高效液相-质谱联用法对盐酸关附甲素在大鼠尿中代谢物的研究

目的研究盐酸关附甲素在大鼠尿中的代谢产物。方法大鼠iv盐酸关附甲素后收集尿,用高效液相-质谱联用方法测定。通过与标准化合物的色谱保留时间、分子离子峰、碎片离子峰对照从而鉴定I相代谢物。通过用葡糖醛酸酶和硫酸酯酶酶解鉴定其水解产物(苷元)从而确定II相结合物。结果大鼠尿中发现I相代谢物关附醇胺和关附壬素;尿经过葡糖醛酸酶和硫酸酯酶酶解后,产生关附甲素和关附壬素。结论盐酸关附甲素在大鼠体内可以转化为关附壬素、关附醇胺、关附甲素葡糖醛酸和硫酸结合物、关附壬素葡糖醛酸和硫酸结合物。经过生物转化,代谢产物的极性增加,药效下降。     

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。     

高效液相色谱-四级杆飞行时间串联质谱法分析瑞香素在大鼠肠壁的代谢产物

研究瑞香素在大鼠肠壁产生的代谢产物。采用大鼠在体肠灌流模型,分别收集瑞香素0～2 h内的十二指肠、空肠、回肠和结肠灌流液,以液相色谱-四级杆飞行时间串联质谱法分析肠道灌流液中瑞香素的代谢产物。在大鼠十二指肠、空肠、回肠灌流液中共发现瑞香素原形药物和4个代谢产物,分别推测为瑞香素-7-硫酸酯、瑞香素-8-硫酸酯、瑞香素-7-葡糖醛酸结合物和瑞香素-8-葡糖醛酸结合物;而在结肠灌流液中未发现代谢产物。在瑞香素的4个代谢产物中,瑞香素-7-硫酸酯和瑞香素-8-硫酸酯(m/z 257)为首次发现的瑞香素在大鼠体内的Ⅱ相代谢产物,揭示了其在大鼠体内代谢的新途径。     

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.     

LC-MS~n法研究曲马多在大鼠尿中的代谢产物

研究曲马多在大鼠尿中的主要代谢产物。健康大鼠腹腔注射盐酸曲马多(剂量为9.0 mg.kg-1),收集0～36 h不同时间段的尿样,经固相萃取法预处理后,直接采用LC-MSn法对曲马多和代谢产物进行分析。通过比较空白尿样和给药后尿样的总离子流色谱图和选择离子监测色谱图,与3个代谢产物对照品比较,并分析各个色谱峰的色谱保留时间、准分子离子和多级碎片离子,在大鼠尿样中共检测到13种代谢产物,包括9种I相代谢产物,4种II相代谢产物,其中1种为首次在生物体内发现的曲马多代谢产物,证明曲马多在大鼠体内的主要代谢途径为O-去甲基化、N-去甲基化、羟基化、N-氧化和葡糖醛酸化反应。本文所建立的LC-MSn方法可进一步研究曲马多在其他动物和人体内的代谢产物。     

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。     

大鼠灌胃四逆散提取物后血浆、尿液、粪便、胆汁中主要代谢产物的鉴定

采用UPLC-Q-TOF/MS研究四逆散提取物在大鼠体内的代谢产物,利用碰撞能量梯度(MSE)和质量亏损过滤(MDF)技术进行分析,鉴定大鼠灌胃四逆散提取物后血浆、尿液、粪便、胆汁中的代谢产物。四逆散提取物中柚皮苷、柚皮素、橙皮苷、新橙皮苷、甘草苷、甘草素、甘草酸、甘草次酸、柴胡皂苷a、柴胡皂苷d在大鼠不同代谢途径中推测出原形、羟基化、葡糖醛酸化、硫酸化、葡糖醛酸化与硫酸化结合、羟化葡糖醛酸化等共41个代谢产物。     

