LC-MS法测定人尿液中依非巴特的浓度

目的：建立测定人尿液中依非巴特浓度的方法。方法：尿液样品经蛋白沉淀后,采用液-质联用法进样测定,以甲醇-0.008mol·L-1甲酸铵水溶液（含0.1%甲酸）（35∶65）为流动相,AgilentZorbaxEclipseXDB-C18为色谱柱进行分离;采用电喷雾电离源,以多反应监测（MRM）方式进行正离子检测,用于定量分析的离子对依非巴特为m/z832.6→m/z646.4,内标为m/z931.6→m/z745.6。结果：依非巴特尿药浓度在5.0～5000.0ng·mL-1范围内线性关系良好（r=0.9996）,最低定量限（LLOQ）为5.0ng·mL-1;日内和日间RSD均〈7%,方法回收率为99.4%～103.1%,绝对回收率≥93%。结论：本方法选择性强、灵敏度高、重现性好,能快速、准确测定人尿液中依非巴特的浓度,并且成功用于依非巴特尿药排泄的研究。     

LC-MS/MS法测定老年房颤患者血浆中利伐沙班的浓度

目的：建立LC-MS/MS法检测老年房颤患者血浆中利伐沙班血药浓度,用于患者服用利伐沙班后血药浓度监测及药动学研究。方法：血浆样品经含内标利伐沙班-D4乙腈沉淀蛋白,使用色谱柱为Agilent Eclipse XDB-C18柱（2.1 mm × 100 mm,3.5 μm）,流动相为0.1%甲酸水（A）∶甲醇（B）,采用梯度洗脱分离,流速0.3 mL·min-1,柱温为40 ℃。采用电喷雾离子化电离源（ESI）,正离子方式检测,利伐沙班和内标的离子对分别为m/z 436.4→m/z 145.2和m/z 440.7→m/z 145.3。结果：血浆中利伐沙班在5~500 ng·mL-1范围内线性关系良好（r=0.999 7）。最低检测浓度为5 ng·mL-1,提取回收率与基质效应分别为90.8%~101.3%和95.2%~103.3%。质控样本日内和日间准确度分别为98.6%~112.8%和99.7%~112.9%,相对标准差（RSD）均<10%。样本稳定性符合要求。结论：本研究建立的方法快速、准确,适用于血浆利伐沙班的血药浓度检测和药动学研究。     

小儿风叶咳喘平合剂3个成分在大鼠体内的药代动力学研究

目的:对小儿风叶咳喘平合剂中3个主要药效成分在大鼠体内的代谢产物以及药代动力学进行研究。方法:基于超高效液相色谱-电喷雾飞行时间质谱(UHPLC-ESI TOF MS)方法，采用ACQUITY UPLC?HSS T3色谱柱(100 mm×2.1 mm, 1.8μm),以0.1%甲酸水和乙腈为流动相进行梯度洗脱，流速为0.3 mL·min^-1,质谱采用电喷雾离子源(ESI),以正负离子模式采集多级质谱碎片信息，对大鼠灌胃小儿风叶咳喘平合剂后的血清、胆汁、尿液和粪便进行分析，结合离子碎片进行代谢产物鉴定分析；同时采用超高效液相色谱串联质谱(UHPLC-MS/MS)的技术进行药代动力学研究。以格列齐特为内标物，采用ACQUITY UPLC?BEH C₁₈色谱柱(50 mm×2.1 mm, 1.7μm),以0.1%甲酸水和乙腈为流动相进行梯度洗脱。血浆样品采用甲醇沉淀蛋白，采用ESI及多反应监测(MRM),选择监测离子(麻黄碱m/z 166.2→148.1、甘草次酸m/z 471.2→453.2、对香豆酸m/z 162.9→119.1、内标m/z 3...     

