Name: a convenient method for extraction and analysis with high-pressure liquid chromatography of catecholamine neurotransmitters and their metabolites
Uploaded: 2018-03-01T13:00:00.000+00:00
Duration: 13 min 35 s
Description: Watch this Scientific Journal Video about A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites at JoVE.com

这项研究得到了中国国家科学基金会 (81172720, 81673230 号) 的支持, 江苏省科技部社会发展研究项目 (no。BE2016741), 中国质量监督检验检疫总局科学 & 技术项目 (2015QK055), 教育部儿童发展与学习科学重点实验室开放项目方案,东南大学 (CDLS-2016-04)。我们真诚地感谢元宋和萍柳协助我们收集样品。

获得了父母的知情同意, 并获得了机构审查委员会的批准。这项研究是按照世界医学协会 (赫尔辛基宣言) 的道德准则进行的, 涉及人类的实验。所有参与者的照顾者都提供了书面同意, 让他们参加这项研究。还获得了东南大学附属中大医院的伦理委员会批准。
1. 为提取和测定儿茶酚胺所需的柱和溶液的制备
<ol><li>准备 PFSPE 列。将1-2 毫克的四氯乙烯-PS 纳米纤维分成5-6 整除数, 并使用直径为0.5 毫米的细钢棒将它们有序地压缩到吸管尖端的末端, 体积为200µL。</li>
	<li>用2.427 克尿素, 0.034 克尿酸, 0.09 克肌酐, 0.297 克柠檬酸三钠, 0.634 克氯化钠, 0.45 克氯化钾, 0.161 克氯化铵, 0.089 克氯化钙二水合物, 0.1 克硫酸镁制备人工尿液水合物, 0.034 克碳酸氢钠, 0.003 克草酸钠, 0.258 克硫酸钠, 0.1 克磷酸二氢钠, 0.011 克磷酸氢钠, 溶解上述化学物质为200毫升去离子水。</li>
	<li>通过将2毫克的化合物溶解到1毫升蒸馏水中, 制备出2毫克/毫升的 diphenylborate (DPBA) 溶液。在4摄氏度的黑暗中存储解决方案。</li>
	<li>
分析标准 注: 儿茶酚胺的化学结构和性质不稳定, 易于分解。标准的制备过程必须非常快, 并且要防止暴露在阳光直射下。<ol>
<li>1.0 毫克的 NE, E, DA, MHPG, DOPAC 和内部标准 34-dihydroxybenzylamine 氢溴酸盐 DHBA 在单独1.5 毫升离心管。在使用前将水中的 DHBA 溶液稀释至100毫升。</li>
		<li>在黑暗中以高速振荡准备好的标准, 直到完全溶解。这是主要的股票;贮存在-20 摄氏度, 长达数周。</li>
		<li>准备1000次/毫升分析物的储存。对于 NE, E, DA, DOPAC 和 MHPG, 将每一个主要分析物的5µL 转移到4975µL 的蒸馏水中5毫升离心管, 并存储在黑暗中4°c, 直到使用。每天新鲜地准备这些解决方案。对于 DHBA, 将5µL 的原库存转移到5毫升离心管中的4995µL 蒸馏水中, 并分别在4摄氏度处存放在黑暗中。</li>
		<li>进一步稀释二次分析物库存, 以创建标准曲线 (e. g,补充表 2)。存储解决方案在黑暗中的4°c, 并准备新鲜的每日。</li>
		<li>用合适浓度的标准库存测试幼儿早期检测器的最佳电压。改变电压, 以找到一个值, 其中的表现出最好的峰值外观。</li>
	</ol>
</li>
	<li>制备含有30% 磷酸、15% 乙腈和55% 蒸馏水的淋洗剂组成。对于10毫升的淋洗剂组成溶剂, 使用5.5 毫升蒸馏水, 并加入1.5 毫升的乙腈和3毫升的磷酸滴入水中。</li>
