UPLC/Q-TOF MS鉴定沃尼妙林在大鼠体内的代谢产物

利用超高效液相色谱/四级杆-飞行时间质谱（UPLC/Q-TOF MS）分析鉴定了VLM在大鼠体内的代谢产物,并讨论了VLM在大鼠体内的代谢途径。按20 mg/kg体重的剂量,给Sprague-Dawley大鼠口服灌食沃尼妙林（VLM）,分别采集给药前及给药后的尿液样品。采用UPLC/Q-TOF MS对样品进行分析,代谢物数据经MetaboLynx XS软件处理后,根据常见代谢途径的准确质量数变化对代谢产物进行筛选,利用代谢物的MS/MS信息鉴定了五种代谢产物,并确定VLM代谢途径包括母核羟基化和侧链硫醚键氧化等。研究结果表明,VLM在大鼠体内代谢迅速,母核的羟基化是主要的代谢途径。     

喹赛多及其两种代谢产物在鸡体内消除规律研究

通过喹赛多(Cyadox,CYX)的不同给药方式,研究CYX及其两种重要代谢产物在鸡体内消除规律,了解其对食品安全的影响,并为今后的药理学和毒理学研究提供较为详细的数据基础。建立并优化了喹赛多、脱二氧喹赛多(BDCYX)和喹啉-2-羧酸(QCA)在鸡血浆、胆汁、可食性组织和粪便中的提取、纯化及HPLC检测方法,并通过按推荐剂量连续混饲给药7 d和一次性灌胃给药两种不同给药方式,研究喹赛多及其两种代谢产物在鸡体内消除规律。结果表明,连续混饲给药7天后在血浆、肝脏、肾脏、肌肉、脂肪中均未检测到CYX,停药后6 h和24 h的肝脏样品中检测到BDCYX。除了胆汁以外,所检测的其它上述5种组织中均检测到QCA,肝脏和肾脏中持续检测到72 h。一次性灌服CYX后的鸡排泄物中CYX原形和BDCYX在给药后2 d便无法检出,QCA可检测到第3天。研究结果为喹赛多药理学和毒理学研究提供了可直接借鉴的参考数据。     

乙酰甲喹在斑马鱼中的代谢研究

为了阐明乙酰甲喹在水产动物中的代谢过程,本试验采用超高效液相色谱串联四级杆/飞行时间质谱(UPLC-Q/TOF-MS)技术及代谢软件MetabolynxXS自动分析采集功能,研究其在斑马鱼中的主要代谢产物。斑马鱼经药浴摄入乙酰甲喹后,分别采用乙酸乙酯和乙腈提取组织中的乙酰甲喹及代谢物,过0.22μm滤膜后经UPLC-Q/TOF-MS分析,通过比较试验组和对照组的色谱图新增色谱峰,确定代谢产物的数量;通过比较乙酰甲喹标准品及代谢产物的精准MS/MS质谱图,确定代谢产物的化学结构,并推测其可能的代谢途径。结果显示,乙酰甲喹在斑马鱼中的代谢产物较少,主要是脱氧代谢物,包括单脱氧代谢物N1-脱氧乙酰甲喹(1-DMEQ)、N4-脱氧乙酰甲喹(4-DMEQ)及双脱氧代谢物N1,N4-双脱氧乙酰甲喹(1,4-BDMEQ),代谢途径主要为N→O基团还原。残留消除规律研究发现,乙酰甲喹消除较快,4h降至初始浓度一半以下,并呈现逐渐降低的趋势;其3种代谢产物浓度均呈现先增高后降低的趋势,其中2种单脱氧代谢物在给药后2h浓度达到最高,8h降至最高浓度一半以下,双脱氧代谢物在给药后4h药物浓度达到最高,12h降至最高浓度一半以下,结果表明,乙酰甲喹在斑马鱼中代谢消除速率较快。上述研究结果可为乙酰甲喹在其他水产食品动物中的代谢研究提供参考,并为水产动物源性食品安全的监控及药代动力学研究提供技术支持。     

