LC-MS/MS法测定骨肉瘤患者血清中甲氨蝶呤及其 代谢产物7-羟基甲氨蝶呤的浓度

目的:建立测定人血清中甲氨喋呤及其代谢产物7-羟基甲氨喋呤浓度的LC-MS/MS法,并应用于骨肉瘤患者大剂量甲氨蝶呤化疗后的治疗药物监测,为临床合理用药提供依据.方法:以氨基蝶呤作为内标,血清样品经含内标甲醇沉淀蛋白处理.色谱柱为Agilent Zorbax Eclipse Plus C18;柱温35℃;流速0.4 mL· min-1;流动相为含0.1％甲酸的水溶液和含0.1％甲酸的乙腈溶液,梯度洗脱.质谱检测方式为电喷雾离子阱正离子模式,MRM扫描,监测甲氨蝶呤m/z 455.098→308.000,7-羟基甲氨喋呤m/z 471.187→324.100,氨基蝶呤m/z 441.198→294.100.结果:甲氨蝶呤浓度在2～1000 ng·mL-1 (r=0.9985),7-羟基甲氨蝶呤浓度在20～10000 ng·mL-1(r=0.9984)与峰面积线性关系良好.甲氨蝶呤、7-羟基甲氨蝶呤日内及日间RSD≤6.21％.甲氨蝶呤、7-羟基甲氨蝶呤的平均回收率分别为102.83％～119.34％、88.41％～95.37％.结论:本方法简便、准确、快速,适用于甲氨喋呤及其代谢产物7-羟基甲氨喋呤的血药浓度测定.     

UPLC-MS/MS测定犬血浆中的氢溴酸加兰他敏

目的采用超高效液相色谱-串联质谱法测定犬血浆中氢溴酸加兰他敏(GH)的含量。方法采用BEH C18色谱柱(50 mm×2.1 mm,1.7μm),流动相为乙腈-0.01 mol·L-1醋酸铵(含0.1%甲酸)(40∶60),流速0.1 mL·min-1,柱温40℃,进样量3μL。GH及内标盐酸克仑特罗的监测离子分别为:m/z 288.23→198.06,m/z 277.08→202.93。结果 GH的线性范围为2.38～609.60μg·L-1(r=0.998,n=5),在人血浆基质中,高、中、低浓度(4.76、38.10、304.80μg·L-1)的日内、日间RSD均小于15%,方法的平均回收率为102.3%;血浆基质对测定无干扰。结论所建方法处理简单、快速、灵敏、特异性高,定量准确,适用于血浆中GH的测定。     

Determination of Galanthamine hydrobromide in the dog plasma by UPLC - MS/MS

目的 采用超高效液相色谱-串联质谱法测定犬血浆中氢溴酸加兰他敏(GH)的含量.方法 采用BEH C18色谱柱( 50 mm×2.1 mm,1.7 μm),流动相为乙腈-0.01 mol·L-1醋酸铵(含0.1％甲酸)(40∶60),流速0.1 mL·min-1,柱温40℃,进样量3μL.GH及内标盐酸克仑特罗的监测离子分别为:m/z 288.23→198.06,m/z 277.08→202.93.结果 GH的线性范围为2.38～609.60μg·L-1(r=0.998,n=5),在人血浆基质中,高、中、低浓度(4.76、38.10、304.80 μg·L-1)的日内、日间RSD均小于15％,方法的平均回收率为102.3％;血浆基质对测定无干扰.结论 所建方法处理简单、快速、灵敏、特异性高,定量准确,适用于血浆中GH的测定.     

电喷雾离子阱二级质谱法测定人血浆中利培酮浓度

目的 建立电喷雾离子阱二级质谱法(LC-ESI-MS/MS)测定人血浆中利培酮浓度.方法 色谱柱DiamonsilTM C18(4.6mm×150mm,5μm);流动相:乙腈(1%甲酸):0.02mol·L-1醋酸铵=(60:40,V:V),流速:0.8 mL·min-1;柱温:25℃;进样体积:10μL;质谱条件为电喷雾离子源,检测方式为正离子多离子反应监测(MRM),用于定量分析的离子为利培酮m/z411→191,内标替米沙坦m/z516→497,生物样本采用醋酸乙酯:二氯甲烷(4:1)液液萃取处理.结果 利培酮血药浓度线性范围为0.5～50 μg·L-1,线性方程为C=2.38F-0.24,r=0.999(n=7),最低检测浓度为0.1μg·L-1,高中低三个浓度的提取回收率分别为71.70%,64.58%,64.93%,日内、日间RSD均小于15%.结论 本法灵敏、准确、快速,可用于临床常规血药浓度测定和药动学研究.     

