高效液相色谱-荧光检测流速梯度法测定去卵巢小鼠脑内单胺类神经递质

 建立了同时检测去卵巢(OVX)小鼠脑内的去甲肾上腺素(NE)、多巴胺(DA)、 5-羟色胺(5-HT)3种神经递质的高效液相色谱方法。采用的色谱条件为：C18柱分离,荧光检测器检测,流动相为0.1　mol/L的 KH2PO4缓冲液-甲醇(体积比为9∶1),设置单泵流速梯度洗脱。方法的检测限(S/N=3)均在10 nmol/L以下,绝对回收率均在80％以上,样品日内测定峰面积的相对标准偏差在3.03%以下。该方法具有检测灵敏度高、样品制备简便等优点,对小鼠脑内的3种单胺类递质的检测具有实用价值。     

毛细管电泳法测定单胺氧化酶活性

应用毛细管电泳技术,建立了快速测定单胺氧化酶（MAO）活性的方法。研究对分离缓冲液pH值、浓度、毛细管表面改性剂十四烷基三甲基溴化铵（TTAB）浓度等影响因素进行优化,探讨了方法的可行性,确立了最佳分离条件。以70cm×50μm（i．d．）未涂敷熔融石英毛细管为色谱分离柱,运行电压15kv,运行缓冲液：0．5mmol／LTTAB,磷酸盐缓冲液（50mmol／L,pH 10．5）;紫外检测波长：214nm。MAO催化犬尿胺（Kyn）反应的产物4-羟基喹啉（4-HQ）的浓度和峰面积的线性范围为5～1000μmol／L,相关系数为0．9997,相对标准偏差（RSD）小于5．6％（n=5）,检出限为2μmol／L（S／N=3）。实验证明,此方法可以用于生物样品中MAO催化活性的检测。     

高效液相色谱法测定鼠脑谷氨酸脱羧酶活性

应用高效液相色谱-电化学检测技术建立了鼠脑谷氨酸脱羧酶(GAD)活性的测定方法.方法快速,灵敏.总分析时间为9min.γ-氨基丁酸的最低柱上检测限为50fmol.天内及天间相对标准差分别小于4.3%和9.8%.测得CAD最大催化速率为0.723nmol/min/mg蛋白.讨论了最佳衍生化pH及色谱条件.     

急性酒精中毒的鼠脑内乙醛及其单胺神经递质缩合物的测定

应用高效液相色谱法测定了急性酒精中毒后新生鼠脑中乙醛的含量,以及乙醛分别与多巴胺、5-羟色胺的缩合产物1-甲基-6,7-二羟基-1,2,3,4-四氢异喹啉(Salsolinol,Sal)和6-羟基-1-甲基-1,2,3,4-四氢-β-咔啉(6-OH-MTHβC)的含量。采用2,4-二硝基苯肼(DNPH)的乙腈溶液在60℃水浴中衍生15min提取脑匀浆中的乙醛,离心后进行色谱分离,检测波长为360nm,本方法简单准确,灵敏度高。应用本方法测定鼠脑组织中的乙醛含量时发现,急性酒精中毒后新生鼠脑内乙醛的含量在24h内显著高于盐水对照组(P<0.05)。同时,采用HSF5柱分离和库仑阵列电化学检测的方法对Sal和6-OH-MTHβC进行分析表明,乙醛浓度升高后,急性酒精中毒鼠脑内Sal和6-OH-MTHβC的含量显著升高(P<0.05)。乙醛的非正常代谢及内源性神经毒素的生成可能在酒精的神经毒性机理中发挥了重要作用。     

小鼠肝组织中单胺氧化酶碱解产物(次黄嘌呤、腺嘌呤)的高效液相色谱分析

用HPLC法检测小鼠肝组织中单胺氧化酶碱解产物(次黄瞟呤、腺嘌呤)方法。该方法快速简单,提取样品所用缓冲溶液KOH-KHPO_4(pH=7.2)为最佳。碱解量KOH 2ml,温度36℃,时闻24h,所用色谱条件为: HPLC柱C_(18) 4.6mm×25cm 5μm,流动相为)甲醇:水10:90(V/V,检测波长249nm,灵敏度0.05AuFs,该方法只需12min将完成一次色谱分析。     

