′-二甲氧羰基联苯(简称4-去甲联苯双酯)。此外还证实,大鼠灌胃后在肝、肾组织中均以葡萄糖醛酸结合物为主,仪有少量非结合形代谢物和原形药物。     

雷公藤甲素和雷公藤红素在大鼠体内的代谢产物分析

大鼠灌胃给予雷公藤甲素与雷公藤红素混合液.采用液相色谱-串联质谱法,在负离子模式下,以一级、二级全扫描方式检测大鼠血清、尿与粪中的代谢产物.在大鼠血清中仅发现原形药物.在尿样中检测到了雷公藤甲素的3种代谢产物,推测分别为其单羟基化代谢产物、环氧化物水解开环代谢产物及谷胱甘肽结合物.在粪中则检测到1种代谢产物,推测为雷公藤甲素的另一单羟基化代谢产物.在尿样中检测到雷公藤红素的1种代谢产物,推测为葡萄糖醛酸结合物;在粪中检测到硫酸结合物.     

大鼠体内大豆苷元及其代谢物分布影响因素研究

目的研究大鼠灌胃给予大豆苷元后,多剂量与性别对大豆苷元及其代谢物在大鼠血浆中分布的影响。方法雌、雄大鼠分别单次或多次灌胃给予大豆苷元1.0 mg·kg-1,血浆样品经沉淀蛋白处理,采用LC-MS/MS方法测定血浆样品中大豆苷元及其II相代谢物的质量浓度,采用Winnolin6.4计算各化合物的主要药动学参数,并对获得的药动学参数进行统计分析。结果大鼠多次灌胃给予大豆苷元后,血浆中大豆苷元及其代谢物的AUC_(0-24)明显高于单次给药。多次给药后雌鼠血浆中大豆苷元主要以大豆苷元-7-葡萄糖醛酸结合物和大豆苷元-7-硫酸结合物形式存在,雄鼠血浆中主要以大豆苷元-7-葡萄糖醛酸结合物、大豆苷元-7-硫酸结合物和大豆苷元-7-葡萄糖醛酸-4'-硫酸结合物形式存在。结论多次给药及性别差异明显影响大豆苷元在大鼠血浆中的分布。     

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。     

用液相质谱法鉴定盐酸半附甲素在大鼠胆汁中I相和Ⅱ相代谢产物

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。     

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。     

UPLC-Q-Exactive Plus Orbitrap-MS鉴定桑色素在大鼠体内的代谢产物

目的 利用UPLC-Q-Exactive PlusSOrbitrap-MS技术对大鼠灌胃给予桑色素后体内的的主要代谢产物进行研究。方法 大鼠灌胃给予桑色素20 mg/kg后,分别收集血浆、尿液和粪便样品,采用超高压液相色谱串联高分辨质谱技术测定桑色素的体内代谢物。结果 根据一级质谱分子离子信息和二级质谱碎裂离子信息,在大鼠血浆和尿液中均发现2个葡萄糖醛酸代谢产物,在粪便中发现脱氢产物。结论 桑色素在大鼠体内的主要代谢途径为葡萄糖醛酸化反应。本研究初步阐明了桑色素在大鼠体内的代谢情况,为进一步药理作用机制研究提供依据。     

URINARY EXCRETION OF ARSENIC METABOLITES AFTER LONG-TERM ORAL ADMINISTRATION OF VARIOUS ARSENIC COMPOUNDS TO RATS

The metabolism of arsenic compounds in rats was studied by comparing urinary metabolites of arsenic compounds administered for 1 wk or 7 mo. Male F344/DuCrj rats were given 100 mg As/L as monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), trimethylarsine oxide (TMAO), or arsenobetaine (AsBe), or 10 mg As/L as arsenite \[As(III)] via drinking water for 7 mo. Urine was collected by forced urination after 1 wk or 7 mo. Arsenic metabolites in urine were analyzed by ion chromatography with inductively coupled plasma mass spectrometry. In the case of As(III) ingestion, a small portion of all arsenic excreted in urine (about 6% ) was excreted in inorganic form, while most arsenic was excreted as methylated arsenic metabolites. Following MMA treatments for 1 wk or 7 mo, the predominant products excreted were unchanged MMA and DMA accompanied by small amounts of TMAO and tetramethylarsonium (TeMA). In the case of DMA treatment the urinary compounds found were mainly the parent DMA and TMAO with minute amounts of TeMA. TMAO was methylated to TeMA to a slight extent after 1 wk and 7 mo of administration, although most TMAO was excreted in the form of unchanged TMAO. AsBe was predominantly eliminated in urine without any transformation. Two unidentified metabolites were detected in urine after 7 mo of arsenic species exposure; the amounts of these metabolites increased in the order DMA > MMA > TMAO with only small quantities of these detected in the As(III)-treated group. These results suggest that these unidentified metabolites are formed during a demethylation process, and not during methylation. Our findings indicate that long-term exposure to As(III), MMA, or DMA decreases the proportion of TMAO elimination in urine and increases that of DMA, M-1, and M-2, and that further methylation to TMAO to TeMA does occur to a slight extent following long-term exposure to arsenical compounds in rats.     

