LC-MS/MS测定大鼠口服芫花粗提物后芹菜素、3’-羟基芫花素和芫花素的血药浓度

目的建立一个灵敏、快速的液相色谱-质谱联用法同时测定大鼠口服芫花粗提物后芹菜素、3’-羟基芫花素和芫花素在血浆样品中的浓度。方法以秦皮甲素为内标,大鼠血浆样品经液液萃取后,以甲醇-0.5%甲酸(80∶20)为流动相,采用Lichrosorb Rp-C18(4.5 mm×150 mm,5μm)色谱柱分离,通过ESI三重四级杆串联质谱,以多反应监测(MRM)方式进行检测。结果芹菜素、3’-羟基芫花素和芫花素测定方法的线性范围均为10~2 000 ng·m L-1;定量下限为10 ng·m L-1。该方法被成功应用于芫花粗提物在大鼠体内的药动学研究。结论该方法灵敏度高,操作简便,分析速度快,适用于含有芹菜素、3’-羟基芫花素或芫花素的药物测定或药动学研究。     

人EC-SOD在大肠杆菌中表达的发酵条件研究

对含人EC-SOD重组质粒的工程菌进行诱导表达形成包涵体.通过SDS-PAGE电泳分析,考察了诱导时间和诱导剂种类、浓度对EC-SOD表达量的影响.同时考察了细胞培养温度对产物表达形式的影响,并对菌体密度不同时诱导表达的结果进行了比较.     

LC-MS-MS法同时测定人血浆中阿戈美拉汀及其2种代谢产物的浓度

目的建立同时测定人血浆中阿戈美拉汀及其代谢产物7-去甲阿戈美拉汀和3-羟基-阿戈美拉汀浓度的方法。方法采用LC-MS/MS法(液相色谱串联质谱)进行检测,以非那西汀为内标、Phenomenex ODS-3为色谱柱,A(0.2%甲酸铵+100%水)-B(甲醇)=30∶70为流动相进行色谱分离。采用ESI(电喷雾电离源)和MRM(多反应监测)的方式进行正电荷检测,用作定量分析的阿戈美拉汀、7-去甲-阿戈美拉汀、3-羟基-阿戈美拉汀和非那西汀的离子反应对m/z分别为244.1→185.1、230.1→171.1、260.1→201.1、180.1→110.1。结果阿戈美拉汀、7-去甲-阿戈美拉汀、3-羟基-阿戈美拉汀分别在0.045 7~100 ng/m L、0.137 2~300 ng/m L、0.457 2~1000 ng/m L范围内线性关系良好;最低检测限分别为0.045 7、0.137 2、0.457 2 ng/m L;所有待测样本的日内及日间精密度和准确度均符合相关生物样品分析的要求,且RSD<10%。在稳定性试验中,血浆样品在室温放置4 h、在4℃放置24 h、经历3次冷冻-解冻循环及在-80℃中放置3个月等条件下均可保持稳定。结论该方法具有操作简便、检测快速及灵敏性好等特点,能够用于临床相关药动学研究。     

Determination of itopride and its N-oxide in biological fluids by high-performance liquid chromatography with positive ion electrospray ionization tandem mass spectrometric detection

Itopride, which is a dopamine D2 antagonist with acetylcholinesterase inhibitory for patients with functional actions, is often prescribed dyspepsia. The primary metabolite in humans is the N-oxide, generated by oxidation of the tertiary amine N-dimethyl group. The urinary excretions of itopride and its N-oxide were 3.7% and 75.4%, respectively, in healthy subjects after a single oral administration at a therapeutic dose. Studies support a predominant role of FMO3 in the formation of itopride N-oxide in human liver microsomes. In addition, itopride appears to be a suitable probe for human liver FMO3. Our aim was to establish a simple method adopting a one-step liquid-liquid extraction with dichloromethane followed by high-performance liquid chromatography （HPLC） with positive ion electrospray ionization tandem mass spectrometric （ESI- MS/MS） detection to simultaneously determine itopride and its N-oxide in human plasma and urine for FMO3 pharmacogenetics study, using sulpiride as an internal standard （IS）.METHODS： Acquisition was performed in multiple reaction monitoring （MRM） mode, by monitoring the transitions： m/z 360.1 〉 166.4 for itopride, m/z 376.1 〉 165.5 for itopride N-oxide and m/z 342.9 〉 112.2 for IS, respectively. Analytes were chromatographed on an Intersil ODS-3 reverse-phase chromatographic column （ 2.1 nun × 150 mm, 5 μm） by isocratic elution with water （ 1000 mL water added 1 mL formic acid）-acetonitrile （60 ： 40, v/v）, the flow rate was 0.2 mL/min with a total analysis time of 3 min per run.[第一段]     