LC-MS/MS法研究1-[1-(6-甲氧基-2-萘基)乙基]-2-(4-硝基苄基)-6,7-二甲氧基-1,2,3,4-四氢异喹啉氢溴酸盐在大鼠体内的代谢产物

采用液相色谱-串联质谱法对大鼠灌胃1-［1-(6-甲氧基-2-萘基)乙基］-2-(4-硝基苄基)-6,7-二甲氧基-1,2,3,4-四氢异喹啉氢溴酸盐(编号P91024)后粪便、尿液、胆汁和血浆中的主要代谢产物进行研究。通过比较给药样品和空白样品的全扫描总离子流色谱和选择离子扫描色谱图差别寻找I相代谢产物；根据其一级和二级质谱图，确定I相代谢产物的分子结构。完全提取I相代谢产物后的样品溶液，再用葡糖醛酸酶酶解，得II相结合物的苷元部分，采用与I相代谢产物鉴定同样方法寻找和鉴定II相代谢产物苷元的结构，进而确证II相代谢产物的分子结构。从大鼠粪便中鉴定出P91024的2个I相代谢物，从胆汁中鉴定出1个I相和5个II相代谢产物，从尿液中鉴定出1个I相和3个II相代谢产物，从血浆中鉴定出4个I相和1个II相代谢产物；并分别分析推测出它们的结构。P91024在大鼠体内被代谢转化为多种产物，利用LC-MS/MS可以快速寻找和鉴定。     

不同剂量染料木黄酮及其代谢产物在大鼠胆汁中的排泄动力学

目的研究不同剂量染料木黄酮及其葡糖醛酸化代谢产物在大鼠胆汁内的排泄动力学。方法将染料木黄酮制成混悬液，分别按6.25，12.5和50 mg·kg^-1给大鼠灌胃，于灌胃后不同时间收集胆汁，用葡糖醛酸酶溶液处理胆汁。采用高效液相色谱法测定胆汁中染料木黄酮及其葡糖醛酸化代谢产物的浓度。结果3种剂量6.25，12.5和50 mg·kg^-1分别给药后，累积以原形从胆汁排泄的药物分别为(42.56±6.54)，(75.17±18.87)和(126.60±34.78) μg，累积经胆汁排泄的总药物(原形药物+葡糖醛酸化药物)分别为(108.46±35.23)，(423.46±158.31)和(853.74±320.84) μg，葡糖醛酸化代谢产物分别占胆汁排泄总量(原形药物+葡糖醛酸化药物)的60.76%，82.25%和85.17%。结论染料木黄酮在大鼠胆汁中主要以葡糖醛酸结合形式排泄，原形药物及其葡糖醛酸化代谢产物在大鼠胆汁中的排泄呈现明显的非线性剂量依赖性特征。     

LC/DAD/MSD技术研究大鼠胆汁中盐酸非洛普的II相代谢产物

目的研究大鼠服药后胆汁中盐酸非洛普(DDPH)II相代谢产物.方法收集大鼠空白胆汁及给药后12h内的胆汁,以LC/DAD/MSD技术判断II相代谢产物峰位.以HPLC法制备II相代谢产物馏分并以β-葡糖醛酸酶水解,再进行分析;同时将与II相代谢产物相应的I相代谢产物对照品按相同条件进行分析对照.结果大鼠给药后胆汁色谱图中峰M₇,M₈和M₉为DDPH的II相代谢产物,它们的β-葡糖醛酸酶水解产物分别为M₃,M₂和M₁.结论β-1-O-{3,5-二甲基-4-[-2-甲基-2-(3,4-二甲氧基苯乙氨基)-乙氧基]-苯基}-葡糖醛酸(M₇),β-1-O-{2,4-二甲基-3-[2-甲基-2-(3,4-二甲氧基苯乙氨基)-乙氧基]-苯基}-葡糖醛酸(M₈)和β-1-O-{2-甲氧基-4-[1-甲基-2-(2,6-二甲基苯氧基)-乙氨基-乙基]-苯基}-葡糖醛酸(M₉)为大鼠ipDDPH后产生的II相代谢产物.     