Further structural analysis of urinary metabolites of suprofen in the rat

The urinary metabolites of 2-(4-(2-thienylcarbonyl)phenyl)propionic acid (suprofen, S) in rats were analyzed by radio-GC, GC/MS, or 1H NMR technique. Radio-GC analysis of trimethylsilylated materials after TLC separation of intact urine showed the presence of three radioactive peaks with the retention times corresponding to the authentic S, 2-(4-(2-thienylhydroxymethyl)phenyl)propionic acid, and 2-(4-carboxyphenyl)propionic acid. About 40% of the total radioactivity appearing in the 0-24-hr urine was accounted for by the three metabolites and their conjugates. The identification of these metabolites was confirmed by comparison of the MS spectra of urine, in which rats were administered an equimolar mixture of S and S[phenyl-d4], with those of synthetic standards. The labile metabolites of S, corresponding to about 32% of the total radioactivity appearing in the 0-24-hr urine, were isolated and purified by ether extraction from the fresh urine and GC/MS or HPLC. GC/MS of the methylated metabolite revealed the consistent presence of the ion peaks at m/z 304, 245, 217, and 141, indicative of a dimethylated product with monohydroxy group on the thiophene ring. Analysis of the 1H NMR spectrum demonstrated the metabolite to be 2-(4-(5-hydroxy-2-thienylcarbonyl)phenyl)propionic acid.     

Confirmation of a carboxylic acid metabolite of polychlorotrifluoroethylene and a method for its GC-ECD analysis in biological matrices.

3.1 Oil, referred to as polychlorotrifluoroethylene (pCTFE), is a polymeric mixture consisting primarily of trimers and tetramers of chlorotrifluoroethylene (CTFE) end-capped with chlorine. Inhalation studies have associated dose-related body weight loss, increased organ weights, and abnormal hepatic enzyme activities with exposure to pCTFE. The carboxylic acid metabolites of pCTFE have been shown to cause hepatotoxicity in rats, which is manifested by increased liver weights and the proliferation of hepatic peroxisomes. A method was developed to derivatize these carboxylic acid metabolites. Tissue homogenates and feces were extracted with methanol, and urinary metabolites were extracted on octadecylsilane (ODS) solid-phase extraction columns. Aliquots of the extracts and whole blood were methylated with 3N methanolic HCl to transesterify the carboxylic acid metabolites to volatile methyl esters. The pCTFE methyl esters were analyzed by gas chromatography (GC) with electron capture detection (ECD). The on-column limit of detection was 5 pg for each methyl ester. Solid-phase extraction of spiked urine gave extraction efficiencies of 90.4% for the trimer acid and 84.7% for the tetramer acid. This method was successfully applied to toxicity studies in rats and nonhuman primates. The identities of the derivatized metabolites were confirmed by GC/MS.     

Investigation of the metabolic fate of dihydrocaffeic acid

The antioxidant dihydrocaffeic acid is a dietary constituent and a microbial metabolite of flavonoids. Orally administered to rats, dihydrocaffeic acid was very rapidly absorbed most probably by the gastric or duodenal epithelium and excreted in urine as free and conjugated forms. LC-MS2 analysis of plasma and urine samples allowed confident identification of the dihydrocaffeic acid metabolites. The parent compound was glucuronidated, sulphated or methylated, on one of the hydroxyl groups present on its phenyl ring. All the dihydrocaffeic acid metabolites peaked in plasma within the first 30min following ingestion, suggesting a metabolism possibly by the gastric or duodenal cells and by the liver. Using in vitro and ex vivo models of the intestinal epithelium and the liver, the identity and source of the metabolites detected in vivo were examined. The data obtained suggest that, in rats, intestinal cells are more able to glucuronidate dihydrocaffeic acid, whereas liver favours sulphation. Moreover, glucuronidation, sulphation and methylation seem to be regio-selective, preferably on the 3-OH of dihydrocaffeic acid. The methyl conjugate, dihydroferulic acid, was shown to be oxidized into ferulic acid by intestinal and hepatic cells, which were also able to perform the reverse reaction, the reduction of ferulic acid into dihydroferulic acid. As a conclusion, the main form of dihydrocaffeic acid circulating in plasma after its ingestion is a mixture of different primary and secondary metabolites.     

