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。     

硫酯缩合反应中的消旋问题研究

用反相高效液相色谱检测了Ｂｏｃ－Ａｌａ－Ａｌａ－ＳＣＨ２ＣＨ２ＣＯ－ＧｌｙＯＢｚｌ与ＧｌｙＯＢｚｌ在银离子作用下缩合反应的消旋问题，发现四氢呋喃是较好的溶剂，当硫酯：Ｎ－羟基丁二酰亚胺：Ｎ－甲基吗啡啉摩尔比为１：１５：１２时，反应２ｈ转化率达１００％，消旋度低于２％。     

1─（2─吡啶偶氮）─2─萘酚─6─磺酸衍生化反相高效液相色谱法测定铁、钴、镍

本文研究了水溶性试剂１－（２－吡啶偶氮）－２－萘酚－６－磺酸（ＰＡＮ－Ｓ）与铁（Ⅱ）、钴（Ⅱ）和镍（Ⅱ）之螯合物的衍生和液相色谱分离条件。在Ｎｏｖａ－ＰａｋＴＭＣ１８柱上，用含１０ｍｍｏｌ／Ｌ的ｐＨ５．０的醋酸－醋酸钠缓冲溶液的甲醇－水溶液（５０：５０，Ｖ／Ｖ）作流动相，溴化四丁基铵（ＴＢＡ·Ｂｒ）作离子对试剂，流动相流速为１．０ｍＬ／ｍｉｎ，在５５０ｎｍ波长处进行光度检测。在Ⅱｍｉｎ内用高效液相色谱分离测定了Ｆｅ（Ⅱ）、Ｃｏ（Ⅱ）和Ｎｉ（Ⅱ）与ＰＡＮ—Ｓ的螯合物，提出了离子对反相高效液相色谱快速分离测定痕量铁、钴、镍的新方法。信噪比（ＳＮＲ）为２时，检测下限分别为０．０４３、０．００７和０．０１２ｍｇ／Ｌ。     

氯化血红素模拟过氧化物酶高效液相色谱柱后衍生法测定过氧化氢和水溶性有机过氧化物

研究了氯化血红素（Ｈｅｍｉｎ）模拟辣根过氧化物酶（ＨＲＰ），对羟基苯乙酸（ＰＨＰＡＡ）作 底物，高效液相色谱（ＨＰＬＣ）柱后衍生荧光法测定Ｈ２Ｏ２和水溶性有机过氧化物的方法。采用 Ｈｅｍｉｎ作催化剂柱后衍生反应的最佳ｐＨ值约为１１，与荧光检测ｐＨ值一致，使得以往ＨＰＬＣ 柱后衍生方法所需的高压泵从３台减少到２台，而一些不能作为ＨＲＰ底物的羟烷基过氧化 物在ｐＨ值≥１０的溶液中迅速水解为Ｈ２Ｏ２从而得到测定。优化了测定Ｈ２Ｏ２和甲基过氧化 氢（ＣＨ３ ＯＯＨ， ＭＨＰ）的条件。最佳条件下 Ｈｅｍｉｎ方法测定 Ｈ２Ｏ２的检测限为 ９．０ × １０－９ ｍｏｌ／Ｌ， 测定 ＭＨＰ的检测限为 ２．０ × １０－７ ｍｏｌ／Ｌ。     

固定化谷氨酸脱羧酶柱式反应器测定L—谷氨酸

用羧甲基化的Ｎ,Ｎ’－甲叉双丙烯酰胺交联的烯丙基葡萄糖（简称ＣＭ－ＣＡＤＢ）凝胶树脂为新型载体,研究了谷氨酸脱羧酶（ＧＤＣ）在ＣＭ－ＣＡＤＢ上的固定化与环境的依赖头等确定了酶固定化最佳条件,研究了固定化ＧＤＣ的性状与底物浓度,ｐＨ温度的依存性,动态响应特性,热稳定性的寿命,求算了米氏常数。将固定化ＧＤＣ酶柱与进样系统具ＣＯ２气敏电极的离子活度分析器－计算机数据采集系统匹配,构成酶反应谷氨酸检测装置     