液-质联用测定Nrf2野生型和基因敲除型小鼠肝脏中16种胆汁酸

目的:建立测定小鼠肝脏中16种胆汁酸浓度的LC-MS/MS法,并将测定结果应用于比较Nrf2野生型和基因敲除型小鼠胆汁酸代谢谱。方法:小鼠肝脏样品加入70%乙腈匀浆,用甲醇沉淀匀浆液中的蛋白并高速离心,取上清液置进样瓶, 用Xtimate^TM C₁₈柱分离,以甲醇-醋酸铵及甲酸水溶液为流动相进行梯度洗脱,流速0.25 ml·min^-1,进样5 μl分析。结果:16种胆汁酸在0.013~8.000 μmol·L^-1范围内,线性关系良好,定量下限浓度为0.013 μmol·L^-1,肝脏匀浆液加样回收率在94.9%~112.8%之间,日内、日间RSD均小于10.8%。结论:该方法准确可靠,灵敏度高,处理简便,适用于肝脏中胆汁酸浓度的测定,为生物样品中胆汁酸含量测定提供参考。     

LC-MS/MS测定大鼠血浆中阿托伐他汀及其活性代谢产物

目的 建立LC-MS/MS同时测定大鼠血浆中阿托伐他汀、邻羟基阿托伐他汀及对羟基阿托伐他汀的方法,并应用于CYP3A酶诱导模型大鼠和正常大鼠体内阿托伐他汀的药动学研究。方法 采用甲基叔丁基醚-乙酸乙酯（50︰50）液液萃取法提取大鼠血浆中药物。使用XBridge C₁₈（2.1 mm×250 mm,3.5 μm）色谱柱,柱温35℃;流动相为0.1%甲酸-乙腈（40：60）,等度洗脱4.4 min,流速0.2 mL·min^-1;进样体积10 μL。质谱采用电喷雾离子（ESI）源,以正离子多反应监测（MRM）模式进行定量分析,选择m/z 559.1→440.1（阿托伐他汀）,m/z 575.3→440.2（邻羟基阿托伐他汀/对羟基阿托伐他汀）,m/z 564.3→445.3（阿托伐他汀-d₅,内标）作为检测离子对。以地塞米松80 mg·kg^-1·d^-1连续灌胃给药4 d,建立CYP3A酶诱导模型,取正常及模型大鼠给药后0,0.083,0.17,0.25,0.33,0.5,0.75,1,1.5,2,3,4,6 h血样于肝素抗凝管中,离心收集血浆,冷冻保存直到进行测定。结果 阿托伐他汀及其2种代谢产物在0.49~500.00 ng·mL^-1内均有良好的线性关系（r²>0.99）;批内、批间精密度RSD<15%（n=6）;方法的提取回收率和基质效应均满足生物样品的检测要求;含药血浆在室温放置4,24 h、4℃放置3 d稳定。诱导组大鼠血浆中阿托伐他汀血药浓度达峰时间（T_max）提前,血药浓度-时间曲线下面积（AUC_0-t）显著低于正常组,消除速率常数K和清除率CL略高于正常组。结论 新建立的方法简便、稳定、灵敏,能够用于大鼠血浆内阿托伐他汀及其活性代谢产物的浓度测定和药动学研究。进入CYP3A酶诱导模型大鼠与正常大鼠体循环的活性药物成分差异显著。     

LC-MS/MS测定大鼠血浆中K15的浓度及其在药动学研究中的应用

目的 建立SD大鼠血浆中（3AS,4S,6AR）-四氢-4-甲氧基-呋喃并[3,4-B]呋喃-2（3H）-酮（K15）的LC-MS/MS测定方法,并进行K15的药动学研究。方法 分别灌胃及静脉注射给予大鼠不同剂量K15后,从眼眶静脉丛取血,并采用LC-MS/MS测定大鼠血浆中K15的浓度变化。利用DAS药动学软件拟合主要药动学参数AUC、C_max、T_max、T_1/2等。结果 大鼠血浆中的内源性杂质不干扰K15的测定,日内精密度4.53%~6.60%,日间精密度5.19%~8.14%,准确度为96.10%~102.49%。K15的线性范围为25~1 000 ng·mL^-1,r=0.998 5。K15的定量下限为25 ng·mL^-1（RSD=12.7%,n=6）。150,450和1 000 mg·kg^-1灌胃给药的生物利用度分别为79.5%,44.4%和57.0%。结论 该方法适用于K15在大鼠中的药动学研究。K15在大鼠中的药动学为非线性动力学,灌胃给予大鼠后在其体内具有一定的系统暴露量,但没有随给药剂量呈线性增加。在给药剂量为100和450 mg·kg^-1时,其在体内的C_max没有显著性增高,但是在1 000 mg·kg^-1时,C_max显著增加。随着给药剂量的增加,K15在大鼠体内的T_1/2显著增加,提示K15在大鼠体内的暴露时间随剂量的增加显著延长。     