Phase I and phase II metabolite identification of rutaecarpine in freshly isolated hepatocytes from male Sprague–Dawley rats

Rutaecarpine, an alkaloid originally isolated from Evodia rutaecarpa, has been used for the treatment of gastrointestinal disorders in Asia. In the present study, the phase I and phase II metabolites of rutaecarpine were investigated in freshly isolated hepatocytes from male Sprague–Dawley rats. The individual metabolites were characterized via liquid chromatography-tandem mass spectrometry. The incubation of rutaecarpine with freshly isolated hepatocytes for 2 h yielded five major phase I metabolites. In addition, three glucuronide conjugates and four sulfate conjugates were observed. Because the majority of metabolites observed in vivo were identified, freshly isolated hepatocytes might be useful for the identification of certain metabolites formed from drug candidates from a reduced number of experimental animals.     

Studies related to the metabolism of anabolic steroids in the horse: the phase I and phase II biotransformation of 19-nortestosterone in the equine castrate

The metabolism of 19-nor[4-14C]testosterone has been studied in the equine castrate. Following XAD-2 extraction of aliquots of the 0-24 h urine samples, the glucuronic acid and sulphate conjugates were separated by Sephadex LH-20 column chromatography. After hydrolysis of the conjugates, the neutral phase I metabolites of 19-nortestosterone were extracted, purified and identified by g.l.c.-mass spectrometry. In phase I metabolism stereospecificity was observed in the reduction of the A-ring with the formation of the 5 alpha, 3 beta-isomers of estranediol. Epimerization at C-17 and hydroxylation at C-16 were the other major pathways. In phase II metabolism the C-17 alpha steroid epimers were predominantly conjugated with glucuronic acid and the C-17 beta epimers with sulphuric acid. One animal showed a slight variation in metabolism with a tendency towards the formation of polar metabolites.     

Metabolism of the nephrotoxicant N-(3,5-dichlorophenyl)succinimide in rats: evidence for bioactivation through alcohol-O-glucuronidation and O-sulfation

The agricultural fungicide, N-(3,5-dichlorophenyl)succinimide (NDPS), was shown to be nephrotoxic in rats. Previous studies have indicated that the metabolism of NDPS contributes to its mechanism of toxicity and both phase I and phase II pathways may be involved. In the current report, we investigated the detailed biotransformation of [(14)C]NDPS in rats using HPLC-ESI-MS. The chemical reactivity of the phase II NDPS metabolites was also evaluated. In vivo studies were conducted by administering [(14)C]NDPS to male Fischer 344 rats. Urine, tissue (liver and kidney), and plasma samples were analyzed. The mechanism of formation and chemical reactivity of the glucuronide and sulfate metabolites of NDPS were investigated in vitro using liver subcellular preparations. Major in vivo metabolites of NDPS were identified as the oxidative [N-(3,5-dichlorophenyl)-2- and 3-hydroxysuccinamic acid, 2-/3-NDHSA] and hydrolytic products [N-(3,5-dichlorophenyl)succinamic acid]. N-Acetylcysteine and cysteine (with intramolecular aminolysis) conjugates were also detected in rat urine and fecal extracts, respectively, suggesting the formation of reactive intermediate(s) in the metabolism of NDPS. Small amounts of the alcohol-O-glucuronide and O-sulfate of 2-/3-NDHSA were detected in rat urine, plasma, and tissue homogenates. The formation of these phase II metabolites was found to be mediated through the initial conjugation of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) followed by hydrolysis. As compared to NDHS, NDHS-O-sulfate is approximately 500-fold more reactive toward GSH conjugation. In rat liver S9, fortifying phase II cofactors (UDPGA or PAPS) in incubation mixtures with NDHS also significantly increased the amount of GSH adducts produced. Results of this research demonstrate that phase II metabolites of NDPS were produced in rats. The formation of the alkyl alcohol-O-glucuronide and O-sulfate conjugates represents bioactivation pathways in the metabolism of NDPS that could potentially contribute to its mechanism of nephrotoxicity.     

