高效液相色谱-柱后固定化酶反应器-电化学检测法分析大鼠脑微透析液中的乙酰胆碱和胆碱

建立了用高效液相色谱分离－柱后固定化酶反应器酶解－电化学检测器检测酶解最终产物Ｈ２Ｏ２的方法，分析了麻醉和自由活动大鼠脑微透析液中乙酰胆碱（ＡＣｈ）和胆碱（Ｃｈ）的含量。至少在０．２～１００μｍｏｌ／Ｌ范围内ＡＣｈ和Ｃｈ的浓度与其响应的线性关系良好，它们的检测极限都可达５０ｆｍｏｌ。对高效液相色谱结合固定化酶反应器的分析方法作了简要的讨论。     

高效液相色谱-柱后固定化酶反应器-电化学检测法分析大鼠脑微透析液中的乙酰胆碱和胆碱

 建立了用高效液相色谱分离－柱后固定化酶反应器酶解－电化学检测器检测酶解最终产物Ｈ２Ｏ２的方法，分析了麻醉和自由活动大鼠脑微透析液中乙酰胆碱（ＡＣｈ）和胆碱（Ｃｈ）的含量。至少在０．２～１００μｍｏｌ／Ｌ范围内ＡＣｈ和Ｃｈ的浓度与其响应的线性关系良好，它们的检测极限都可达５０ｆｍｏｌ。对高效液相色谱结合固定化酶反应器的分析方法作了简要的讨论。     

微柱液相色谱和电化学检测法测定鼠脑微透析液中神经传递物质

报道了使用微柱色谱和电化学检测器测定鼠脑微透析液中神经传递物质的方法。儿茶酚胺，５－羟色胺和代谢物的检测限为 ０． １～０． ５ ｐｇ。含有乙酰胆碱酯酶和胆碱氧化酶的柱后反应器使分离后的乙酰胆碱和胆碱转化成过氧化氢．再用铂电极或过氧化物酶修饰过的玻碳电极检测过氧化氢。乙酰胆碱的检测限当使用怕电极时是５０× １０－１５ｍｏｌ，当使用酶修饰电极时是１０×１０－１５ｍｏｌ。     

同位素编码衍生-分散液液微萃取/超高效液相色谱-串联质谱检测大鼠脑微透析液中儿茶酚胺

为实现微量大鼠脑微透析液中3种儿茶酚胺化合物的高灵敏检测,采用d0/d3-10-甲基-吖啶酮-2-磺酰氯(d0/d3-MASC)为同位素编码衍生化试剂,联合使用超声辅助分散液液微萃取技术,建立了超高效液相色谱-串联质谱(UHPLC-MS/MS)快速测定多巴胺(DA)、肾上腺素(EP)和去甲肾上腺素(NE)的分析方法。分别以d0-MASC和d3-MASC衍生微透析液样品和对照品,混合后采用分散液液微萃取技术富集净化,再进行UHPLC-MS/MS检测,以d3-MASC衍生物作为相应d0-MASC衍生物的内标物进行定量。结果表明,在p H 10.8乙腈水溶液中于37℃衍生反应3 min,即可实现待测物的完全分离与检测,3种儿茶酚胺化合物的线性范围为0.02~10.0 nmol/L,相关系数大于0.995,检出限为0.005~0.010 nmol/L,定量下限为0.018~0.040 nmol/L。该方法与已报道方法相比具有显著优势,能够满足大鼠脑微透析液中儿茶酚胺的检测要求。     

