液相微萃取-后萃取光化学荧光高效液相色谱法测定生物样品中甲氨蝶呤的含量

目的:采用液相微萃取-后萃取光化学荧光高效液相色谱法测定生物样品中甲氨蝶呤的含量。方法:应用自制的液相微萃取装置,在0.05mol.L-1盐酸酸性介质中,甲氨蝶呤以分子状态首先被聚醚砜中空纤维孔壁中的正丁醇萃取,继而被25μL0.05mol.L-1氨水溶液后萃取。后萃取液用等体积的0.2mol.L-1盐酸酸化后,经紫外光照射45min,发生光化学反应,取样20μL进行高效液相色谱分析,在λex=275nm,λem=370nm荧光检测,建立了液相微萃取-后萃取-光化学荧光高效液相色谱法测定生物样品中甲氨蝶呤含量的方法。结果:该方法在血浆和尿液中的浓缩倍数可达20~24倍,线性范围分别为0.05~50mg.L-1和0.01~50mg.L-1,检出限分别为10μg.L-1和5μg.L-1,RSD<11%。通过液相微萃取-后萃取,能有效地去除生物样品中干扰甲氨蝶呤测定的内源性杂质,提高了选择性。结论:该方法将液相微萃取和光化学荧光-高效液相色谱法相结合,为生物样品中甲氨蝶呤的检测提供了一种有效的分离分析方法。     

离子对-反相高效液相色谱法测定人胎盘和羊水中利凡诺

目的:运用离子对-反相高效液相色谱法测定人胎盘和羊水中利凡诺的含量。方法:采用 Spherisorb C_(18)色谱柱(250mm×4.6 mm,5μm);柱温:30℃;流动相:甲醇-乙腈-0.05%十二烷基磺酸钠溶液(35:35:30,pH=3);流速:1.0 mL·min~(-1);荧光检测器:激发波长360 nm,发射波长505 nm。结果:人胎盘、羊水样品分别在10 ng·g~(-1)～1μg·g~(-1)、10 ng·mL~(-1)～1μg·mL~(-1)浓度范围内,均呈现良好线性,检测限分别为3 ng·g~(-1)、3 ng·mL~(-1),准确度和精密度好。结论:本方法操作简便,结果准确可靠。     

液相微萃取-非水后萃取-高效液相色谱法测定大鼠体内厚朴酚与和厚朴酚的浓度

目的:建立液相微萃取-非水后萃取(LPME/NBE)-高效液相色谱法同时测定大鼠体内厚朴酚及和厚朴酚浓度的方法.方法:利用自制的液相微萃取装置,以正丁醇为萃取溶剂,以600 r·min-1转速萃取10 min,对大鼠血浆、肝脏和肾脏生物样品中的厚朴酚与和厚朴酚进行分离、萃取、纯化.色谱条件:ODS为色谱柱,甲醇-水(82∶18)为流动相,294 nm为检测波长.结果:厚朴酚与和厚朴酚线性范围分别为0.15～30.0 mg·L-1和0.10～30.0 mg·L-1,r＞0.900;RSD＜7.5%;平均回收率分别为93.7%～114%和90.5%～109%,血浆中检出限分别为30 μg·L-1和20μg·-1,肝脏中分别为25μg·L-1和15 μg·L-1,肾脏中均为10μg·L-1.结论:首次提出液相微萃取-非水后萃取方法,并将其成功应用于中药厚朴酚与和厚朴酚在大鼠体内的浓度测定.液相微萃取-非水后萃取能有效去除生物样品中干扰厚朴酚与和厚朴酚测定的内源性杂质,提高选择性.     

高效液相色谱-荧光法测定人血浆中非索非那定

目的:建立测定人血浆中非索非那定的高效液相色谱-荧光法。方法:血浆样品经液-液萃取后,以乙腈-水-磷酸-三乙胺(32∶68∶0.8∶0.1)为流动相,流速1.2 mL·min~(-1),采用 Diamonsil C_(18)柱(4.6 mm×150 mm,5μm)分离,以荧光检测器进行检测,激发波长为230 nm,发射波长为290 nm。结果:标准曲线线性范围为0.01～1.0μg·mL~(-1),定量下限为0.01μg·mL~(-1),日内和日间精密度(RSD)均小于9.6%,准确度(RE)在±2.8%以内。结论:该法操作简便、快速、灵敏,适用于非索非那定的临床药动学研究。     

固相萃取高效液相色谱内标法测定冬虫夏草中甘露醇的含量

目的:研究用固相萃取预分离,高效液相色谱内标法测定冬虫夏草中甘露醇的含量。方法:冬虫夏草样品中的甘露醇用水超声振荡浸取,浸取液用 Waters Sep-Pak-C_(18)固相萃取小柱预分离,以海藻糖为内标物,Waters Sugar-Pak-1(6.5 mm×300 mm)钙型阳离子交换柱为固定相,0.05 g·L~(-1)EDTA 钙钠水溶液为流动相,流速0.5 mL·min~(-1),示差折光仪为检测器测定冬虫夏草样品中的甘露醇含量。结果:最低检测限为2.0μg·mL~(-1);回收率在97%～102%之间,RSD 在0.86%～1.2%之间。结论:方法简便、灵敏、准确、适用于冬虫夏草样品的甘露醇测定。     