采用UHPLC-MS和质量亏损过滤技术分析二苯乙烯苷在大鼠体内的代谢产物和代谢途径

目的:分析二苯乙烯苷在大鼠体内的代谢产物并推测代谢途径.方法:将雄性SD大鼠随机分为血浆组(n=3)、尿液组(n=3)、胆汁组(n=3)和组织组(n=9),各组大鼠均单次灌胃二苯乙烯苷200 mg/kg,分别收集给药后10、30 min和1、1.5、2、4 h的血浆,给药后0～6 h的尿液,给药后0～4 h的胆汁以及给...     

Glucuronidation of hydroxylated polychlorinated biphenyls (PCBs)

Polychlorinated biphenyls (PCBs) may be metabolized to hydroxylated compounds. While many of these metabolites are further converted to either the glucuronic acid or the sulfate conjugates by phase II enzymes, which facilitates their excretion, some hydroxylated PCBs persist in the body. This may reflect their inability to be conjugated. A possible role of uridine diphosphate glucuronosyl transferase (UGT) in the elimination of hydroxylated metabolites of PCBs was therefore investigated. Glucuronidation studies of PCB metabolites included ones which are eliminated with relative ease and also ones which are reported to be retained in blood. Liver microsomes, prepared from male Wistar rats treated by intraperitoneal injections of phenobarbital for 3 days (400 micromol/kg/day), were used as the source of UGT. Enzyme kinetics (V(max) and K(m)) were determined for each of the metabolites. The efficiency of glucuronidation (V(max)/K(m)) was found to vary from <3 to 116 microL/min/mg and was dependent on the structure of the metabolites. Substitution of chlorine atoms on the nonhydroxylated ring greatly lowered the V(max) of the enzyme, with substitution in the meta and para positions being least favorable for enzyme activity. Steric hindrance around the hydroxyl group by chlorines on adjacent carbon atoms did not play a major role. A weak relationship between the calculated dihedral angle (planarity), pK(a), log D, and enzyme activity was determined (r(2) < 0.5). However, a stronger relationship for the surface area and surface volume of the molecule was observed (r(2) >or= 0.5). This study explains in part why some PCB metabolites persist in the body.     

Phase II metabolism of warfarin in primary culture of adult rat hepatocytes.

We used adult rat hepatocytes in primary culture (HPC) as a model system to study the hepatic phase II metabolism of the anticoagulant warfarin. Hepatocytes were isolated by a collagenase perfusion technique and maintained for 24 hr in Waymouth's medium containing 0.1 mM (R)-warfarin. When HPC medium was analyzed by reverse phase high performance liquid chromatography with diode-array detection, 4'-, 6-, and 7-hydroxywarfarin were identified. Several putative conjugates were observed eluting between 13 and 18 min. Treatment of hepatocyte medium with beta-glucuronidase and sulfatase resulted in the loss of five putative conjugates and concomitant increases in 4'-, 6-, and 7-hydroxywarfarin and warfarin, suggesting that these metabolites and warfarin were conjugated. Use of the beta-glucuronidase inhibitor saccharic acid 1,4-lactone enabled the determination of the relative extents of conjugation of each metabolite by glucuronic acid and sulfate. Glucuronidation was the predominant pathway for 4'-hydroxywarfarin, whereas 6-hydroxywarfarin and warfarin occurred mainly as sulfate conjugates. In contrast, 7-hydroxywarfarin was converted to both glucuronide and sulfate conjugates. Exposure of HPC to phenobarbital resulted in a decrease in cytochrome P-450-mediated production of hydroxylated warfarin metabolites; however, an increase in the production of 8-hydroxywarfarin was observed when HPC were exposed to beta-naphthoflavone. Unique conjugation patterns were found when hydroxylated warfarins were substituted for warfarin in HPC medium. Both 7- and 8-hydroxywarfarin were converted to one sulfate and two glucuronide conjugates, whereas 4'-hydroxywarfarin was converted to a single glucuronide conjugate. A spectral library of these conjugates was used to identify the major conjugates of warfarin formed by rat HPC.     