稳定同位素编码衍生-分散液液微萃取-超高效液相色谱-三重四极杆质谱检测大鼠脑微透析液中左旋多巴和多巴胺

帕金森病(PD)大鼠脑微透析液中左旋多巴(L-DOPA)和多巴胺(DA)的高灵敏检测技术,是PD相关的临床医学及L-DOPA减毒增效协同药物筛选必不可少的手段。采用d₀/d₃-10-甲基-吖啶酮-2-磺酰氯(d₀/d₃-MASC)作为稳定同位素编码衍生试剂,联合超声波辅助-分散液液微萃取(UA-DLLME)技术,建立并验证了超高效液相色谱-串联质谱(UHPLC-MS/MS)快速检测L-DOPA和DA的分析方法。分别使用d₀-MASC和d₃-MASC衍生微透析液样品和混合对照品,将衍生溶液混合后采用UA-DLLME技术富集净化,继而进行UHPLC-MS/MS检测(多反应监测模式),以d₃-MASC衍生物作为d₀-MASC衍生物的内标物进行定量。实验表明,在乙腈/水(pH 10.8碳酸钠-碳酸氢钠缓冲液)溶液中37 ℃下反应3.0 min后衍生化反应完成,梯度洗脱条件下2.0 min可完成分离检测,线性范围为0.20~1500.0 nmol/L,相关系数大于0.994, L-DOPA和DA的检出限(S/N=3)分别为0.005和0.009 nmol/L。分析方法评价结果良好,与已报道方法相比在灵敏度、分析速度和抗基质干扰等方面具有优势,本方法已成功应用于测定中药方剂首乌方对PD大鼠脑微透析液中L-DOPA和DA浓度波动的影响。     

高效液相色谱法测定大鼠给药盐酸氟西汀后脑渗析液中的5-羟色胺

建立了高效液相色谱柱前衍生化法测定大鼠腹腔注射(i.p.)给药盐酸氟西汀前后脑渗析液中5-羟色胺(5-HT)的浓度变化情况。在大鼠给药盐酸氟西汀前和给药后不同时间点取其脑渗析液,加入衍生化试剂反应后,以乙腈-20 mmol/L醋酸盐缓冲液(pH 5.0)(体积比为45∶55)（含20 mmol/L辛烷磺酸钠）作为流动相,在Hypersil C18色谱柱 (250 mm×2.0 mm,5 μm)上进行分离,荧光检测波长为λex=330 nm,λem=455 nm。5-HT在浓度为0.25～5.0 nmol/L 范围内线性关系良好(r=0.9991);5-HT的最低定量限为0.25 nmol/L。本法准确可靠、简便,适用于生物样品中5-HT的浓度测定。     

液相色谱-串联质谱法测定大鼠脑透析液中CTN986及其脱糖产物的含量

建立了测定CTN986及其脱糖产物芦丁和陆地棉苷在大鼠脑透析液中含量的高效液相色谱-串联质谱(HPLC-MS/MS)法. 采用微透析技术结合HPLC-MS/MS, 以甲醇-异丙醇-水-甲酸(体积比20∶10∶70∶0.1)为流动相, 用Zorbax C8柱分离, 待测物通过电喷雾离子化四极杆串联质谱, 以多反应监测方式(MRM)进行检测. 采用外标法分析给予CTN986后大鼠脑透析液中CTN986及其脱糖产物的浓度, 经过体内回收率校正后, 计算出脑透析液中待测物的浓度. CTN986、芦丁和陆地棉苷的线性范围为2~500 ng/mL, 日内及日间精密度(RSD)均小于15%, 准确度(RE)在-4%~13%之间. 本方法专属性强、灵敏度高, 适用于脑透析液中CTN986的药代动力学分析, 为药代动力学研究提供了新的方法学参考.     

基于计时库仑技术的可再生型三磷酸腺苷适配体电化学传感器的研究

构建了一种可再生型三磷酸腺苷(ATP)适配体计时库仑电化学传感器.将一条短链DNA通过AuS键自组装固定在电极表面, ATP的核酸适配体与该短链DNA杂交而结合在电极表面.带负电的DNA通过静电吸引结合电解液中的六氨合钌(RuHex)阳离子.当传感器和靶分子ATP孵育后,ATP与核酸适配体结合,使适配体链从电极表面解离,电极表面吸附的DNA量减少,结合RuHex的量随之降低.通过计时库仑技术检测RuHex响应信号降低的量 ,可以对ATP进行定量测定.此传感器的电化学响应信号与ATP浓度对数值呈线性关系,线性检测范围为0.001~100 μmol/L,检出限(S/N=3)为0.5 nmol/L.此传感器检测靶分子ATP后,可以通过简单的操作步骤再生,再生5次后的响应信号为初始信号的90%以上.采用此传感器检测大鼠脑透析液中ATP的含量为(19.2±3.7) nmol/L (n=3).     