乙酰甲喹在斑马鱼中的代谢研究

为了阐明乙酰甲喹在水产动物中的代谢过程,本试验采用超高效液相色谱串联四级杆/飞行时间质谱（UPLC-Q/TOF-MS）技术及代谢软件Metabolynx^XS自动分析采集功能,研究其在斑马鱼中的主要代谢产物。斑马鱼经药浴摄入乙酰甲喹后,分别采用乙酸乙酯和乙腈提取组织中的乙酰甲喹及代谢物,过0.22 μm滤膜后经UPLC-Q/TOF-MS分析,通过比较试验组和对照组的色谱图新增色谱峰,确定代谢产物的数量;通过比较乙酰甲喹标准品及代谢产物的精准MS/MS质谱图,确定代谢产物的化学结构,并推测其可能的代谢途径。结果显示,乙酰甲喹在斑马鱼中的代谢产物较少,主要是脱氧代谢物,包括单脱氧代谢物N1-脱氧乙酰甲喹（1-DMEQ）、N4-脱氧乙酰甲喹（4-DMEQ）及双脱氧代谢物N1,N4-双脱氧乙酰甲喹（1,4-BDMEQ）,代谢途径主要为N→O基团还原。残留消除规律研究发现,乙酰甲喹消除较快,4 h降至初始浓度一半以下,并呈现逐渐降低的趋势;其3种代谢产物浓度均呈现先增高后降低的趋势,其中2种单脱氧代谢物在给药后2 h浓度达到最高,8 h降至最高浓度一半以下,双脱氧代谢物在给药后4 h药物浓度达到最高,12 h降至最高浓度一半以下,结果表明,乙酰甲喹在斑马鱼中代谢消除速率较快。上述研究结果可为乙酰甲喹在其他水产食品动物中的代谢研究提供参考,并为水产动物源性食品安全的监控及药代动力学研究提供技术支持。     

喹烯酮在鸡体内的代谢及药物动力学研究

以HPLC-MS/MS为定量手段,研究了喹烯酮经静脉注射(2.5 mg/kg)、口服(30 mg/kg)两种给药途径在鸡体内的代谢及药物动力学特征.鸡静脉注射喹烯酮后,血浆中检测到喹烯酮原药和1-脱氧喹烯酮;口服灌注喹烯酮后,血浆中检测到喹烯酮原药和3-甲基喹噁啉-2-羧酸(MQCA).喹烯酮在鸡体内的药动学数据采用统...     

喹赛多及其两种代谢产物在大鼠体内消除规律研究

通过喹赛多(Cyadox,CYX)在大鼠体内消除规律研究,了解其对食品安全的影响,并为今后的药理学和毒理学研究提供较为详细的数据基础,研究建立了喹赛多及其两种主要代谢产物脱二氧喹赛多(BDCYX)和喹噁啉-2-羧酸(QCA)的提取和HPLC检测方法,并以大鼠作为研究载体,按推荐剂量连续混饲给药7 d后,研究喹赛多及其两种代谢物在血浆、肌肉、肝脏中消除规律;一次性灌胃给药后研究喹赛多及其两种代谢物在排泄物中的消除规律特点。结果表明,CYX和BDCYX在0~24 h和24~48 h时间段的粪便中可大量检出,在血浆、肌肉、肝脏和尿液中未检出;QCA在6 h的肌肉中有少量残留,在肝脏中一直到72 h还有一定量的残留,在血浆和粪便中未发现其存在。本研究结果为今后喹赛多在体内处置研究提供了可直接借鉴的技术手段和理论基础。     