</ol>2. 实际尿样和流动相的制备
<ol><li>让母亲用无菌尿杯收集婴儿的第一个清晨尿液。立即将样品转成聚丙烯管和标签。然后, 把样品存放在-20 摄氏度的冰箱里。</li>
	<li>涡流和离心机的尿样在 1510 x g 10 分钟室温 (RT), 以摆脱大多数微粒干扰。丢弃沉积物, 收集上清液进行进一步实验。为了有效地提取样品, 在离心后立即进行 PFSPE 预处理 (步骤 3)。</li>
	<li>
准备移动阶段
	<ol>
<li>准备一个干净的瓶子, 至少1升。移动阶段的构成列在补充表 1中;1 L 流动相, 测量6.7242 克柠檬酸, 93.06 毫克乙烯二胺四乙酸钠盐, 7.02 克 monometallic 钠磷酸, 404.5 毫克的 1-heptanesulfonic 酸钠盐和3.5 克钠水合物进入瓶子。加入40毫升乙腈和蒸馏水到1000毫升。搅动和震动超声15分钟, 直到溶液中的物质全部溶解。</li>
		<li>使用带有玻璃电极的 ph 计, 用饱和氢氧化钠溶液将移动相的 ph 值调整为4.21。</li>
		<li>用0.45 µm 聚偏氟乙烯微孔膜和真空吸尘装置过滤移动相, 去除杂质。</li>
		<li>每次使用前, 使用超声波振动15分钟, 以加气移动相。</li>
	</ol>
</li>
</ol>3. PFSPE 提取和高效液相色谱分析
<ol><li>激活纳米纤维。按100µL 甲醇和100µL 的水依次通过 PFSPE 柱使用5毫升注射器缓慢, 滴状的方式。</li>
	<li>混合100µL 尿样与100µL 2 毫克/毫升 DPBA 溶液和30µL 100 ng/毫升 DHBA 溶液 (是, 内部标准) 在0.5 毫升 EP 管, 然后将混合溶液转移到 PFSPE 柱。通过 PFSPE 柱上的混合样品溶液, 用5毫升气密塑料注射器, 使用气压的力量。</li>
	<li>通过将100µL 的 DPBA 溶液 (2 毫克/毫升) 装入 SPE 柱中, 将该柱浸出三次, 并使用5毫升气密塑料注射器将溶液缓慢地通过带气压的墨盒。</li>
	<li>将淋洗剂组成的50µL 加载到 PFSPE 柱上, 并将其推入柱上, 用0.5 毫升 EP 管收集洗脱液。</li>
	<li>打开 HLPC degasser, 把系统里的空气加起来。在样品分析之前, 系统应运行超过0.5 小时的移动相平衡和减少基线噪音。请参见辅助表 1 , 其中显示了 HPLC 系统的设置参数。</li>
	<li>样品20µL 的洗脱液使用自动取样器, 然后注入到 HPLC-幼儿系统。</li>
	<li>当运行完成后, 使用探测器接口关闭探测器单元。不要用开关在探测器背面关闭单元格, 因为这会损坏仪器。</li>
	<li>手动将移动相组成改为10% 甲醇和90% 水。至少运行30分钟。然后, 手动将移动相转化为高效液相色谱级甲醇。运行约15分钟, 以保护系统在甲醇。如果在建议的运行时间之后无法运行此步骤, 可能会导致列和检测器的损坏。关闭流, 然后关闭 degasser。</li>
</ol>4. 苯硼酸碳粉盒 (PBA) 提取
PBA 墨盒提取过程类似于库马尔et al中的方案。(2011)25. 所有解决方案都通过 PBA 墨盒 (100 毫克, 1 毫升) 通过注射器强制的空气进行推送。
<ol><li>将墨盒与1毫升80:20 乙腈-水 (v/v), 含有1% 甲酸和1毫升50毫米磷酸盐缓冲 (pH 10) 顺序。</li>
	<li>按下缓冲尿样 (1 毫升尿液和2毫升磷酸盐缓冲器, pH 值 8.5) 通过 PBA 墨盒。</li>
	<li>用1毫升 50:50 v/v 乙腈-磷酸盐缓冲液清洗墨盒 (10 毫米, pH 8.5)。</li>
	<li>洗脱1毫升乙腈-水 (80:20 伏/v), 含有1% 甲酸的墨盒。</li>
</ol>5. 对儿茶酚胺的鉴定和量化
<ol><li>
线性
	<ol>