Metabonomic identification of two distinct phenotypes in Sprague-Dawley (Crl:CD(SD)) rats.

Genetic drift in animal populations has been a recognized concern for many years. Less understood is the potential for phenotypic "drift" or variation that is not related to any genetic change. Recently, stock Sprague-Dawley (Crl:CD(SD)) rats obtained from the Charles River Raleigh facility demonstrated a distinct endogenous urinary metabonomic profile that differed from historical control SD urine spectral profiles obtained over the past several years in our laboratory. In follow-up studies, the origin of the variant phenotype was narrowed down to animals of both sexes that were housed in one specific room (Room 9) in the Raleigh facility. It is likely that the two phenotypes are related to distinct populations of gut flora that particularly impact the metabolism of aromatic molecules. The most pronounced difference between the two phenotypes is the relative amounts of hippuric acid versus other aromatic acid metabolites of chlorogenic acid. Though both molecular species are present in either phenotype, the marked variation in levels of these molecules between the two phenotypes has led to the designation of high hippuric acid (HIP) and high chlorogenic acid metabolites (CA) phenotypes. Specific urinary components that distinguish the phenotypes have been thoroughly characterized by NMR spectroscopy with additional, limited characterization by LC-MS (high performance liquid chromatography coupled with mass spectrometry). Co-habitation of rats from the two phenotypes rapidly facilitated a switch of the CA phenotype to the historical Sprague-Dawley phenotype (HIP). The impact of these variant phenotypes on drug metabolism and long-term safety assessment studies (e.g., carcinogenicity bioassays) is unknown.     

Pharmacokinetics,metabolism, and excretion of cycloastragenol,a potent telomerase activator in rats

1.?The objective of this study was to investigate the pharmacokinetics, excretion, and metabolic fate of cycloastragenol (CA) in rats.

2.?An LC-MS method was developed and used to quantify CA in biological samples. Rats were orally administrated with CA at 10, 20, and 40?mg/kg or intravenously administrated at 10?mg/kg to determine pharmacokinetic parameters of CA. For excretion experiment, urine, feces, and bile were collected at 24?h after oral administration (40?mg/kg), also at 12?h after intravenous administration (10?mg/kg). An LC-MS/MS method was developed to identify the metabolites of CA.

3.?The results showed that the oral bioavailability of CA was about 25.70% at 10?mg/kg. CA was excreted through bile and feces and eliminated predominantly by the kidney in rats. It also might exist an enterohepatic circulation of CA in rats. CA could be metabolized widely in vivo in rat, seven, six, and one phase I metabolites were found in feces, urine, and bile samples respectively, but no phase II metabolite was found.

4.?In summary, this study defined pharmacokinetics characteristics of CA, described its excretion, and established its in vivo metabolism in rats.     

丹酚酸B在大鼠体内的代谢产物及代谢途径研究

目的 建立大鼠血浆、胆汁、尿液与粪便中丹酚酸B代谢产物的测定方法,并考察其代谢途径。方法 取SD大鼠18只,平分为灌胃和静注2组,每组再分成3个小组,分别采集血浆、胆汁、尿液及粪便,每小组2只大鼠分别单剂量灌胃（500 mg·kg^-1）和尾静脉注射（16.5 mg·kg^-1）给予丹酚酸B,另一只分别灌胃水和尾静脉注射生理盐水作为空白对照。采用高效液相色谱串联四极杆飞行时间质谱（HPLC-Q-TOF-MS/MS）联用法测定血浆、胆汁、尿液与粪便中的丹酚酸B代谢产物,同时推断代谢途径。结果 根据一级质谱分子离子信息和二级质谱碎裂离子信息,在大鼠体内共发现8个丹酚酸B的代谢产物,其中胆汁、粪便中均含有这8个代谢产物,尿液中发现3个代谢产物,血浆中发现2个代谢产物。由丹酚酸B及其代谢产物结构推断丹酚酸B体内主要通过甲基化反应、酯键水解反应代谢。结论 建立的分析方法准确、灵敏、快速,符合生物样品分析要求,适用于大鼠血浆、胆汁、尿液与粪便中丹酚酸B代谢产物的分析,并分析鉴定了8个代谢产物。代谢实验结果表明,胆汁排泄是丹酚酸B最主要的排泄方式。     

Metabolism of isobutylnaphthyl acetic acid in rats: determination of the chemical structures of metabolites

After oral and intravenous administration of radiolabelled isobutylnaphthyl acetic acid (INAA) to rats two metabolites were isolated from urine and plasma by HPLC. Field desorption, high resolution electron impact mass spectrometry as well as GC-MS after derivatization were used for structure elucidation and identification of the metabolites. The main biotransformation product in rat urine was found to be 5-(2'-hydroxy-2'-methyl-propyl)-1-naphthyl acetic acid (M1). The main metabolite in plasma was derived and was found to be 5-(2'-carboxypropyl)-1-naphthyl acetic acid (M2).     