用液相色谱-串联质谱法测定人血清和尿液中非那西丁及其代谢物浓度

目的:建立测定人体血清和尿液中非那西丁及其代谢产物的液相色谱-串联质谱法。方法:用乙酸乙酯萃取血清和尿液中非那西丁及其代谢产物。色谱柱为Restek Allure PFPP柱(100 mm×2.1 mm,5μm),流动相为乙腈-0.1%甲酸水溶液(30∶70,V/V),流速0.3 mL/min。以磺胺甲氧嘧啶为内标,非那西丁、对乙酰氨基酚和内标的多反应监测(MRM)离子对分别为m/z180→138、152→110和281→156。测定13名健康受试者口服非那西丁1.25 g后血清和尿液中非那西丁及其代谢产物的浓度。结果:非那西丁、对乙酰氨基酚和内标的保留时间分别为4.1、1.4和2.8 min。在0.2~50μg/mL范围内,非那西丁和对乙酰氨基酚的浓度与峰面积相关性良好(r>0.999 0)。非那西丁和对乙酰氨基酚的准确度分别为87.1%~101.5%和89.5%~107.3%,精密度分别为0.85%~7.83%和2.53%~8.74%,提取回收率为53.0%~76.0%,基质效应为92.0%~106.3%,均符合生物样本分析方法的有关要求。除万古霉素和甲泼尼龙外,其他临床常用药物对测定无干扰。13名健康受试者血清中非那西丁和总对乙酰氨基酚浓度为(5.94±4.79)和(8.81±2.91)μg/mL;尿液中非那西丁和总对乙酰氨基酚浓度为(3.25±1.10)和(570.45±187.13)μg/mL。结论:该方法简便、灵敏、特异性强,具有临床实用价值。     

咖啡因及代谢产物的尿药浓度测定方法建立及临床应用

目的 建立同时测定尿液中咖啡因及3种代谢产物茶碱、副黄嘌呤和可可碱浓度的方法并应用于临床。方法 以咖啡因-¹³C₃-d3为内标,尿液样本经乙腈沉淀蛋白后,采用高效液相色谱-串联质谱(HPLC-MS/MS)技术测定咖啡因及3种代谢产物的浓度。以Waters ACQUITY UPLC^? BEH HILIC为色谱柱,60 mmol/L乙酸铵溶液(A)-乙腈(B)为流动相进行梯度洗脱,流速为0.5mL/min,柱温为38℃,进样量为2μL。采用电喷雾离子源,以多反应监测模式进行正离子扫描,用于定量分析的离子对分别为m/z 195.1→110.0(咖啡因)、m/z 181.1→124.0(茶碱)、m/z 181.1→124.0(副黄嘌呤)、m/z 181.1→138.0(可可碱)、m/z 198.1→140.1(内标)。采用上述方法测定19例早产儿呼吸暂停(AOP)患儿尿液中咖啡因及3种代谢产物的浓度。结果 咖啡因、茶碱、副黄嘌呤、可可碱检测质量浓度的线性范围分别为0.200～200、0.050～50.0、0.050～50.0、0...     

LC-MS/MS法测定人血浆及尿液中缬沙坦的浓度及其药动学研究

目的:建立测定人血浆及尿液中缬沙坦浓度的方法。方法:血浆样品经酸化后用乙醚提取浓度后进行分析;尿液样品直接稀释进样,均采用液相色谱-串联质谱(LC-MS/MS)法测定,色谱柱为Aglient ZORBAX SB-C18,流动相为0.1%甲酸-乙腈(梯度洗脱),流速0.2 ml/min。电喷雾离子源(ESI),正离子多反应监测模式测定,缬沙坦定量分析的离子对为m/z 436.4→253.2,定性分析离子对为m/z 436.4→291.3;内标氯沙坦定量分析的离子对为m/z 423.4→207.1,定性分析离子对为m/z 423.4→180.2。结果:血浆和尿液中缬沙坦的线性范围分别为4~5 000 ng/ml(r=0.998 5)、20~50 000 ng/ml(r=0.998 8);最低定量限分别为4、20 ng/ml;血浆萃取回收率为61.21%~70.30%;内标归一化基质效应因子的RSD分别为3.20%、11.21%;日内和日间RSD均≤8.34%。结论:所用方法快速、灵敏度高,适用于人血浆及尿液中缬沙坦的浓度测定及药动学研究。     