<li>用人工尿液稀释二次分析物, 以六浓度 (1.5、3、12、25、50和 100 ng/毫升);人工尿液稀释量在补充表 2之后。用每浓度三个平行样品得到18个分析物的实验解来构造定标曲线。</li>
		<li>用人工尿稀释 DHBA 二次库存10倍, 得到100的实验溶液。</li>
		<li>根据步骤 3 (PFSPE 提取程序), 预处理所有来自5.1.1 的分析解。在步骤3中, 将每个相应洗脱液的20µL 注入 hplc-幼儿系统, 得到 hplc 色谱图谱。</li>
		<li>通过绘制峰值面积 (目标/是) 与 Y 轴的比值 (目标/是) 作为 X 轴, 构造五分析物的校准曲线, 如补充图 1所示。</li>
	</ol>
</li>
	<li>
灵敏度的 LOD 和 loq 矿用值
	<ol>
<li>将20µL 的空白人工尿液注入 hplc-幼儿系统 (如步骤 3), 获得样品的 hplc 色谱。</li>
		<li>在5.2.1 的色谱中, 收集11个空白信号值, 并计算平均值 Xb和标准偏差的b。计算可在一定置信度下检测到的物质的最小信号, xl, 作为 xl = xb+ K Sb (K 是由置信度确定的系数, Sb反映了测量方法和机器噪声的水平)。因此, LOD = (xL-xb)/s = (K sb)/s (s 代表工作曲线的斜率值)。</li>
		<li>将 3:1 (K=3) 的 s/n 定义为检测的极限 (LOD), 并将 10:1 (K=10) 的 s/n 作为量化 (loq 矿用) 的极限。</li>
	</ol>
</li>
	<li>
评估恢复
	<ol>
<li>准备真实的和有刺的尿样。用真正的尿液稀释二次分析仪, 以三浓度 (5, 50, 100 ng/毫升), 以获得尖刺尿样。为每种分析溶液准备三个平行样品。当靶化合物的数量被注入尿样中时, 数出尖刺浓度。将此值定义为A的。</li>
		<li>稀释 DHBA 库存至100毫升, 如步骤5.1.2。</li>
		<li>根据步骤 3 (PFSPE 提取程序) 从5.3.1 处理每个样本溶液, 并将每个相应淋洗剂组成的20µL 注入 HPLC-幼儿系统以获得色谱结果。分析物的价值将被算作定量的靶化合物数量在被刺的尿样品。将此值定义为t。</li>
		<li>将20µL 的尿样注入 HPLC-幼儿 (如步骤 3) 系统以获得色谱结果。分析物的价值将被计算为尿样中量化的靶化合物的初始数量。将此值定义为Ai。</li>
		<li>从标准曲线方程计算样品中目标化合物的数量。百分比恢复估计为方法恢复% = (At - i) x 100/(s)。平均值显示在表 1中。</li>
	</ol>
</li>
	<li>
评估不精确性
	<ol>
<li>在步骤5.3.1 中, 将含有的人工尿标本准备成5、50和 100 ng/毫升浓度。为每种分析溶液准备六个平行样品。每天准备新鲜的实验样品。</li>
		<li>稀释 DHBA 库存至100毫升5.1.2。</li>
		<li>评估日内精度 (n=6)。按照步骤3对5.4.1 中的每个样本溶液进行处理, 并将每个淋洗剂组成的20µL 注入到 HPLC-幼儿系统中以获得色谱。在同一天执行相同的操作六次。</li>
		<li>从标准曲线方程计算样品中目标化合物的数量。在同一化合物浓度相同的情况下, 一天内六种化验的相对标准差 (RSD) 确定为日内精度。平均值显示在表 1中。</li>
		<li>评估日间精度 (n=6)。同时每天在六连续的天, 准备尖刺的人工尿样品为三浓度 5, 50, 100 ng/毫升, 如在5.4.1 和 5.4.2, 并处理每个分析样品解决方案根据步骤3。</li>
		<li>将每个相应洗脱液的20µL 从5.4.5 注入 HPLC-幼儿系统, 以获得每一天的色谱结果。从标准曲线方程计算样品中目标化合物的数量。在六个连续天数内, 从三浓度的尿液样本中检测量的 RSD 表示日间精度。平均值显示在表 1中。</li>
	</ol>
</li>
</ol>