高效液相色谱检测反－2－苯基－邻－硝基肉桂酸

初探了反－２－苯基－邻－硝基肉桂酸（ＰＮＣＡ）的高效液相色谱分离，优选出了最佳分离条件。所建立的方法简单、快速，适用于工业生产中的控制及分析。     

高效液相色谱检测反—2—苯基—邻—硝基肉桂酸

初探了反－２－苯基－－肖基肉桂酸（ＰＮＣＡ）的高效液相色谱分离。优选出了最佳分离条件。所建立的方法简单、快速，适用于工业生产中的控制及分析。     

四种神经肽的反相高效液相色谱法分离

以反相高效液相色谱法（ＲＰ－ＨＰＬＣ）分离甲硫脑啡肽（ＭＥＫ）、亮脑啡肽（Ｌｅｕ－ＥＮＫ）、强啡肽Ａ１－１７（ＤｙｎＡ１－１７）、胆囊收缩素（ＣＣＫ－８ｓ）。色谱柱为ＺＯＲＢＡＸＣ１８（２．１ｍｍＩＤ×１５０ｍｍ）；流动相：乙腈／水／三氟乙酸（ＴＦＡ），梯度洗脱，紫外检测器检测：ＵＶ２１５ｎｍ。上述条件下，四种神经肽在微克水平得到较好分离。     

反相液相色谱法测定微量铬(Ⅵ)苯酚、4-硝基酚和十二烷基苯磺酸钠

１引言铬（Ⅵ）、苯酚和４－硝基酚是我国环境优先监测的工业污染物，其测定方法一直是环境分析中研究的重要课题。十二烷基苯磺酸钠（ＳＤＢＳ）是我国使用量最大的洗涤剂，它对环境造成的污染不容忽视。这４种物质有许多检测方法，但大多是用不同方法分别测定。本文建立了以甲醇、乙醚和ＨＡｃ－ＮａＡｃ缓冲溶液为流动相，反相高效液相色谱法分离和测定铬（Ⅵ）、苯酚、４－硝基酚和ＳＤＢＳ４种污染物的分析方法，并应用于环境水样中。２实验部分２．１仪器与试剂ＳＳＩ２２２Ｄ型高效液相色谱仪，ＵＶ－５００型紫外－可见检测器，Ｃｈ…     

氯化血红素模拟过氧化物酶高效液相色谱柱后衍生法测定过氧化氢和 …

研究了氯化血红素（Ｈｅｍｉｎ）模拟辣根过氧化物酶（ＨＲＰ），对羟基苯乙酸（ＰＨＰＡＡ）作底物，高效液相色谱（ＨＰＬＣ）柱后衍生物荧光法测定Ｈ２Ｏ２和水溶性有机过氧化物的方法，采用Ｈｅｎｍｉｎ作催化剂柱后衍生反应的最佳ｐＨ值约为１１，与荧光检测ｐＨ值一致，使得以往ＨＰＬＣ柱后衍生方法所需要的高压泵认３台减少到２台，而一些不能作为ＨＲＰ底物的羟烷基过氧化物在ｐＨ值≥１０的溶液中迅速水解为Ｈ２Ｏ２从而得     

微柱高效液相色谱法测定大鼠脑微透析液中 的乙酰胆碱和胆碱

用所建立的微柱高效液相色谱分离、柱后固定化酶反应器酶解、电化学检测器检测的方法, 对用不同浓度异氟醚麻醉前后的大鼠脑微透析液中的乙酰胆碱和胆碱的浓度进行了测定。在 乙酰胆碱和胆碱的浓度为10～ 2 000 nmol/L 时,其浓度分别与各自相应峰高的线性 关系良好,两者的检测限(以信噪比为3计)均可达5 nmol/L 。微透析液中乙酰胆碱和胆碱的含量在大鼠清醒时最高,随着异氟醚吸入浓度的增加,乙酰胆碱和胆碱含量明显降低。在大鼠海马和脑皮层中乙酰胆碱浓度的降低与大鼠吸入异氟醚的浓度相关( P ＜     

微柱液相色谱-双电极电化学法测定脑微透析液中单胺类递质的含量

用微柱液相色谱－双工作电极电化学检测法,分析了麻醉大白鼠纹状体微透析样品中的单胺类递质及其代谢产物的含量。无论在上游的氧化电位或下游的还原电位进行检测,至少在２～２００ｐｇ范围内,浓度与响应的线性关系良好,检测极限可达１ｐｇ以下。对于检测的优点和需要注意的问题作了扼要的讨论。     

高效液相色谱法测定大鼠神经组织中的硝酸盐和亚硝酸盐

 建立了大鼠神经组织中亚硝酸盐和硝酸盐的高效液相色谱测定方法,以用于研究一氧化氮在糖尿病慢性神经病变中的作用。在1 μmol/L～25 μmol/L的浓度范围内,NO2-和NO3-的峰面积与浓度的线性相关系数>0.991;最低检测浓度分别为0.2 μmol/L和0.5 μmol/L;日内、日间相对标准偏差<14%。对各实验组大鼠的初步测定结果表明,糖尿病组及糖尿病胰岛素(IGF)治疗组的NO2-和NO3-水平均低于对照组。 关键词：高效液相色谱法;一氧化氮;硝酸盐;亚硝酸盐;糖尿病神经病变     