Studies related to the metabolism of anabolic steroids in the horse: The phase I and phase II biotransformation of 19-nortestosterone in the equine castrate

1. The metabolism of 19-nor[4-¹⁴C]testosterone has been studied in the equine castrate.

2. Following XAD-2 extraction of aliquots of the 0—24 h urine samples, the glucuronic acid and sulphate conjugates were separated by Sephadex LH-20 column chromatography. After hydrolysis of the conjugates, the neutral phase I metabolites of 19-nortestosterone were extracted, purified and identified by g.l.c.-mass spectrometry.

3. In phase I metabolism stereospecificity was observed in the reduction of the A-ring with the formation of the 5α,β-isomers of estranediol. Epimerization at C-17 and hydroxylation at C-16 were the other major pathways.

4. In phase II metabolism the C-17α steroid epimers were predominantly conjugated with glucuronic acid and the C-17β epimers with sulphuric acid.

5. One animal showed a slight variation in metabolism with a tendency towards the formation of polar metabolites.     

Glucuronides of hydroxylated metabolites of amitriptyline and nortriptyline isolated from rat bile

Polar conjugates were isolated from the bile of rats given amitriptyline (AT, unlabeled or labeled with 14C), nortriptyline (NT), or 10-hydroxy (10-OH) derivatives of the drugs. The procedure involved extraction on a column of polystyrene resin, elution with methanol, and separation by preparative TLC followed by reversed phase HPLC. Individual metabolites were characterized by NMR spectroscopy and fast atom bombardment mass spectrometry and by enzymatic or acid deconjugation with subsequent identification of aglycones and glucuronic acid. Conversely, they were compared with conjugates obtained from hydroxy compounds by incubation with rat liver microsomes and UDP-glucuronic acid. Glucuronides isolated from the bile of rats given AT were derived from 2-OH-AT, (E)- and (Z)-10-OH-AT, 2-hydroxy-3-methoxy- (or 3-hydroxy-2-methoxy) AT, 10, 11-(OH)2-AT, and some of the N-demethylated analogues of these compounds. In most cases, 10-OH compounds form two diastereoisomeric glucuronides produced from the enantiomeric alcohols; 10, 11-(OH)2 metabolites occur as cis- and trans-isomers that are conjugated with glucuronic acid. Administration of synthetic (E)- and (Z)-10-OH-AT and -NT leads to the excretion of their glucuronides along with conjugates formed after demethylation and/or introduction of a second OH group. NT gives rise to 2-OH-NT glucuronide besides those conjugates derived from (E)-10-OH-NT. No glutathione conjugates could be detected.     

Deconjugation of N-glucuronide conjugated metabolites with hydrazine hydrate--biomarkers for exposure to the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

The metabolism of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) has been investigated in rabbit liver S9. Two phase I metabolites, N(2)-OH-PhIP and 4'-OH-PhIP were identified based on UV and mass spectra and co-elution with reference standards. Fortification of the incubation with UDGPA resulted in a complete glucuronidation of PhIP and N(2)-OH-PhIP, while 4'-OH-PhIP was only partly glucuronidated. Also, the PhIP metabolite 5-OH-PhIP was completely glucuronidated by rabbit liver S9, while 5-OH-PhIP was a poor substrate for CYP mediated hydroxylation. The glucuronic acid conjugates of PhIP metabolites were unsusceptible to treatment with beta-glucuronidase indicating that these are N-glucuronides. Treatment of the conjugates with hydrazine hydrate, however, resulted in complete hydrolysis of the glucuronic acid conjugates as well as in reduction to the parent amine of metabolites hydroxylated in the exocyclic amino group. Urine was collected from a male volunteer following consumption of fried chicken. Treatment of the urine with beta-glucuronidase/sulfatase resulted in release of 4'-OH-PhIP, while treatment with hydrazine hydrate in addition resulted in release of substantial amounts of PhIP and 5-OH-PhIP. The data show that hydrazine hydrate can hydrolyse N-glucuronides of metabolites of PhIP, glucuronides that are unsusceptible to enzymatic hydrolysis. In addition the data indicate that humans metabolise a large fraction of ingested PhIP to genotoxic metabolites. The chemical hydrolysis of glucuronide conjugates of PhIP metabolites with hydrazine hydrate observed in this study may also be a useful approach in the development of biomarkers for exposure and effect of other xenobiotics.