提交人证明, 与任何金融组织对本文所讨论的材料没有利益冲突。

本文提出的 PFSPE 方法在快速、简便、方便等方面具有重要意义。该协议中所用的吸附剂是静电纺丝纳米纤维, 具有较高的表面面积到体积比, 并吸附的方法, 高效。该程序只需要几毫克的碳纤维和少量的淋洗剂组成溶剂, 不需要蒸发步骤集中的分析。在这里, 我们详细介绍了一种基于 HPLC-幼儿发展的协议, 这将允许新用户建立有效的方法来检测和量化三儿茶酚胺及其两个代谢产物 (NE, E, DA 和 MHPG, DOPAC...

儿茶酚胺类神经递质及其代谢物在体液中的含量可以影响神经功能, 并在很大程度上影响反应刺激状态的平衡1。异常可能导致多种疾病, 如 pheochromacytoma、肾上腺、神经母细胞瘤和神经系统疾病1,2。体液中儿茶酚胺的提取和测定对相关疾病的诊断有重要意义。然而, 生物样品中的儿茶酚胺存在于低浓度, 易于氧化。此外, 由于介质3中的大量干扰, 它们很难洗脱。因此, 同时检测生物液中的儿茶酚胺仍然是一个挑战。
有评论表明, 尿儿茶酚胺可以是一种压力的量度, 他们的水平是重要的生物标记反应的触觉刺激处理新生儿5。根据这项研究, 所有患有过早事件的婴儿都有患脑损伤的风险4,5,6, 伤害可能导致儿茶酚胺的异常释放和相关物质进入体液。在早期的阶段7,8中, 存在能够检测大脑损伤的先进磁共振技术。然而, 在前48小时内, 异常神经发育过程将导致永久性脑损伤, 这在医学图像11中是不明显的。此外, 高昂的成本和稀缺的仪器资源, 连同其他因素, 使得所有新生单位都无法获得这些专门的神经成像技术。然而, 使用易于接近和实用的生物标志物 (如儿茶酚胺及其代谢物) 可以克服这些缺点, 并筛选人体体液中的生物标记可能有助于早期诊断脑损伤, 并导致提示新生儿需要神经保护的识别9。尿中的儿茶酚胺可以是一个容易和明显的指标, 因为它的数量之间的直接关系释放到液体和 neuroactivity 功能。
在生物体液中, 脑脊液 (CSF) 和血浆样本不易通过现有的创伤程序, 而且由于粘附蛋白和其他杂质也很难消除干扰, 导致了麻烦和耗时的取样过程不适合重复检测。此外, 对于儿童来说, 很难以创伤的方式获取样本。因此, 尿液取样优于其他形式的取样, 因为它是无创、易操作的, 可以反复进行。尿样丰富, 易于贮存, 比其他生物样品具有更大的优越性。
定量测定生物液中儿茶酚胺的主要方法包括 radioenzymic 化验10、酶联免疫吸附剂化验11、伏安12和热透镜光谱分析13。但存在着复杂的操作和难以检测的多目标等缺点。目前, 主要的分析技术是高效液相色谱 (HPLC)14, 加上敏感的电化学15或荧光检测16, 因为其灵敏度高, 选择性好。采用串联质谱技术, 如液相色谱/质谱 (lc/ms) 和液相色谱/质谱/质谱 (lc/ms), 分析和定量的神经递质可以达到高准确性和特异性17,18。然而, MS 技术需要昂贵的仪器和大量的合格的人力, 使这一方法难以普遍适用于大多数传统实验室。高效液相色谱-幼儿早期系统在大多数常规和临床实验室中都有广泛的应用, 因此成为研究小组用于化学测定的一个普遍而又好的选择, 但它们要求将样品引入系统中进行清洁和微型卷19。因此, 在分析之前, 对样品进行提纯和浓缩是非常重要的。纯化步骤的经典方法是液-液萃取14,15,20和离线固相萃取, 包括活化氧化铝柱21,22和 diphenylborate (DPBA) 络合23,24,25,26。
Myeongho 李等。已使用以冠醚化学修饰的高分子树脂作为吸附剂, 自 2007年27以来选择性地从人尿中提取儿茶酚胺。此外, 在 2006年, 海博他et al。演示了一种简便的合成方法, boronate 亲和萃取吸附剂利用 functionalizable 纳米磁性面体寡聚 silsesquioxane (POSS) 的合成, 并将其应用于富集的儿茶酚胺人尿 (去甲肾上腺素, 肾上腺素和肾上腺素)28。他们也利用纳米材料来完成这项工作, 使用一种叫做纳米静电纺丝技术, 在纳米尺度上形成聚合物纤维材料。静电纺丝工艺可以通过控制工作电压和改变纺丝溶液的含量以及其他参数29来调整产品的直径、形貌和空间对准。与传统的 SPE 碳粉相比, 静电纺丝纳米纤维非常适合从复杂基质中提取和丰富目标分析物, 因为它们具有较高的表面积-体积比, 能有效吸附高效的分析分析,展示更易于控制的表面化学性质, 允许方便地附着的目标化合物。这些特性使它们成为 SPE 吸附剂的好选择, 大大减少了固相和解吸溶剂量30,31,32,33。对于尿样中的儿茶酚胺, 采用 apolymeric 冠醚与聚苯乙烯 (四氯乙烯-PS) 组成的静电纺丝纳米纤维, 选择性提取三儿茶酚胺 (NE, E, DA) 34.研究表明, 选择性冠醚吸附了 NE、E、DA 等靶点, 其基础是通过形成氢键来结合儿茶酚胺的正确几何。该结果有效地显示了材料冠醚, 去除了生物样品中含有的其它干扰化合物。通过本报告的启发, 提出了一种新的方法, 用于选择性提取儿茶酚胺的静电纺丝复合纳米纤维组成的四氯乙烯-PS。
本文对以前的34方法进行了改进和应用, 不仅成功地分析了 E、NE 和 DA, 而且在尿液中还有其代谢物、MHPG 和 DOPAC。我们还探讨了吸附过程机理的新可能性。该方法对五种分析物的提取效率和选择性有满意的效果, 并对围产期脑损伤和健康控制的高危儿尿液的检测进行了验证。