喹赛多及其主要代谢物在猪体内的药代动力学研究

试验研究了灌服单剂量喹赛多(40 mg/kg体重)后原药及其代谢物在健康猪体内的药代动力学特征。液相色谱-串联质谱法测定血浆中喹赛多及其代谢物的浓度,通过WinNonlin 5.2药代动力学软件分析,用非房室模型统计矩原理计算喹赛多及其代谢产物的药动学参数。主要药动学参数分别为喹赛多:t_1/2 (7.52±1.77) h,C_max(0.02±0.01) μg/mL,AUC_{(0-36 h)} (0.26±0.24) (h·μg)/mL,MRT(11.37±3.21) h;N1(脱一氧喹赛多):t_1/2 (3.05±1.12) h,C_max(0.35±0.18) μg/mL,AUC_{(0-36 h)} (2.13±2.31) (h·μg)/mL,MRT(11.83±3.34) h。N4(脱一氧喹赛多):t_1/2 (2.91±1.15) h,C_max(0.60±0.32) μg/mL,AUC_{(0-36 h)} (3.78±4.28) (h·μg)/mL,MRT(11.00±2.86) h。脱二氧喹赛多:t_1/2 (3.85±1.30) h,C_max(0.46±0.19) μg/mL,AUC_{(0-36 h)} (4.21±2.47) (h·μg)/mL,MRT(13.35±2.65) h。QCA(喹口恶啉-2-羧酸):t_1/2 (5.08±0.57) h,C_max(0.25±0.11) μg/mL,AUC_{(0-36 h)} (3.05±1.46) (h·μg)/mL,MRT(15.15±1.83)h。结果表明,血浆中主要存在形式为代谢物,各代谢物的血药浓度及AUC_(0-∞)均高于喹赛多,喹赛多消除半衰期最长,QCA平均滞留时间最长。     

乙酰甲喹及其主要代谢物在鸡体内的残留消除规律研究

建立了高效液相色谱串联质谱（HPLC—MS／MS）法用于乙酰甲喹及其6种主要代谢物的检测。将65只健康白羽鸡分为两组,10只为空白对照组,其余为试验组。试验组施以20mg／kgb．w．灌胃乙酰甲喹悬浊液,1d2次,连续3d。在给药结束后2、4、6、12、16、24、30、36、48、72、120h分别宰杀5只鸡,采集血液和可食性组织（肌肉、肝脏、肾脏和皮脂）样品。结果表明：乙酰甲喹原药在组织和血浆中迅速消除;乙酰甲喹的代谢物广泛存在于鸡的可食性组织和血浆中;代谢物的残留消除过程较为复杂,代谢物在肌肉和肝脏中残留量较多,在皮脂中残留时间最长。研究结果将有助干榍示7．酷甲喹存鸡体内的砖留消除期,律．     

脱二氧乙酰甲喹及N4-脱一氧乙酰甲喹的合成及结构表征

乙酰甲喹属于喹喔啉-N,N-二氧化物类兽药,有研究认定3-甲基-2-乙酰基-喹喔啉是乙酰甲喹的残留标示物。N4-脱一氧乙酰甲喹则被认为是乙酰甲喹在大鼠、鸡及猪肝微粒体中的主要代谢产物。为给乙酰甲喹代谢产物及代谢机制的深入研究提供理论依据,本研究采用Na2S2O4还原乙酰甲喹,合成3-甲基-2-乙酰基-喹喔啉和N4-脱一氧乙酰甲喹,经质谱、红外及1 H核磁等光谱技术对二者的化学结构进行了确证,为乙酰甲喹代谢产物及代谢机制的深入研究提供了理论依据。     