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。     

Inhibition of Organic Anion Transporting Polypeptide 1B1 and 1B3 by Betulinic Acid: Effects of Preincubation and Albumin in the Media

Betulinic acid (BA), a plant-derived pentacyclic triterpenoid, may interact with the members of the organic anion transporting polypeptide 1B subfamily. Here, we investigated the interactions of BA and its analogs with OATP1B1/3 and rat Oatp1b2 in vitro and in vivo. BA inhibited the activity of OATP1B1/3 and rat Oatp1b2 in vitro. Systemic exposure of atorvastatin was substantially altered with the intravenous co-administration of BA (20 mg/kg). Preincubation (incubation with inhibitors, followed by washout) with BA led to a sustained inhibition of OATP1B3, which recovered rapidly in the media containing 10% fetal bovine serum. The addition of albumin to the media decreased intracellular concentrations of BA and expedited the recovery of OATP1B3 activity following preincubation. For asunaprevir and cyclosporin A (previously known to inhibit OATP1B3 upon preincubation), the addition of albumin to the media shortened recovery time with asunaprevir, but not with cyclosporin A. Overall, our results showed that BA inhibits OATP1B transporters in vitro and may incur hepatic transporter-mediated drug interactions in vivo. Our results identify BA as another OATP1B3 inhibitor with preincubation effect and suggest that the preincubation effect and its duration is impacted by altered equilibrium of inhibitors between intracellular and extracellular space (e.g., albumin in the media).     

Blood-brain barrier penetration of novel pyridinealdoxime methiodide (PAM)-type oximes examined by brain microdialysis with LC-MS/MS

To develop a new reactivator of inhibited acetylcholinesterase (AChE) that can easily penetrate the blood-brain barrier (BBB), BBB penetration of 6 known and novel pyridinealdoxime methiodide (PAM)-type oximes (alkylPAMs) with relatively high reactivation activities was examined by in vivo rat brain microdialysis with liquid chromatography-mass spectrometry (LC-MS/MS). The 50% lethal dose (LD(50)) of alkylPAMs was intravenously determined for Wistar rats, then the limit of detection, quantification range and linearity of the calibration curve of the alkylPAMs in dialysate and blood were determined by LC-MS/MS. Following 10% LD(50) intravenous administration of the alkylPAMs, 4-[(hydroxyimino) methyl]-1-(2-phenylethyl) pyridinium bromide (4-PAPE) and 4-[(hydroxyimino) methyl]-1-octylpyridinium bromide (4-PAO) appeared in the dialysate. Striatal extracellular fluid/blood concentration ratios were 0.039+/-0.018 and 0.301+/-0.183 (mean+/-SEM), respectively, 1 h after treatment. This is the first report of BBB penetration of 4-PAPE, and the concentration ratio was smaller than that of 2-PAM.The mean BBB penetration of 4-PAO was approximately 30%, indicating that intravenous administration of 4-PAO may be effective for the reactivation of blocked cholinesterase in the brain. However, the toxicity of 4-PAO (LD(50); 8.89 mg/kg) was greater than that of 2-PAM. Further investigation is required to determine the effects of these alkylPAMs in organophosphate poisoning.     

3D disorganization and rearrangement of genome provide insights into pathogenesis of NAFLD by integrated Hi-C,Nanopore, and RNA sequencing

The three-dimensional (3D) conformation of chromatin is integral to the precise regulation of gene expression. The 3D genome and genomic variations in non-alcoholic fatty liver disease (NAFLD) are largely unknown, despite their key roles in cellular function and physiological processes. High-throughput chromosome conformation capture (Hi-C), Nanopore sequencing, and RNA-sequencing (RNA-seq) assays were performed on the liver of normal and NAFLD mice. A high-resolution 3D chromatin interaction map was generated to examine different 3D genome hierarchies including A/B compartments, topologically associated domains (TADs), and chromatin loops by Hi-C, and whole genome sequencing identifying structural variations (SVs) and copy number variations (CNVs) by Nanopore sequencing. We identified variations in thousands of regions across the genome with respect to 3D chromatin organization and genomic rearrangements, between normal and NAFLD mice, and revealed gene dysregulation frequently accompanied by these variations. Candidate target genes were identified in NAFLD, impacted by genetic rearrangements and spatial organization disruption. Our data provide a high-resolution 3D genome interaction resource for NAFLD investigations, revealed the relationship among genetic rearrangements, spatial organization disruption, and gene regulation, and identified candidate genes associated with these variations implicated in the pathogenesis of NAFLD. The newly findings offer insights into novel mechanisms of NAFLD pathogenesis and can provide a new conceptual framework for NAFLD therapy.