<table><tbody><tr><td>200 &amp;micro;L pipette tip</td><td></td><td></td><td>column to contain nanofibers</td></tr><tr><td>PCE-PS nanofibers</td><td></td><td></td><td>material for PFSPE extraction</td></tr><tr><td>steel rod (about 0.5 mm diameter)</td><td></td><td></td><td>fill the nanofibres into the column</td></tr><tr><td>gastight plastic syringe (5 ml)</td><td></td><td></td><td>compress solution into the end of the tip</td></tr><tr><td>methanol</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>67-56-1</td><td></td></tr><tr><td>diphenylborinic acid 2-aminoethyl ester(DPBA)</td><td>Sigma-Aldrich.Inc</td><td>A-106408</td><td>complex reagent</td></tr><tr><td>norepinephrine(NE)</td><td>Sigma-Aldrich.Inc</td><td>A-9512</td><td>analyte</td></tr><tr><td>3-Methoxy-4-hydroxyphenylglycol(MHPG)</td><td>Sigma-Aldrich.Inc</td><td>H1377</td><td>analyte</td></tr><tr><td>epinephrine(E)</td><td>Sigma-Aldrich.Inc</td><td>100154-200503</td><td>analyte</td></tr><tr><td>3, 4-Dihydroxyphenylacetic acid(DOPAC)</td><td>Sigma-Aldrich.Inc</td><td>D-9128</td><td>analyte</td></tr><tr><td>dopamine(DA)</td><td>Sigma-Aldrich.Inc</td><td>H-8502</td><td>analyte</td></tr><tr><td>3, 4-dihydroxybenzylamine hydrobromide(DHBA)</td><td>Sigma-Aldrich.Inc</td><td>858781</td><td>interior label</td></tr><tr><td>acetonitrile</td><td>Sigma-Aldrich.Inc</td><td>75-05-8</td><td>eluriant and mobile phase</td></tr><tr><td>phosphoric acid</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>7664-38-2</td><td>eluriant</td></tr><tr><td>uric acid</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>69-93-2</td><td>artifical urine</td></tr><tr><td>creatinine</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>60-27-5</td><td>artifical urine</td></tr><tr><td>trisodium citrate</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>6132-04-3</td><td>artifical urine</td></tr><tr><td>KCl</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>7447-40-7</td><td>artifical urine</td></tr><tr><td>NH4Cl</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>12125-02-9</td><td>artifical urine</td></tr><tr><td>NaHCO3</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>SWC0140326</td><td>artifical urine</td></tr><tr><td>C2Na2O4</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>62-76-0</td><td>artifical urine</td></tr><tr><td>NaSO4</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>7757-82-6</td><td>artifical urine</td></tr><tr><td>disodium hydrogen phosphate</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10039-32-4</td><td>artifical urine</td></tr><tr><td>urea</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>57-13-6</td><td>artifical urine</td></tr><tr><td>NaCl</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>7647-14-5</td><td>artifical urine</td></tr><tr><td>MgSO4.7H2O</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10034-99-8</td><td>artifical urine</td></tr><tr><td>CaCl2</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10035-04-8</td><td>artifical urine</td></tr><tr><td>HCl</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>7647-01-0</td><td>artifical urine</td></tr><tr><td>citric acid</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>77-92-9</td><td>artifical urine and mobile phase</td></tr><tr><td>EDTA disodium salt</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>34124-14-6</td><td>mobile phase</td></tr><tr><td>monometallic sodium orthophosphate</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>7558-80-7</td><td>artifical urine and mobile phase</td></tr><tr><td>1-heptanesulfonic acid sodium salt</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>22767-50-6</td><td>mobile phase</td></tr><tr><td>sodium hydroxide</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>1310-73-2</td><td>mobile phase</td></tr><tr><td>phenylboronic acid column(PBA column)</td><td>Aglilent</td><td>12102018</td><td>PBA extraction</td></tr><tr><td>Inertsil&amp;reg; ODS-3 5 &amp;micro;m 4.6&amp;times;150 mm column</td><td>Dikma</td><td>5020-06731</td><td>HPLC column for seperation</td></tr><tr><td>SHIMADZU SIL-20AC prominence AUTO SAMPLER</td><td>Shimadzu Corporation, Japan</td><td>SIL-20AC</td><td>auto injection for eluriant</td></tr><tr><td>SHIMADZU LC-20AD High Performance Liquid Chromatography</td><td>Shimadzu Corporation, Japan</td><td>LC-20AD</td><td>HPLC pump</td></tr><tr><td>SHIMADZU L-ECD-60A electrochemical detector</td><td>Shimadzu Corporation, Japan</td><td>L-ECD-60A</td><td>detector for the analytes</td></tr><tr><td>ASAP 2020 Accelerated Surface Area and Porosimetry System</td><td>Micromeritics, USA</td><td></td><td>surface and porosity analyzer&amp;nbsp;</td></tr></tbody></table>