微渗析活体取样-高效液相色谱电化学检测法测定鼠脑中的单胺类神经递质

采用微渗析活体取样技术和高效液相色谱电化学检测法,测定了鼠脑纹状体中的3种单胺类神经递质多巴胺(DA)、3,4二羟基苯乙酸(DOPAC)和5羟吲哚乙酸(5HIAA)。在3.0×10-8～1.0×10-5mol/L浓度范围内,DA、DOPAC和5HIAA的浓度分别与氧化峰的峰电流呈良好的线性关系。通过在灌流液中加入1.0×10-5mol/L的NO释放剂硝普钠(SNP),研究了NO对DA释放的影响,结果表明:受NO刺激后纹状体中DA的量为基础水平的150%。     

高效液相色谱-脉冲安培检测法测定脑微透析溶液中单胺类神经递质

详细对比了脉冲安培与恒电位安培检测单胺类神经递质的响应值,结果表明冲安培检测上述物质的响应值是恒电位安培检测的1.2倍。建立了以3.4 -二羟基苄胺（DHBA）为内标的定量方法。在微透析溶液中的微量神经递质的回收率在74％－101％之间。     

高效液相色谱法测定小鼠脑组织单胺递质及其相关化合物

采用苯基柱等度洗脱分离,自然荧光检测法,直接测定小鼠脑组织匀浆中单胺递质及其相关化合物。优化出了最佳色谱条件,流动相为２０ｍｍｏｌ／Ｌ柠檬酸三钠（ｐＨ４．５０）－甲醇（９５：５,Ｖ／Ｖ）;柱温为３５℃。该方法能同时检测８种化合物。最低检测限为１．０－２．５μｇ／Ｌ,回收率均在９３５以上;线性范围为１．０μｇ／Ｌ－１．０ｍｇ／Ｌ。     

猪肝中单胺氧化酶B的分离纯化

依据单胺氧化酶B(monoamine oxidase B, MAOB)的疏水特性,建立了一种从猪肝中分离纯化MAOB的新方法。用含有1% Triton X-100的膜蛋白裂解液制备粗酶,以饱和度为20%～50%的硫酸铵反抽提进行粗提,再利用自制的配基密度为75.7 μmol/mL的苯基疏水色谱及Sepharose Q High Performance离子交换色谱进一步分离纯化,得到纯化倍数为18.2、酶比活为135 U/mg的MAOB。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析显示为相对分子质量约60 000的单一蛋白质带。采用高效液相色谱-电喷雾串联质谱对该酶进行鉴定,证实为MAOB。本研究所用分离纯化方法可以有效纯化MAOB, 为MAOB的深入研究提供技术支撑。     

柴胡皂苷A对抑郁模型大鼠脑中单胺类神经递质及其代谢产物含量的影响

在酸性条件下, 利用高效液相色谱法研究了柴胡皂苷A对抑郁模型大鼠脑中单胺类神经递质及其代谢物含量的影响. 实验结果表明, 柴胡皂苷A可使抑郁型大鼠脑中的高香草酸、去甲肾上腺素、多巴胺及5-羟色胺的含量升高, 这一结果有利于对柴胡皂苷治疗抑郁症的物质基础及相关机理进行更深入了解.     

Dual Electrochemical Detection of Biogenic Amine Metabolites in Micro High-Performance Liquid Chromatography

Abstract

The dual electrochemical detectors for ordinary and micro high-performance liquid chromatography were briefly reviewed.

The electrochemical behaviors of biogenic amine metabolites were studied by cyclic semi-differential and semi-integral voltammetry with a glassy carbon working electrode. It was found that the electrochemical reactions of many biogenic amine metabolites are quasi-reversible. The dual electrochemical detector based on thin-layer electrolytic cell with two working electrodes (anode and cathode) in series configuration was tested for selective detection of biogenic amine metabolites on their electrochemical quasi-reversibility. The detector was successfully utilized for the simultaneous determination of 3, 4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindole-3-acetic acid in human urine directly injected by micro high-performance liquid chromatography.     

Retention-Mobile Phase Relationships for Methylxanthines and Biogenic Amine Metabolites in Adsorption Thin-Layer Chromatographic Systems

JPC – Journal of Planar Chromatography – Modern TLC - Relationships between RF values and mobile-phase composition have been determined for six methylxanthines and acidic and neutral...     

Effect of Mobile-Phase pH on the Chromatographic Behavior of Biogenic Amine Neurotransmitters and their Metabolites

JPC – Journal of Planar Chromatography – Modern TLC - The dependence on mobile-phase pH of the retention behavior of biogenic amine neurotransmitters and their metabolites in...     