脱二氧喹赛多在大鼠血浆及组织的药代动力学研究

本试验研究单剂量口服喹赛多(200 mg/kg体重)后,其主要代谢物脱二氧喹赛多(1.4-bisdesoxycyadox)在健康大鼠血浆及各组织内的药代动力学特征。灌服后在设定的时间点用剖杀的方法采集血浆及组织,经处理后,利用液相色谱-串联质谱法测定血浆及各组织中的药物浓度。通过Win Nonlin 6.1软件,用非房室模型统计矩原理计算脱二氧喹赛多在血浆及各组织中的药动学参数。主要药动学参数分别为血浆:T1/2β(4.63)h,Cmax(1534.00)μg/L,AUC0→∞(9937.08)h*μg/L,MRT(8.49)h;肝脏:T1/2β(32.45)h,Cmax(1982.50)μg/L,AUC0→∞(15489.71)h*μg/L,MRT(10.04)h;肾脏:T1/2β(18.96)h,Cmax(1286.67)μg/L,AUC0→∞(7671.06)h*μg/L,MRT(9.08)h;肌肉:T1/2β(10.19)h,Cmax(2293.33)μg/L,AUC0→∞(16154.84)h*μg/L,MRT(9.00)h;脂肪:T1/2β(8.47)h,Cmax(711.50)μg/L,AUC0→∞(5107.22)h*μg/L,MRT(8.79)h。脱二氧喹赛多在大鼠血浆及各组织中的消除速率缓慢,达峰浓度高,分布较广泛。     

Excretion mass balance evaluation,metabolite profile analysis and metabolite identification in plasma and excreta after oral administration of [14C]‐meloxicam to the male cat: preliminary study

Grudé, P., Guittard, J., Garcia, C., Daoulas, I., Thoulon, F., Ebner, T. Excretion mass balance evaluation, metabolite profile analysis and metabolite identification in plasma and excreta after oral administration of [¹⁴C]‐meloxicam to the male cat: preliminary study. J. vet. Pharmacol. Therap. doi: 10.1111/j.1365‐2885.2010.01157.x. The objective of this study was to investigate the metabolic pathways and routes of excretion of oral meloxicam in the cat. [¹⁴C]‐meloxicam was administered orally to three fasted male cats. Urine, faeces, vomit and cage washes were collected over the following 144 h period. Blood was collected predosing and at 3 and 12 h postdosing. Metabolites were identified by HPLC/MS/MS. When possible a metabolic structure was proposed for each metabolite detected. Only unchanged meloxicam was identified in plasma. Five major metabolites were detected in urine and four in faeces, which were identified by HPLC/MS/MS as products of oxidative metabolism. No conjugated metabolites were detected. Elimination occurred early (61% during the first 48 h). A total of 21% of the recovered dose was eliminated in urine (2% as unchanged meloxicam, 19% as metabolites) and 79% in the faeces (49% as unchanged meloxicam, 30% as metabolites). The results indicate that after oral administration the major route of excretion of meloxicam in the cat is faecal and that the main pathway of biotransformation of meloxicam in the cat is oxidation.     

Dietary exposure assessment of cyadox based on tissue depletion of cyadox and its major metabolites in pigs,chickens, and carp

The tissue kinetics of cyadox, an antibacterial agent used in food animals, and its major metabolites in pigs, chickens, and carp were investigated followed by a complete dietary exposure assessment to evaluate the food safety of cyadox. Cyadox and its major metabolites, bisdeoxycyadox (Cy1), 4‐desoxycyadox (Cy2), N ‐(quinoxaline‐2‐methyl)‐cyanide acetyl hydrazine (Cy4), quinoxaline‐2‐carboxylic acid (Cy6), and 2‐hydromethyl‐3‐hydroxy‐quinoxaline (Cy12), were simultaneously quantitated with a high‐performance liquid chromatography?ultraviolet (HPLC ‐UV ) method. Pigs, chickens, and carp were fed with 150 mg/kg cyadox in feed for consecutive 60, 40, and 30 days, respectively. The residue amount of cyadox and its major metabolites in liver, kidney, muscle, and fat (skin) tissues was determined. Cy2 was below the limit of quantitation even at the withdrawal time of 6 hr, cyadox, Cy4, Cy6, and Cy12 could be detected at 6–24 hr with low level less than 50 μg/kg. By contrast, Cy1 persisted for 3 days in the kidney of pigs and chickens, and in the liver of carp. Based on these residue depletion data and previous toxicology results, the global estimated chronic dietary exposure assessment of cyadox for general population was conducted, indicating a zero withdrawal time (WDT ) may be appropriate for cyadox in food animals when used in feed for prolonged administration. These results provide analytical techniques and safety standards suitable for residue monitoring of cyadox in food animals.     