a convenient method for extraction and analysis with high-pressure liquid chromatography of catecholamine neurotransmitters and their metabolites

生物液中儿茶酚胺类神经递质的提取和分析对评价神经系统功能和相关疾病具有重要意义, 但其精确测量仍然是一个挑战。许多协议已被描述为神经递质测量的各种仪器, 包括高压液相色谱 (HPLC)。然而, 存在着复杂的操作或难以检测的多目标的缺陷, 但目前仍存在着以高效液相色谱和敏感电化学或荧光检测为主导的分析技术, 由于灵敏度高, 选择性好。本文介绍了用静电纺丝复合纳米纤维组成的高压力液相色谱法在婴幼儿真实尿样中对儿茶酚胺的预处理和检测的详细协议。以聚苯乙烯为吸附剂的高分子冠醚, 也称为填料-纤维固相萃取 (PFSPE) 法。我们展示了如何容易 precleaned 尿样的碳纤维包装固体相柱, 以及如何在样品中的分析物可以迅速丰富, 吸, 并发现在一个幼儿系统。PFSPE 大大简化了生物样品的预处理程序, 从而减少了时间、费用和减少目标损失。
总的来说, 这项工作说明了一个简单和方便的协议, 固相萃取耦合的 HPLC-幼儿系统, 同时测定三单胺类神经递质 (NE), 肾上腺素 (E), 多巴胺 (DA)) 和两个其代谢产物 (3-甲氧基 4 hydroxyphenylglycol (MHPG) 和 34-二羟基苯乙酸 (DOPAC)) 在婴幼儿尿液中。建立的协议用于评估高危婴儿与围产期脑损伤和健康控制之间的尿儿茶酚胺及其代谢物的差异。比较分析发现两组尿 MHPG 有显著差异, 表明儿茶酚胺代谢物可能是早期诊断婴幼儿脑损伤危险病例的重要候选指标。

该协议是一种简便、方便的 PFSPE 方法, 预处理尿样, 通过高效液相色谱-幼儿系统丰富五儿茶酚胺进行检测;进程的图表显示在图 1中。该协议主要包括四步激活, 加载, 冲洗和洗脱-结合少量的四氯乙烯-PS 纳米纤维和一个简单的固相萃取装置。用表面和孔隙度分析仪对四氯乙烯-PS 纳米纤维的形貌进行了评估 (见材料表)。纹理属性-赌注 (布鲁?...