高效液相色谱-荧光检测法在小儿尿蝶呤分析中的新应用

目的:建立高效液相色谱-荧光法测定小儿尿液中新蝶呤(N)和生物蝶呤(B)含量,利用多波长发射光光谱比对及3D数据拟合,准确判断色谱图上各色谱峰的性质和归属。方法:选用 COSMOSIL 5C_(18)-AR-Ⅱ色谱柱(4.6mm×250mm,5μm),甲醇-水(8:92)为流动相,用荧光检测器检测。结果:新蝶呤在0.0625～16μg·mL~(-1)范围内、生物蝶呤在0.0675～17.28μg·mL~(-1)范围内,峰面积与浓度具有良好的线性关系。新蝶呤和生物蝶呤的最低检测限分别为0.0625μg·mL~(-1)和0.0675μg·mL~(-1)。两者测定的日间、日内精密度符合测定要求,日内测定 RSD<1%,日间测定 RSD<5%。回收率均在93%～102%之间。样品处理后在室温或4℃条件下稳定性较好。新蝶呤和生物蝶呤的最大发射光波长均在450nm,通过发射光光谱比对、3D 数据拟合可以准确判断生物蝶呤和新蝶呤峰。结论:本方法灵敏、准确、快速,多波长发射光谱比对可以有效判断色谱峰的归属和纯度,为临床诊断提供可靠的试验数据。     

RP-HPLC法测定人体血浆中芬布芬的浓度

目的:建立测定人血浆中芬布芬浓度的高效液相色谱法。方法:采用 Diamonsil(钻石)ODS C_(18)色谱柱(250 mm×4.6mm,5μm),以甲醇-0.02 mol·L~(-1)磷酸二氢钾-乙腈(75:20:5)(用磷酸调节 pH 3.0)为流动相,流速为1.0 mL·min~(-1),检测波长285 nm。结果:芬布芬的线性范围为0.16～16.0μg·mL~(-1),定量限为0.16μg·mL~(-1),检测限为0.06μg·mL~(-1),平均回收率为(101.2±3.5)%,RSD 为1.8%,日内及日间精密度分别为1.7%和2.7%。结论:该方法简便、快速、准确,适用于临床上芬布芬的体内研究。     

高效液相色谱法测定屈螺酮炔雌醇片的有关物质

目的建立屈螺酮炔雌醇片有关物质的检查方法。方法采用高效液相色谱法,Prontosil ACEeps色谱柱和Chromolith RP 18色谱柱串联,流动相A为乙腈-甲醇-水(203∶150∶550),流动相B为乙腈-甲醇-水(595∶150∶50),流速0.35 mL·min~(-1),进行梯度洗脱。紫外检测器和荧光检测器串联使用,(1)紫外检测波长222 nm(0~60 min);(2)荧光检测在0~37 min和42~60 min,激发波长215 nm,发射波长315 nm;在37~42 min,激发波长215 nm,发射波长344 nm;进样量为20μL。结果屈螺酮、炔雌醇与各杂质能有效分离,进样量在线性范围与峰面积线性关系良好(r为0.9998~0.9999),屈螺酮和炔雌醇的检测限分别为0.137μg·mL~(-1)和0.035μg·mL~(-1)(紫外检测),Δ9,11-炔雌醇和炔雌醇的检测限分别为0.0034μg·mL~(-1)和0.0042μg·mL~(-1)(荧光检测)。结论建立的方法准确,灵敏度高,可用于检查屈螺酮炔雌醇片的有关物质。     

反相高效液相色谱法测定人血浆中马来酸氟伏沙明浓度

目的:建立高效液相色谱法测定人血浆中马来酸氟伏沙明浓度。方法:以安定为内标,待测血浆样品经乙醚萃取后,用高效液相色谱法紫外进行检测。色谱柱为 Discovery~(?)RP Amide C_(16)(5μm,150 mm×4.6 mm),柱温35 ℃;流动相为乙腈-0.05 mol·L~(-1)醋酸铵(40:60),流速1.2 mL·min~(-1);检测波长250 nm。结果:血浆中马来酸氟伏沙明浓度线性范围为1.56～99.6 ng·mL~(-1),最低定量浓度1.56 ng·mL~(-1);回收率为95.7%～106.1%。结论:本法分离效果良好,灵敏度高,回收率高,能满足于人体血药浓度测定及药代动力学研究。     

反相高效液相色谱法测定大鼠血浆及子宫组织中达那唑的药物浓度

目的:建立反相高效液相色谱法测定大鼠血浆及子宫组织样品中达那唑药物浓度。方法:生物样本经沉淀蛋白后,上清液直接进样。色谱条件:甲醇-水(78:22)为流动相,用 Agilent Zorbax Eclipse XDB-C_(18)(250 mm×4.6 mm,5 μm)色谱柱进行分离,284 nm 检测,流速1.0 mL·min~(-1)。结果:血浆中达那唑的线性范围为31.25～2500 ng·mL~(-1),定量限为31.25 ng·mL~(-1);子宫组织中达那唑的线性范围为62.5～2500 ng·g~(-1) ,定量限为62.5 ng·g~(-1);方法回收率均人于90%。结论:本方法简单、准确,专属性强,灵敏度高,可用于临床药代动力学研究。     

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.     

Pharmacological treatment of COVID-19: an opinion paper

The precocity and efficacy of the vaccines developed so far against COVID-19 has been the most significant and saving advance against the pandemic. The development of vaccines has not prevented, during the whole period of the pandemic, the constant search for therapeutic medicines, both among existing drugs with different indications and in the development of new drugs. The Scientific Committee of the COVID-19 of the Illustrious College of Physicians of Madrid wanted to offer an early, simplified and critical approach to these new drugs, to new developments in immunotherapy and to what has been learned from the immune response modulators already known and which have proven effective against the virus, in order to help understand the current situation.     

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.