喹赛多的合成工艺研究

以苯并呋咱-N-氧化物和丙酮醛缩二甲醇为起始原料,N,N-二甲基甲酰胺为溶剂,吡咯烷为催化剂,经过Beirut反应得到喹噁啉-1,4-二氧-2-甲醛缩二甲醇,喹噁啉-1,4-二氧-2-甲醛缩二甲醇在以甲醇为溶剂、浓盐酸为催化剂的条件下与氰乙酰肼反应生成喹赛多.终产物喹赛多经核磁共振氢谱、核磁共振碳谱、质谱、红外光谱进行确证.总收率为55.8％.     

In vivo and in vitro metabolism of dexamethasone in the camel

The metabolism of dexamethasone (DXM) in the camel was assessed by in vivo and in vitro techniques. Liver samples were collected at the abattoir from camels of either sex, and microsomes were isolated and characterized as to their protein and haemoprotein content as well as for their ability to metabolise several cytochrome P450 model substrates. The expression of different P450 enzymes was evaluated by means of immunoblotting, and the glucuronidating capacity was assessed with 1-naphthol as the substrate. The activity of 11 beta-hydroxysteroid dehydrogenase type 1 was assayed using metyrapone as a model substrate. To examine the in vitro metabolism of DXM, microsomes were incubated with the corticoid in the presence of either a NADPH-generating system or of uridindiphosphoglucuronic acid. In vivo metabolism of DXM was studied in two male camels, injected with a bolus intravenous dose of DXM (0.2 mg/kg body weight) and DXM metabolites were evaluated in urine samples collected at different times after the administration. DXM and metabolites were extracted using solid phase and liquid-liquid extraction, and analysed by liquid chromatography mass spectrometry (LC/MS) and by LC/MS/MS. Comparative results were obtained by in vitro and in vivo studies. Two phase I metabolites were detected: the major one resulted from reduction of the 3-carbonyl group in ring A and the minor metabolite from ring hydroxylation of ring A. Glucuronidation involved both phase I metabolites as well as the parent compound.     

喹赛多对山羊生长性能及胴体品质的影响

60只3月龄左右杂交山羊,随机分为6组,空白对照组饲料不添加任何药物,药物对照组饲料添加50 mg/kg喹乙醇,4个试验组饲料中分别添加25、50、150和250 mg/kg喹赛多,试验期90 d.结果表明喹赛多25、50和150mg/kg能改善山羊平均日增重(ADG)4.4%、14.5%和7.2%,提高饲料转化率(FCR)5.9%、12.0%和7.4%.喹赛多250 mg/kg提高FCR 8.6%,但降低ADG 2.1%.喹赛多50 mg/kg组ADG和FCR均高于喹乙醇组.喹赛多50 mg/kg降低山羊腹泻频率39.0%,但随剂量增加,其抗腹泻作用下降.喹赛多25、50、150和250 mg/kg能提高山羊屠宰率2.5%、9.0%、5.0%和1.0%,增加眼肌面积4.8%、34.1%、16.1%和14.0%.喹赛多50 mg/kg组山羊净肉率提高14.0%,高于其他各组.喹赛多各剂量均能显著增高肌肉pH值,平均降低滴水损失值13.7%.可见,喹赛多适宜剂量,尤其50 mg/kg,有较好的促进山羊生长和改善胴体品质的作用.     