Watch this Scientific Journal Video about A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites at JoVE.com

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

我们提出了一种方便的固相萃取与高压液相色谱 (HPLC) 结合电化学检测 (幼儿发展) 同时测定三单胺类神经递质和两个代谢物在婴儿的尿液中。我们还确定代谢物 MHPG 作为早期诊断婴儿脑损伤的潜在生物标志物。

用高压液相色谱法提取和分析儿茶酚胺神经递质及其代谢物的简便方法

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

a-convenient-method-for-extraction-analysis-with-high-pressure-liquid

Universidad San Sebastian

Research

JoVE Journal

Chemistry

14.6K 次观看.  Laboratory of Environment and Biosafety Research Institute of Southeast University in Suzhou. 我们提出了一种方便的固相萃取与高压液相色谱 (HPLC) 结合电化学检测 (幼儿发展) 同时测定三单胺类神经递质和两个代谢物在婴儿的尿液中。我们还确定代谢物 MHPG 作为早期诊断婴儿脑损伤的潜在生物标志物。

视频: A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

科研

化学

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

柱后衍生用反应流高效液相色谱柱

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is ...

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

微型固相萃取和LC-MS / MS检测人尿中3-硝基酪氨酸的临床应用

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital aspect of metabolomics is sample preparation whereby inconsistent techniques generate unreliable results. This technique encompasses protein precipitation, liquid-liquid extraction, and solid-phase extraction as a means of fractionating metabolites into four distinct classes.&#160;Improved enrichment of low abundance molecules with a resulting increase in sensitivity is obtained, and ultimately results in more confident identification of molecules. This technique has been applied to plasma, bronchoalveolar lavage fluid, and cerebrospinal fluid samples with volumes as low as 50 &#181;l. &#160;Samples can be used for multiple downstream applications; for example, the pellet resulting from protein precipitation can be stored for later analysis. The supernatant from that step undergoes liquid-liquid extraction using water and strong organic solvent to separate the hydrophilic and hydrophobic compounds. Once fractionated, the hydrophilic layer can be processed for later analysis or discarded if not needed. The hydrophobic fraction is further treated with a series of solvents during three solid-phase extraction steps to separate it into fatty acids, neutral lipids, and phospholipids. This allows the technician the flexibility to choose which class of compounds is preferred for analysis. It also aids in more reliable metabolite identification since some knowledge of chemical class exists.

The reliability of results in metabolomics experiments depends on the effectiveness and reproducibility of the sample preparation. Described is a rigorous and in-depth method that enables extraction of metabolites from biological fluids with the option of subsequently analyzing up to thousands of compounds, or just the compound classes of interest.

多步制备技术，以恢复多个代谢化合物的种类进行深入和信息化代谢组学分析

Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital ...

生物工程

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

在黑质和腹侧被盖区多巴胺调控的综合性能分析

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

神经科学

Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, penicillin, paclitaxel, rapamycin, or artemisinin, commonly used in medical practice, have been first identified and isolated from natural products. However, the identification and isolation of biologically active specialized metabolites from natural sources is a challenging and time-consuming process. Traditionally, individual metabolites are isolated and purified from complex mixtures, following the extraction of biomass. Subsequently, the isolated molecules are tested in functional assays to verify their biological activity. Here we present the use of cellular membrane affinity chromatography (CMAC) columns to identify biologically active compounds directly from complex mixtures. CMAC columns allow for the identification of compounds interacting with immobilized functional transmembrane proteins (TMPs) embedded in their native phospholipid bilayer environment. This is a targeted approach, which requires knowing the TMP whose activity one intends to modulate with the newly identified small molecule drug candidate. In this protocol, we present an approach to prepare CMAC columns with immobilized tropomyosin kinase receptor B (TrkB), which has emerged as a viable target for drug discovery for numerous nervous system disorders. In this article, we provide a detailed protocol to assemble the CMAC column with immobilized TrkB receptors using neuroblastoma cell lines overexpressing TrkB receptors. We further present the approach to investigate the functionality of the column and its use in the identification of specialized plant metabolites interacting with TrkB receptors.