In vivo microdialysis and reverse phase ion pair liquid chromatography/tandem mass spectrometry for the determination and identification of acetylcholine and related compounds in rat brain

A method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed for the determination of basal acetylcholine (ACh) in microdialysate from the striatum of freely moving rats. A microdialysis probe was surgically implanted into the striatum of the rats and Ringer's solution was used as the perfusion medium at a flow rate of 2 microL per minute. The samples were then analyzed off-line by LC/MS/MS experiments. The separation of ACh and choline (Ch) was carried out using reverse phase ion pair liquid chromatography with heptafluorobutyric acid as a volatile ion pairing reagent. Analytes were detected by electrospray ionization tandem mass spectrometry in the positive ion mode. The detection limit for ACh was 1.4 fmol on column, which is at least three times lower than previously reported. Three quaternary ammonium compounds in the rat brain microdialysate were also identified by tandem mass spectrometry experiments in which the unknown mass spectra were compared with standard reference compounds. These compounds were identified as carnitine, acetylcarnitine and (3-carboxypropyl)trimethylammonium. This is the first known report of the compound (3-carboxypropyl)trimethylammonium being found in rat brain.     

Determination of choline and acetylcholine levels in rat brain regions by liquid chromatography with electrochemical detection

Regional choline (Ch) and acetylcholine (ACh) in rat brain were clearly determined by high-performance liquid chromatography with electrochemical detection. The method is based on that of Potter et al.: the hydrogen peroxide that is enzymatically produced from both compounds is measured and a successful improvement of the method, particularly for purification, is described. Recoveries were 96.1 +/- 1.4% for Ch and 95.6 +/- 2.2% for ACh and amounts as low as 10 pmol could be determined. Prior to measuring the compounds, a newly developed magnetic field microwave instrument (10 kW) was utilized for the rapid inactivation of brain enzymes. The levels of Ch and ACh in brain regions were compared with those reported elsewhere.     

Simultaneous determination of choline and acetylcholine based on a trienzyme chemiluminometric biosensor in a single line flow injection system.

A detector for the simultaneous determination of choline (Ch) and acetylcholine (ACh) based on a sensitive trienzyme chemiluminometric biosensor in a single line flow injection (FI) system is described. Immobilized choline oxidase (ChOx), immobilized peroxidase (POx), immobilized acetylcholinesterase, and coimmobilized ChOx/POx were packed, in turn, in a transparent ETFE tube (1 mm i.d., 75 cm) and the tube was placed in front of a photomultipier tube as a flow cell. Two-peak response was obtained by one injection of the sample solution. The first and second peaks were dependent on the concentrations of Ch and ACh, respectively. The influence of some experimental parameters such as flow rate, amounts of immobilized enzymes on the behavior of the sensor was studied in order to optimize the sensitivity, sample throughput and resolution. Calibration curves were linear at 1 - 1000 nM for Ch and 3 - 3000 nM for ACh. The sample throughput was 25/h without carryover. The FI system was applied to the simultaneous determination of Ch and ACh in rabbit brain tissue homogenates.     

Determination of thiols and disulfides in normal rat tissues and hamster pancreas treated with N-nitrosobis(2-oxopropyl)amine using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate

Biological thiols and disulfides in rat and hamster tissues were simultaneously determined by HPLC-fluorescence detection using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) and ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). The coefficients of variation (CV) of the method for reduced glutathione (GSH) and oxidized glutathione (GSSG) in liver and for cysteine (CySH) and cystine (CySSCy) in kidney were less than 3.1%. In 11 tissues of Wistar rats (liver, spleen, heart, lung, stomach, bladder, ovary, uterus, adrenal, kidney and pancreas), only CySH, CySSCy, GSH and/or GSSG were detected. Other thiols and disulfides were at extremely low levels in all samples. Both concentrations of CySH and CySSCy in the livers of old rats (111 weeks old, F344) were significantly higher than those of young rats (8 weeks old) (CySH, 0.246 +/- 0.099 vs 0.130 +/- 0.020 mumol/g; CySSCy, 0.051 +/- 0.027 vs 0.013 +/- 0.002 mumol/g). Administration of N-nitrosobis(2-oxopropyl)amine (BOP), a selective carcinogen of hamster pancreatic cancer, to Syrian golden hamsters (38 weeks old) resulted in the increase in the pancreas of GSH to a level 19 times as high and of GSSG to a level 14 times as high as those in untreated hamsters (GSH, 1.173 +/- 0.272 vs 0.062 +/- 0.017 mumol/g; GSSG, 0.155 +/- 0.063 vs 0.011 +/- 0.001 mumol/g).     