UPLC-MS/MS法测定动物源性食品中氯苯胍及其代谢物残留

为快速有效地检测动物源性食品中氯苯胍及其代谢物残留,研究建立一种在鸡蛋、鸡肉、牛肉、鱼肉和猪肉5种动物源性食品中,同时检测氯苯胍及其代谢物（对氯苯甲酸、对氯苯甲酰氨基乙酸）残留的超高效液相色谱-串联质谱（UPLC-MS/MS）法。样品经过2%（V/V）甲酸乙腈溶液提取,无水硫酸钠去除水分,氮吹浓缩后甲醇复溶,正己烷除脂,高速冷冻离心,得到净化后的样品进行上机测定。选用Waters ACQUITY UPLC BEH C18色谱柱（2.1 mm×100 mm,1.7 μm）,将甲醇-0.1%甲酸水溶液作为流动相进行梯度洗脱。通过多反应检测（MRM）,在正/负离子模式下,采用基质匹配外标法,同时对3种化合物进行定性和定量分析。结果显示,氯苯胍、对氯苯甲酸和对氯苯甲酰氨基乙酸在各自浓度范围内线性关系良好,相关系数R²>0.999。氯苯胍的检出限（LOD）和定量限（LOQ）分别为0.5和1.0 μg/kg,对氯苯甲酸的LOD和LOQ分别为2.5和5.0 μg/kg,对氯苯甲酰氨基乙酸的LOD和LOQ分别为1.0和2.5 μg/kg。不同基质中,3种化合物在4个添加水平（氯苯胍：1.0、25、50、100 μg/kg;对氯苯甲酸：5.0、25、50、100 μg/kg;对氯苯甲酰氨基乙酸：2.5、25、50、100 μg/kg）的平均回收率为76.0%~95.9%,相对标准偏差（RSDs,n=6）为2.6%~10.6%。基质效应|ME|为0.2%~26.2%,其中氯苯胍在鸡蛋中,对氯苯甲酰氨基乙酸在鸡肉、牛肉和鱼肉中存在较强的基质效应（|ME|>20%）,空白猪肉可作为代表基质用于3种化合物的定量分析。本方法前处理简单,灵敏度较高,重现性好,可用于动物源食品中氯苯胍及其代谢物残留的测定。     

The inability of tapeworm Hymenolepis diminuta and fluke Dicrocoelium dendriticum to metabolize praziquantel

Biotransformation enzymes can, to a certain extent, protect parasitic worms against the toxic effects of anthelmintics and can contribute to drug-resistance development. The objective of our work was (1) to find and identify phase I and II metabolites of the anthelmintic praziquantel (PZQ) formed by the lancet fluke (Dicrocoelium dendriticum) and the rat tapeworm (Hymenolepis diminuta) and (2) to compare PZQ metabolites in helminths with PZQ biotransformation in rat as host species. Ultra high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) was used for this purpose. During in vitro incubations, mitochondria-like and microsomes-like fractions (prepared from homogenates of adult worms or from rat liver homogenate) were incubated with 10 and 100 μM PZQ. Liquid/liquid extraction was used for samples during in vitro experiments. In the ex vivo study, living D. dendriticum and H. diminuta adults were incubated in RPMI-1640 medium in the presence of 50 nM or 100 nM PZQ for 24h. After incubation, the worms were removed from the medium and homogenized. Homogenates of worms, medium from the incubation of worms or rat hepatocytes and rat urine (collected during 24h after oral PZQ administration) were separately extracted using solid-phase extraction. The results showed that both D. dendriticum and H. diminuta enzymatic systems are not able to metabolize PZQ. On the other hand, thirty one different phase I and four phase II PZQ metabolites were detected in rat samples using UHPLC/MS/MS analyses. These results show that our experimental helminths, as the members of tapeworm and fluke groups of parasites, are not able to deactivate PZQ, and that the biotransformation enzymes of the studied helminths do not contribute to PZQ-resistance.