The protocol describes the preparation of cell membrane affinity chromatography (CMAC) columns with immobilized cell membrane fragments containing functional transmembrane tropomyosin kinase receptor B proteins. The use of CMAC columns in the identification of specialized plant metabolites interacting with these receptors and present in complex natural mixtures is also explained.

细胞膜亲和色谱柱鉴定与固定化托肌球蛋白激酶受体B相互作用的特化植物代谢物

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, ...

生物化学

Semi-Targeted Ultra-High-Performance Chromatography Coupled to Mass Spectrometry Analysis of Phenolic Metabolites in Plasma of Elderly Adults

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss of muscle mass). Plasma samples were taken at the beginning of the intervention and after 30 days of consuming the functional meals. A semi-targeted ultra-high-performance chromatography coupled with tandem mass (UPLC-MS/MS) analysis was carried out to identify phenolic compounds and their metabolites. Plasma proteins were precipitated with ethanol and the samples were concentrated and resuspended in the mobile phase (1:1 acetonitrile: water) before injection into the UPLC-MS/MS instrument. Separation was carried out with a C18 reverse-phase column, and compounds were identified using their experimental mass, isotopic distribution, and fragment pattern. Compounds of interest were compared to those of data banks and the internal semi-targeted library. Preliminary results showed that the major metabolites identified after the intervention were phenylacetic acid, glycitin, 3-hydroxyphenylvaleric acid, and gomisin M2.

The goal of this protocol is to detect phenolic metabolites in plasma using a semi-targeted chromatography-mass spectrometry method.

老年人血浆中酚类代谢物的半靶向超高效色谱耦合质谱分析

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss ...

<meta charset="utf8"></head><body>使用 LC-MS/MS 对植物代谢物中的莨菪烷生物碱进行分类

Applications of Liquid-Chromatography Tandem Mass Spectrometry in Natural Products Research: Tropane Alkaloids as a Case Study

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and can yield promising leads for resistant or emerging diseases. The challenge, however, is twofold: not only must researchers find natural products and elucidate their structures, but they must also identify what is worth isolating and assaying (and what is already known - a process known as dereplication). With the advent of modern analytical instrumentation, the pace of natural product discovery and dereplication has accelerated. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an especially valuable technique for identifying and classifying chemical structures. Tropane alkaloids (TAs) are plant-derived compounds of great medicinal and toxicological significance. In this study, we developed an LC-MS/MS-based screening workflow utilizing the multiple MS/MS configurations available on a triple-quadrupole (QQQ) mass spectrometer to annotate and classify TA structures based on their distinct fragmentation patterns. By using a combination of data-dependent (DD) product ion scans, precursor ion scans (PrIS), and neutral loss scans (NLS), we applied this method to TA-rich extracts of the nightshades Datura stramonium and Datura metel. This method is rapid, sensitive, and was successfully employed for both preliminary dereplication of complex TA-containing samples and for the discovery of a novel candidate for isolation, purification (and eventual bioassay).

<meta charset="utf8"></head><body>作者聚焦：使用先进的质谱技术在植物中发现新的生物碱

We present a method for rapid mass spectrometry (MS)/mass spectrometry (MS)-based annotation and classification of tropane alkaloids, useful for both preliminary dereplication of tropane-containing samples and discovery of novel alkaloids for isolation.

液相色谱串联质谱法在天然产物研究中的应用：以莨菪烷生物碱为例

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and ...

用高压液相色谱法提取和分析儿茶酚胺神经递质及其代谢物的简便方法

摘要

探索更多视频

此视频中的章节

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

柱后衍生用反应流高效液相色谱柱

微型固相萃取和LC-MS / MS检测人尿中3-硝基酪氨酸的临床应用

多步制备技术，以恢复多个代谢化合物的种类进行深入和信息化代谢组学分析

在黑质和腹侧被盖区多巴胺调控的综合性能分析

细胞膜亲和色谱柱鉴定与固定化托肌球蛋白激酶受体B相互作用的特化植物代谢物

老年人血浆中酚类代谢物的半靶向超高效色谱耦合质谱分析

液相色谱串联质谱法在天然产物研究中的应用：以莨菪烷生物碱为例

液相色谱串联质谱法在天然产物研究中的应用：以莨菪烷生物碱为例

液相色谱串联质谱法在天然产物研究中的应用：以莨菪烷生物碱为例