LC-MS/MS方法研究新型酪氨酸酶抑制剂UP302的大鼠皮肤吸收量和皮肤代谢稳定性

目的建立大鼠皮肤中UP302定量的LC-MS/MS方法,并应用于研究UP302乳膏在大鼠皮肤局部给药24h后的皮肤吸收量,以及离体大鼠皮肤中UP302的代谢稳定性。方法 UP302用甲醇溶解稀释,皮肤样品用2倍体积甲醇沉淀处理,内标法进行定量。采用Hypersil Gold C18色谱柱,柱温30℃;甲醇为流动相A,5mmol.L-1甲酸铵水溶液为流动相B,以0.2mL.min-1梯度洗脱,进行色谱分离,运行时间6min。采用负离子电喷雾离子化电离源和选择反应监测（SRM）模式进行串联质谱分析,用于定量分析的离子反应分别为m/z 301.1→135.2（UP302）和m/z 252.9→132.0（内标大豆苷元）。结果 UP302皮肤标准样品在5～2000ng.mL-1的浓度范围内线性关系良好（r=0.9998）。UP302在大鼠皮肤匀浆中的最低定量限是5ng.mL-1。本方法日内准确度在100.00%～105.23%,日内精密度小于5.82%。结论本LC-MS/MS方法专属性强、灵敏度高、重现性好、操作简便、结果准确,可用于UP302在皮肤组织中含量的测定,以及UP302在大鼠皮肤组织中的代谢稳定性研究。     

LC-MS-MS法测定血浆中多西他赛和紫杉醇浓度及在乳腺癌患者中的应用

目的：建立一种快速、灵敏的液相色谱－串联质谱（LC-MS/MS）法检测乳腺癌患者血浆中多西他赛、紫杉醇的浓度。方法：多西他赛和紫杉醇互为内标,血浆样品100μL加入1 mL叔丁基甲醚萃取,分离有机相,以氮气吹干后流动相复溶进样。色谱柱为Agilent Eclipse XDB-C18（2.1 mm×100 mm,3.5μm）,流动相为0.4%甲酸水溶液-0.4%甲酸乙腈溶液（20∶80,v/v）,流速0.3 mL·min-1,柱温为40℃。采用多反应监测（MRM）进行定量,电喷雾电离源（ESI）正离子方式进行检测,多西他赛与紫杉醇用于定量分析的检测离子对分别为m/z 808.5→m/z 527.2和m/z 854.3→m/z 569.4。结果：多西他赛和紫杉醇的线性范围分别为5.0~1000 ng·mL-1和1.0～500 ng· mL-1,最低检测浓度分别为5.0 ng·mL-1和1.0 ng·mL-1。两药低、中、高三个浓度的批内和批间RSD均<15%,平均提取回收率分别为65.9%~84.3%和90.4%~106.5%。结论：本法快速、准确、灵敏、专属性强,适用于同步测定多西他赛和紫杉醇血药浓度及其在中国乳腺癌患者中的药动学研究。     