液-质联用法测定人血浆伊潘立酮的药物浓度

目的:建立高效液相串联质谱方法测定人血浆中伊潘立酮的药物浓度。方法:方法色谱柱为Kromasil 60-5CN(100mm×2.1mm,5μm),流动相:乙腈与5mmol·L-1醋酸铵缓冲液(含0.1%甲酸)体积比为35∶65,流速为0.3mL·min-1,吡格列酮为内标,采用电喷雾离子源,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分别为m/z 427.2→m/z261.2(伊潘立酮),m/z357.2→m/z133.8(吡格列酮,内标)结果:伊潘立酮的血浆浓度在20～20 000pg·mL-1范围内线性良好,定量下限为20pg·mL-1,日内精密度<3%,日间精密度<9%,回收率为96.9%～101%。结论:该法操作简单,灵敏,准确,重现性好,适用于伊潘立酮人体药动学研究及生物等效性研究。     

LC-MS/MS法测定犬血浆中华法林钠的浓度及其应用

目的建立LC-MS/MS联用的方法测定犬血浆中华法林钠的浓度并进行药动学研究。方法以卤米松作为内标,犬血浆经甲醇蛋白沉淀处理后稀释进样。色谱柱为Venusil XBP苯基柱(100 mm×2.1 mm,5μm),流动相为0.1%甲酸水溶液(含2 mmol·L-1醋酸铵)-甲醇(含0.1%甲酸)(25∶75),流速0.3 m L·min-1;进样体积5μL。采用电喷雾离子源,负离子方式多反应监测(MRM)扫描分析。检测离子通道分别为m/z 307.1→161.3(华法林钠)和m/z 489.5→413.1(卤米松)。结果华法林钠的线性范围为5~1 500μg·L-1(r=0.997 8),批内和批间精密度RSD均<8%,高、中、低浓度的方法回收率为103.33%~107.29%。结论本方法操作简捷,灵敏度高,专属性好,适用于华法林钠的药动学研究。     

固相萃取LC-MS/MS法测定人血浆中头孢呋辛的浓度

目的建立人血浆中头孢呋辛浓度的LC-MS/MS检测方法。方法以头孢拉定为内标,血浆样品酸化后用固相小柱萃取处理;分析柱为Thermo Hypurity C18柱(2.1 mm×150 mm,5μm),流动相为甲醇-20mmol·L-1甲酸铵水溶液(65:35);流速为0.2 m L·min-1,柱温30℃;使用电喷雾离子源(ESI),负离子多反应监测(MRM)方式进行检测。结果头孢呋辛监测离子为m/z 423.1→m/z 207.1,内标头孢拉定监测离子为m/z 348.2→m/z 235.2,样品分析时间为3 min,血浆中内源性物质对测定无干扰,头孢呋辛线性范围为0.1024~20.00μg·m L-1(r2=0.998 8),定量下限为0.102 4μg·m L-1。批内、批间精密度(RSD)均<8.5%,头孢呋辛与内标的平均回收率分别为104.4%和95.5%,平均基质效应为95.8%和93.9%,且均不存在浓度依赖性。结论该方法特异性强,灵敏度高,测定结果可靠,适用于血浆样品的高通量分析。     

液相色谱串联质谱法测定人血浆中硫酸黏菌素的浓度

目的建立液相色谱串联质谱(LC-MS/MS)法测定人血浆中硫酸黏菌素的浓度。方法内标选用多黏菌素B,血浆样品采用甲醇-5%三氯乙酸(50∶50,V/V)沉淀蛋白。色谱柱为Dikma C_(18)(4.6 mm×150 mm,5 μm),流动相A为水(含0.1%甲酸),流动相B为乙腈(含0.1%甲酸),梯度洗脱,流速为0.8 mL·min-~1,柱温为40℃,进样量为10 μL。采用电喷雾离子化,正离子模式下进行多反应监测多黏菌素E_1(m/z 585.7 → 101.2)、多黏菌素E_2(m/z 578.8 → 101.2)和内标多黏菌素B(m/z 602.7 → 241.4)。结果硫酸黏菌素在0.1～10 μg·mL-~1内与峰面积线性关系良好(r=0.9982),定量限为0.1 μg·mL-~1,日内与日间精密度、准确度、提取回收率、基质效应、稳定性均符合方法学要求。结论本方法简便、快速、灵敏,可用于测定人体内硫酸黏菌素的浓度。     

HPLC-/MS/MS法测定健康人血浆中普拉克索浓度

目的 建立高效液相串联质谱法测定人血浆中普拉克索浓度的方法.方法 在碱性条件下,用乙酸乙酯提取浓缩后,进样用LC-MS/MS,固定相为AQ-C18柱(4.6 mm× 150 mm,10 μm),流动相为乙腈-20 mmol·L-1醋酸铵水溶液=60:40,质谱条件为电喷雾离子源、正离子方式、多级离子反应监测,离子反应分别为m/z:212.2→152.9(普拉克索)和m/z 273.2→109.6(吡西卡尼).结果 普拉克索的血浆浓度在5 ～ 1000 pg·mL-1内线性关系良好,Y=1.33×103X+0.05(r=0.9979),定量下限可达5 pg·mL-1.结论 建立的检测方法准确、稳定,可满足血浆中普拉克索含量测定.     

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.     

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.     

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.