鼠脑尾核和海马区微透析液中儿茶酚胺类神经递质的高效液相色谱分析

采用反相液相色谱分离-脉冲安培检测和微透析取样技术,测定了取自鼠脑尾核和海马区微透析液中儿茶酚胺类神经递质的含量。对不同神经活动状态下取得微透析液中肾上腺素含量作了比较,将该法用于测定肾上腺素,灵敏度为25μg/L.     

基于聚苯乙烯微球放大的凝血酶化学发光检测新技术

本文以羧基96孔板为分离载体,核酸适配体作为分子特异性识别元件,聚苯乙烯微球作为放大载体,辣根过氧化物酶为标记物,构建了化学发光（CL）高灵敏度凝血酶检测新技术.实验结果表明：该放大技术不但灵敏度高,且抗干扰能力强,其他蛋白质如IgG、IgM、IgA、IgE、IFN均无明显干扰.聚苯乙烯微球放大体系中凝血酶的线性范围为7.8～250pmol/L,最低检测浓度可达3.9pmol/L;而不放大检测技术的线性范围为0.94～30nmol/L,最低检测浓度为0.46nmol/L,放大体系将检测灵敏度提高100多倍.综合而言,基于适配体识别和聚苯乙烯微球放大的凝血酶CL检测新技术具有通量大、简单快速和灵敏度高的特点,有望在凝血酶高通量检测领域获得应用.     

脑部磁共振结合静息态fMRI方法在卒中患者认知障碍中的应用

本研究主要探讨脑部磁共振结合静息态fMRI方法在卒中患者认知障碍中的应用。本研究将符合卒中认知障碍患者25例设为患者组,将25例健康者设为对照组。采用脑部静息态核磁共振对实验者进行扫描,以脑部双侧海马为种子点进行功能连接分析,并通过软件分析两组实验者静息态双侧海马差异。结果发现,与对照组相比,患者组右侧海马与左右侧额中回、左侧额下回等的连接减少;右侧海马与左侧颞上中回、双侧丘脑的连接增加;左侧海马与左侧颞额中回、右侧颞额上回、右侧额下回的连接减少;左侧海马与右侧顶下回连接增加。总之,本研究发现卒中患者海马脑区与颞叶皮层、额叶皮层之间的连接减少,可能是由卒中患者脑损害导致。     

A highly sensitive and stable detection of acetylcholine by HPLC-osmium-horseradish peroxidase redox polymer electrode coated on a gold radial flow ring disk

To detect a low level of acetylcholine (Ach) output in the dialysate of rat frontal cortex, osmium-peroxidase redox polymer modified gold-ring disk electrode (Os-HRP-RDE) was fabricated in high-performance liquid chromatography (HPLC)-electrochemical detection (ECD). In comparison with platinum electrode, the sensitivity of acetylcholine detection by Os-HRP-RDE was stable during a day, the coefficient of variation was 0.95%, and the decrease of the sensitivity for 1 week was 5.4%. The Os-HRP-RDE could detect lower than 3 fmol of acetylcholine. The present technique, in the absence of acetylcholinesterase (AchE) inhibitor, is useful to facilitate physiological investigations of cholinergic neuronal activity in the brain.     

Application of liquid chromatography-tandem mass spectrometry method for the simultaneous quantitative analysis of propentofylline and its chiral metabolite M1 in rats

A sensitive and selective liquid chromatographic–electrospray ionization mass spectrometric method for the simultaneous determination of propentofylline and enantiomers of its active metabolite M1 in rat serum, cortex and hippocampus was developed and validated according to GLP procedures. Sample preparations were carried out by liquid–liquid extraction using diethyl ether after the addition of the internal standard (pentoxifylline). The dried residue was reconstituted in mobile phase and injected onto a Chiralpak AD column (10 µm, 250 × 4.6 mm i.d.). The limit of quantification for propentofylline in serum, cortex and hippocampus was set at 0.25 ng/mL and for enantiomers of its metabolite M1 at 1.25 ng/mL. The established LC/ESI‐MS/MS method has been successfully applied to an initial pharmacokinetic study of propentofylline and also to assessment of distribution of parent drug and enantiomers of its pharmacologically active metabolite M1 to cortex and hippocampus after intravenous administration of propentofylline to rats at a dose of 5 mg/kg. Copyright © 2010 John Wiley & Sons, Ltd.     