Microbiosensor for acetylcholine and choline based on electropolymerization/sol–gel derived composite membrane

Amperometric enzyme biosensors for the determination of acetylcholine (ACh) and choline (Ch) have been described. For the fabrication of the biosensors, N-acetylaniline (nAN) was first electropolymerized on a Pt electrode surface to be served as a permselective layer to reject interferences. Bovine serum albumin (BSA) and choline oxidase (CHOD) were co-immobilized in a zinc oxide (ZnO) sol–gel membrane on the above modified Pt electrode for a Ch sensor, or CHOD, acetylcholinesterase (AChE) and BSA immobilized together for an ACh/Ch sensor. The poly (N-acetylaniline) (pnAN) film was the first time used for an ACh/Ch sensor and found to have excellent anti-interference ability, and the BSA in the sol–gel can improve the stability and activity of the enzymes. Amperometric detection of ACh and Ch were realized at an applied potential of +0.6 V versus SCE. The resulting sensors were characterized by fast response, expanded linear range and low interference from endogenous electroactive species. Temperature and pH dependence and stability of the sensor were investigated. The optimal ACh/Ch sensor gave a linear response range of 1.0 × 10⁻⁶ to 1.5 × 10⁻³ M to ACh with a detection limit (S/N = 3) of 6.0 × 10⁻⁷ M and a linear response range up to 1.6 × 10⁻³ M to Ch with a detection limit of 5.0 × 10⁻⁷ M. The biosensor demonstrated a 95% response within less than 10 s.     

The Role of Acetylcholine in the Cognitive Function of Frontal Neurons in Monkeys

To study the role of acetylcholine(ACh) in the cognitive function of frontal lobes, ACh and atropine were applied iontophoretically to the task-related neurons recorded from an area around the superior ramus of arcuate sulcus in the frontal cortex of 4 rhesus monkeys during the performance of two tasks. The spontaneous discharges and taskrelated responses of most of the neurons which increased their discharge rate in the delay and differentiation periods of the discrimination response go/no-go task and in the cue and delay periods of the delayed discrimination go/no-go task, were further increased during the application of ACh and suppressed during atropine. These results suggest that ACh may be involved in the excitatory process of the activity of the neurons in the above-mentioned area of frontal cortex to play an important role in the cognitive functions of attention, discrimination and short-term memory.     

High-performance liquid chromatographic separation of malondialdehyde-thiobarbituric acid adduct in biological materials (plasma and human cells) using a commercially available reagent.

The assay of malondialdehyde (MDA) is widely used in clinical chemistry laboratories to investigate lipid peroxidation in oxidative pathologies. In the present work, the thiobarbituric acid (TBA) reaction was carried out on plasma, human erythrocytes and fibroblasts. The reagents used were those of the fluorimetry MDA kit manufactured by Sobioda. We have defined the application of this kit to high-performance liquid chromatography. This adaptation satisfied the criteria of good analytical practice. The detection limit was 2.5 pmol per injection. The retention time of the MDA-TBA2 peak (4.96 +/- 0.07 min) led to excellent resolution of the complex. The within-assay (6-12%) and between-assay (11-12%) precisions were satisfactory. The analytical recovery of MDA after spiking samples of human plasma with tetraethoxypropane standards varied from 70 to 100%. The mean lipoperoxide concentration determined in 32 healthy adults (20-40 years) was 1.04 +/- 0.23 mumol l-1 in plasma. Applied to the erythrocytes of fifteen laboratory workers, the method furnished physiological values of 0.59 +/- 0.21 mumol l-1. Concentrations were significantly higher in chronic renal dialysis patients (4.15 +/- 2.35 mumol l-1. The MDA content of fibroblasts cultured in standard medium was 0.38 +/- 0.04 mumol per g of protein and increased (5.78 +/- 1.38 mumol per g of protein) if the cells were grown in an iron-enriched medium. This accurate high-performance liquid chromatographic method for detection of MDA is the first one which can be applied to plasma, red blood cells and cultured cells. This technique will prevent false positives and should make inter-laboratory comparisons possible.