Determination of Total Monoamines in Rat Brain via Nanotubes Based Human Monoamine Oxidase B Biosensor

A specific and sensitive electrochemical biosensor with human monoamine oxidase B (hMAO B) as biological receptor and a MnO₂ modified nanomaterial based transducer system has been developed and optimised. Best results for the biosensor were achieved when using enzyme immobilisation with a dialysis membrane (regenerated cellulose, molecular weight cut‐off 14000) and a 20 % (m/m) MnO₂ modified multi‐walled carbon nanotubes (MWCNTs, ratio of fluid to solid compounds of 1 : 0.7 (m/m)) paste electrode smoothed with a glassy carbon paste (GCP, ratio 1 : 3.6 (m/m)) containing the same mediator concentration. The biosensor was operated in a flow injection analysis (FIA) system with Sørensen phosphate buffer (33 mM, pH 7.5) and amperometric detection at a fixed potential of +400 mV vs. Ag/AgCl. The developed sensor underwent validation using phenylethylamine (PEA) as standard substrate showing linearity between 5.0 and 400 µM PEA and limits of detection and quantification of 1.5 and 5.0 µM PEA, respectively. The sensor was successfully tested for the determination of total monoamines in rat brain calculated as PEA equivalents showing a result of 1.2 µg/g brain tissue (n=3, relative standard deviation 4.4 %).     

A New Technique for Simultaneous Assay of Biogenic Amines and Their Metabolites in Unpurified Mouse Brain

Abstract

We developed a chromatographic system which separates cate-cholamines and indolamines as well as a large number of their metabolites (acids, aminoacids and glycols). Waters Radial Pak columns compressed radially to avoid formation of cavities in the packing, were used. These columns must be washed with methanol in order to obtain reproducible results when an ion-pairing mobile phase is employed. The conditioning of bhe columns was not affected by washing with triethylamine or variations in radial pressure. Good resolution of 13 samples was obtained in 28 minutes with a mobile phase composed of 0.1 M KH²PO⁴, 0.1 mM EDTA and 5 mM heptane sulfonic acid in water and Me OH (85:15), pH = 3.6. Products were identified with the use of a Metrohm 64l electrochemical detector.

The technique was used for rapid, simultaneous determination of NA, DA, DOPAC, HVA, 5HT and 5HIAA in deproteinated mouse brain samples.     

HPLC Determination of Monoamines in Rat Brain after Enzymatic Treatment with Ascorbate Oxidase and Sulfatase

Abstract

The simultaneous analysis of norepinephrine, dopamine and serotonin along with their respective metabolites, MHPG, DOPAC and 5HIAA is accomplished in rat brain by using a recently developed HPLC technique. Sulfated MHPG is decongegated with sulfatase for detection purposes and endogenous ascorbate is diminished with ascorbate oxidase to reduce the front.     

Determination of Semicarbazide-Sensitive Amine Oxidase Activity in Blood Plasma by a Light Scattering Technique

A novel method for the determination of semicarbazide-sensitive amine oxidase (SSAO) activity in blood plasma has been developed. The method was based on the change of light scattering (LS) intensity resulting from the derivative product of the interaction of 2,4-dinitrophenylhydrazine (DNPH) with benzaldehyde produced by catalyzing of SSAO to benzylamine. In Britton-Robinson (B-R) buffer solution, the intensity of system's LS at 514.6 nm was significantly enhanced and was directly proportional to the concentration of benzaldehyde. In this method, SSAO enzyme activity is defined as the concentration of benzaldehyde (nmol) formed per mL plasma per hour. The range of determination of SSAO enzyme activity was 6.40 × 10^?3 ?0.340 nmol mL^?1 h^?1 with a detection limit of 1.92 × 10^?3 nmol mL^?1 h^?1. The relative standard deviation was 2.8–4.1% and the average recovery was 67.9% (n = 6).     

Synthesis and Monoamine Oxidase Inhibitory Activity of 3-Substituted 5H-Indeno[1,2-c]pyridazines

The one-pot synthesis of nine 5H-indeno[1,2-c]pyridazines is described. These compounds are shown to be potent, reversible inhibitors of monoamine oxidase B (MAO-B) with little or no effect on monoamine oxidase A (MAO-A). Qualitative structure-activity relations indicate that the MAO-B inhibitory activity is strongly influenced by electronic and bulk properties of substitutents.