Quality control in large sample analysis

To investigate the effects of lanthanum exposure on regional distribution of inorganic elements in rat brain. Wistar rats were exposed to lanthanum chloride through oral administration at 0, 0.1, 2, and 40 mg/kg concentration for 6 months. The elements such as Cl, K, Ca, Fe, Cu, and Zn were identified in the brain slices by synchrotron radiation X-ray fluorescence (SRXRF) analysis. Differences of brain elemental distributions were noticed. Cl, Ca, and Zn were primarily concentrated in hippocampus of the controls. With the increase of the lanthanum dosage, the Ca and Zn levels significantly decreased, while the Cu levels significantly elevated in cortex, hippocampus and thalamus. Our results suggest that subchronic lanthanum exposure in rats appears to change elemental distributions in brain.     

Sustained release of acetylcholine in rat hippocampus using a polyanhydride drug-delivery system

A biodegradable polymer drug-delivery system has been developed for the selective localized application of agents to brain parenchyma. The copolymer of poly[bis(p-carboxyphenoxy)propane] anhydride and sebacic acid (PCPP–SA) was impregnated with [³H]-acetylcholine (ACh) to form 1–5 μm microspheres. Drug-loaded microspheres were implanted into hippocampus bilaterally in 25 rats, and brain sections processed for autoradiography in groups of five animals at 2, 5, 10, 20 and 40 days, respectively. By densitometric analysis, the concentration of radiolabelled ACh in polymer and adjacent hippocampus rapidly decreased between 2 and 5 days, after which a gradual decrease in [³H]-ACh was observed up to 40 days. Between 2 and 40 days the concentration of radiolabelled ACh was reduced by 25.8% in polymer matrix and 40.1% in hippocampus. The spread of [³H]-label into adjacent brain parenchyma showed a similar temporal relationship, with initially wider dispersion at 2 days (44±3 μm), then a linear decrease in dispersion over the remaining period (10±0.9 μxm at 40 days), suggesting bulk flow of the radiolabel into hippocampus. Brain parenchyma showed only a minimal inflammatory reaction to the polyanhydride implants over all time periods. Polyanhydrides can provide localized continuous release of ACh to brain parenchyma, and may potentially be used to deliver various agents to brain in a number of clinical and experimental applications.     

Neuroprotective effects of sonochemical- synthesized SiO2 nanoparticles in vivo models of ischemic/reperfusion injury in stroke

Cerebral ischemic injury is one of the debilitating diseases that showed inflammation plays an essential role in aggravating ischemic damage. After synthesizing silica nanoparticles (SiO₂ NPs) by sonochemical method, serum parameters in the presence of different concentrations of SiO₂ NPs are measured for toxicity assay. Rats were separated randomly into control, ischemia/reperfusion, and ischemia/reperfusion + SiO₂ NPs groups. Transient forebrain ischemia induced with bilateral occlusion of both common carotid arteries followed by 60minuts of reperfusion. SiO₂ NPs were administered (500 mg/kg/day p.o.) 21 days before ischemia/reperfusion time. Animals sacrificed and frontal cortex and hippocampal tissues used to determine malondialdehyde (MDA) level, nitric oxide (NO), glutathione (GSH) levels, an essential antioxidant, superoxide dismutase (SOD), alterations in the level of cytokines, TNFα, IL-1β, MCP-1, and phosphor Ik-кB. We also revealed the involvement of NF-κB downregulation by using western blotting. We reported on a histological investigation. The results showed that SiO₂ NPs with a diameter of around 50 nm in dose of 500 mg/kg didn't change the level of liver enzyme (including ALT, AST and ALP) and hematological parameters. 500 mg/kg SiO₂ NPs showed significant effects on remission of behavioral impairment. Ischemia/reperfusion oxidative injury in the rat hippocampus demonstrated a significant increase in MDA, TNFα, MCP-1, IL-1β, phosphor Ik-кB, NO levels, and a significant decrease in GSH contents and SOD activities in the hippocampal tissue compared to the control group. Pretreatment of ischemic rats with SiO₂ NPs decreased the elevated levels of MDA, TNFα, MCP-1, IL-1β, phosphor Ik-кB, and NO levels. A significant alteration observed in SOD activities and GSH content results between treated and untreated ischemia/reperfusion brains in rats. Decreased protein level of NF-κB also measured in SiO₂ NPs-treated animals. Untreated ischemia/reperfusion brains had significantly decreased in number of cells in CA1 hippocampus, nevertheless SiO₂ NPs increase the normal cell and decrease the neurodegeneration in hippocampus but it was not significant alteration. SiO₂ NPs reduced the damage caused by cerebral ischemia/reperfusion in rats and its molecular mechanism attributed to the downregulation of NF-κB signaling pathway.     