Structural elucidation of in vivo and in vitro metabolites of anisodine by liquid chromatography-tandem mass spectrometry

Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMSn) was employed to investigate the in vivo and in vitro metabolism of anisodine. Feces, urine and plasma samples were collected after ingestion of 20 mg anisodine to healthy rats. Feces and urine samples were cleaned up by liquid-liquid extraction and solid-phase extraction procedures (C18 cartridges), respectively. Methanol was added to plasma samples to precipitate plasma proteins. Anisodine was incubated with homogenized liver and intestinal flora of rats in vitro, respectively, followed by extraction with ethyl acetate. LC-MSn was used for the separation and identification of the metabolites using C18 column with mobile phase of methanol/0.01% triethylamine solution (2 mM, adjusted to pH 3.5 with formic acid) (60:40, v/v). The results revealed that five metabolites (norscopine, scopine, alpha-hydroxytropic acid, noranisodine and hydroxyanisodine) and the parent drug existed in feces. Three new metabolites (dimethoxyanisodine, tetrahydroxyanisodine and trihydroxy-methoxyanisodine) were identified in urine. Four metabolites (norscopine, scopine, hydroxyanisodine and anisodine N-oxide) and the parent drug were detected in plasma. Two hydrolyzed metabolites (scopine and alpha-hydroxytropic acid) were found in rat intestinal flora incubation mixture, and two metabolites (aponoranisodine and anisodine N-oxide) were identified in homogenized liver incubation mixture.     

Arsenic speciation in bile and urine following oral and intravenous exposure to inorganic and organic arsenics in rats.

Although inorganic arsenate (iAsV) and arsenite (iAsIII) are metabolized in liver and excreted into bile and urine, the metabolites in the bile after the oral intake of iAs remain unclear. Male Sprague-Dawley rats were orally (po) or intravenously (iv) exposed to iAs and methylated arsenics, and the arsenic speciation in the urine and bile was analyzed by high performance liquid chromatography-inductively coupled argon plasma mass spectrometry. Arsenic caused induction of multidrug resistance-associated protein 2 (MRP2), and changes of glutathione (GSH) levels in the liver and bile were also determined. The metabolic speciation studies revealed that arsenic was excreted into bile in the methylarsenic-diglutathione (MADG) and/or dimethylarsenic acid (DMAV) forms in iAsIII- or iAsV-po rats, but that MADG and arsenic-triglutathione (ATG) are the main forms excreted into bile both in iAsIII- and iAsV-iv rats. In MADG-po rats, the MADG was excreted into bile in the MADG and DMAV forms. Monomethylarsonic acid (MMAV)- and DMAV-iv rats did not excrete significant amounts of either MMAV or DMAV into bile and mostly excreted into urine in the unchanged chemical forms. Taken together, the DMAV detected in the bile is mostly supposed to be the dissociation of dimethylarsenic-glutathione (DMAG). Urinary arsenic speciation showed that arsenic metabolized to 43% methylated DMAV, 47% unmethylated iAsIII, and 10% iAsV in iAsIII-iv rats, whereas only 3% methylated DMAV, 87% unmethylated iAsV, and 10% iAsIII were detected in iAsV-iv rats. Arsenic was accumulated dose dependently, and arsenic concentration was significantly higher in the iAsIII-po rat liver than in the iAsV-po rat liver. GSH levels in the bile were decreased by relatively higher doses of iAsV-po, but significantly increased by iAsIII- or iAsV-iv. iAs-exposure increased the expression of MRP2 in the liver. Pretreatment with buthionine sulfoximine predominantly inhibited arsenic excretion into bile in iAs-iv rats. In conclusion, our data demonstrated that biliary and urinary arsenic excretion and speciation are affected by the route, dose, and chemical forms of arsenical administration, and GSH plays a key role in arsenic metabolism. We are also first to show that DMAV that probably originated from DMAG is excreted into the bile in iAs-po rats.