UPLC-QTRAP-MS/MS法同时测定大鼠血浆中洛伐他汀和活性代谢产物洛伐他汀酸的浓度及其药动学研究

摘要 目的：建立测定大鼠血浆中洛伐他汀及其活性代谢产物洛伐他汀酸浓度的超高效液相三重四级杆线型离子阱串联质谱检测方法,并将其应用于药动学研究。方法：采用乙酸乙酯萃取血浆样品,以辛伐他汀为内标,采用Agilent ZORBAX C18 2.1 mm × 50 mm, 1.8 μm）为色谱柱,以乙腈（A）-0.1%甲酸（B）为流动相,梯度洗脱流速为0.4 mL·min-1,柱温40°C。以正离子多反应离子监测模式,用于定量分析的离子对分别为m/z 405.1?225.1（洛伐他汀）,m/z 423.2?199.2（洛伐他汀酸）,m/z 419.2?199.1（辛伐他汀）。结果：洛伐他汀和洛伐他汀酸的线性范围分别为0.08-80.0 ng·mL-1和1.60-1600.0 ng·mL-1,其定量下限（LLOQ）分别为0.08 ng·mL-1和1.60 ng·mL-1;洛伐他汀和洛伐他汀酸的日内和日间精密度均小于9.0%,准确度在-5.18%-6.83%范围内;洛伐他汀和洛伐他汀酸的提取回收率均大于72.26%,RSD均小于10.83%;洛伐他汀和洛伐他汀酸的基质效应在91.11%-105.73%,RSD均小于7.46%;洛伐他汀和洛伐他汀酸的Tmax分别为1.58 ± 0.11 h和2.15 ±0.26 h,Cmax分别为136.22 ± 30.25 ng·mL-1和212.57 ± 33.92 ng·mL-1,t1/2分别为35.42 ± 12.67 h和17.86 ± 6.15 h,AUC0?24 h分别为279.92 ± 44.18 ng h·mL-1和390.34 ±20.15 ng h·mL-1。结论：该方法快速、灵敏、准确,可用于洛伐他汀和洛伐他汀酸在大鼠体内的药动学研究。     

高效液相-质谱串联法测定大鼠血浆中辛伐他汀及辛伐他汀酸的浓度

目的:建立HPLC-MS法测定大鼠血浆中辛伐他汀及其代谢物辛伐他汀酸的浓度。方法:血浆样本加入适量内标和醋酸铵缓冲液,以甲基叔丁基醚萃取后采用LC-MS进行分析。色谱柱采用Inertsil ODS-3柱(150 mm×2.1 mm,5.0μm);流动相由乙腈-2.5 mmol.L-1醋酸铵(含0.1%甲酸)(75∶25)组成,柱温35°C;流速0.3 mL.min-1;采用电喷雾离子源(ESI),以多反应监测方式(MRM)进行定量分析。辛伐他汀和内标洛伐他汀在正离子模式下定量分析离子对分别为m/z 419.2→m/z199.2和m/z 405.2→m/z 199.2;辛伐他汀酸和内标洛伐他汀酸在负离子模式下定量分析离子对分别为m/z 435.2→m/z319.2和m/z 421.4→m/z 319.2。结果:辛伐他汀和辛伐他汀酸在5.0～6 400 ng.mL-1内线性关系良好(r>0.999),最低定量限为0.1 ng.mL-1,提取回收率为87.91%～99.77%,日内、日间精密度均不高于8.95%。结论:该方法分析速度快、灵敏、准确,为临床进一步研究辛伐他汀提供了基础。     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.     

Alzheimer’s disease – current trends in basic and clinical research. Highlights from the 16th ECNP

Advances in the molecular biological knowledge of neuronal nicotinic acetylcholine receptors (nAChRs) have led to a growing interest by the pharmaceutical industry in the development of novel compounds that selectively modulate nAChR function. The ability of (-)-nicotine, an activator of nAChRs, to enhance attentional aspects of cognition in animals and humans, to exert neuroprotective and anxiolytic-like effects, and presumably to mediate the negative correlation between smoking and Alzheimer's (and Parkinson's) Disease, has focused interest on the potential therapeutic utility of modulators of nAChR function for treatment of some of the deficits associated with these progressive, neurodegenerative conditions. Numerous compounds are known which activate nAChRs and which might serve as lead compounds toward the development of such agents. The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favourable side-effect profiles than existing agents. This broader class of agents, collectively called cholinergic channel modulators (ChCMs), is anticipated to encompass compounds which would have more favourable side-effect profiles than existing agents, which generally exhibit low selectivity. This selectivity may be achieved by preferentially activating some subtypes of nAChRs (i.e., Cholinergic Channel Activators, ChCAs) or inhibiting the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs). An overview of the biology of nAChRs and the rationale for the use of ChCMs for the treatment of dementia related to neurodegenerative diseases are presented, followed by a discussion of lead compounds and compounds under consideration for clinical evaluation.