Voltammetric Studies on Chromotrope 2B‐Protein Interaction and Its Analytical Application

In this paper the interaction of chromotrope 2B (Ch2B) with proteins was studied by the electrochemical method. Ch2B is an azo dye and shows irreversible electrochemical responses on the mercury electrode in a pH 3.0 Britton‐Robinson (B‐R) buffer solution. After the addition of human serum albumin (HSA) into the Ch2B solution, an interaction took place, and a supramolecular complex was formed in the mixed solution. The electrochemical parameters of the Ch2B‐HSA interaction system were calculated and compared. The results showed that in the absence and presence of HSA in Ch2B solution, the electrochemical parameters such as the formal potential E⁰, the electrode reaction standard rate constant k_s, etc. showed no significant changes, which indicated that an electro‐inactive supramolecular biocomplex was formed. The free concentration of Ch2B in reaction solution was decreased, and this resulted in the decrease of the peak current. The binding constant and the binding ratio were calculated as 7.85 × 10⁹ and 1:2, respectively, and the interaction mechanism was discussed. Based on the decrease of the peak current, this new electrochemical method was proposed for the determination of HSA in the concentration range of 2.0?25.0 mg/L with the linear regression equation as ΔIp′ (nA) = 50.56C (mg/L) — 6.72 (γ = 0.995). This method was further used to determine other different kinds of proteins, such as bovine serum albumin (BSA), oval albumin, etc‥ The new method was successfully applied to detect the content of albumin in healthy human serum samples with the results in good agreement with the traditional Coomassie Brilliant Blue G‐250 spectrophotometric method.     

Elemental concentration in the cortex and hippocampus of Wistar rats by X-ray total reflection fluorescence with synchrotron radiation

Summary The study of trace element levels is of great importance due to their relevance in agingand several neurodegenerative diseases. This work compares the elemental concentrations in different postnatal ages and between the temporal cortex, entorhinal cortex and hippocampus from Wistar rats, using X-ray total reflection fluorescence with synchrotron radiation. Ten elements were determined in brain samples: Ti, Cr, Mn, Fe, Cu, Zn, (at trace level) and P, S, Cl and K (at major levels). The elements that increased with aging in cortical areas were: S, K, Fe, Cu and Zn. Ca and Zn levels decreased with advancing age in the hippocampus. In addition to this, Ti, Mn and Fe levels were more conspicuous in the entorhinal cortex.     

Decreased l-tryptophan concentration in distinctive brain regions of mice treated repeatedly with phencyclidine

It has been reported that repeated phencyclidine (PCP) treatment induces schizophrenia-like behavior in mice. l-Tryptophan (Trp) concentrations in brain tissues of control (n?=?8) and PCP-treated mice (10 mg/kg/day, s.c., 14 days, n?=?10) were determined using high-performance liquid chromatography (HPLC) with fluorescence detection. The HPLC method involved pre-column fluorescence derivatization with (R)-(?)-4-(N,N-dimethylaminosulfonyl)-7-(3-isothiocyanatopyrrolidin-1-yl)-2,1,3-benzoxadiazole (DBD-PyNCS). Eight different parts of the brain, namely, the frontal cortex, nucleus accumbens, striatum, hippocampus, amygdala, thalamus, hypothalamus, and cerebellum, of both groups were investigated. A significant decrease in the l-Trp concentration in the nucleus accumbens (p?=?0.024) and hippocampus (p?=?0.027) was observed in PCP-treated mice, suggesting that alteration of the l-Trp metabolism might occur in these brain parts.