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)。
此协议的优越性主要来源于过程中的四个关键步骤, 如图 1所示: 使用四氯乙烯-PS 纳米纤维捕获目标化合物, 以将吸附剂的尺寸减少到几毫克, 从而快速吸附/解吸, 只需要少量的洗脱液 (1);将 DPBA 加入尿液中, 以改善其疏水性 (2);用含有 DPBA 的溶液冲洗纤维, 以保留分析物并除去杂质 (3);和优化提取和分析条件, 以获得良好的敏感性和选择性, 为分析 (4)。
在步骤 (1) 中, 乙烯-PS 吸附能力的优异性能可能归因于多种因素。在纳米纤维中掺杂的冠醚聚合物, 可以通过 H 键 (例如儿茶酚胺), 与含有氨基基团的来宾分子组成一个宿客复合体。此外, 红优胡 et 等和吉顺 et等。还建议硼酸可以通过 B 氮相互作用与化学结构中的氮原子结合, 并且疏水性骨架可以与苯环和其他脂肪族组进行相互作用36,37。同时, 在解吸过程中, 氢键和 B 氮相互作用在极低的 pH 值下容易断裂;因此, 酸性 pH 的 eluants 通常适用于解吸的探索。此外, 还有几个次要的相互作用, 包括疏水、离子和氢键, 这可能发生在硼酸化学物质和相关化合物之间,36,37,38。所有这些相互作用可能导致对材料的分析的吸附。
对于步骤 (2), 在添加 DPBA 的帮助下, 四氯乙烯-PS 纳米纤维迅速吸附 DPBA 儿茶酚胺配合物。振柳et表明, boronate 亲和材料已成为重要的培养基, 用于选择性分离和分子识别有机化合物, 如苷, 糖类, 多糖, 糖蛋白等38。络合主要发生在硼酸和顺二醇组之间的 boronate 亲和作用, 或两个相邻的羟基, 形成五或六元循环酯, 如补充图 2A所示。辅助图 2B描述了这项工作中对硼酸化合物的五个主要反应部位。当环境变得酸性时, 硼酸-顺二醇复合离解。硼酸与顺二醇组或相邻两羟基的相互作用存在着广泛而相对强烈的影响。
对于步骤 (3), 在分析物和 DPBA 之间形成的复杂可以将它们保存在吸附剂上, 而其他杂质则从四氯乙烯-PS 表面漂洗, 从而达到对目标的最佳预留。对于步骤 (4), 有一份报告显示, 最佳 pH 值为硼酸和邻苯二酚化合物39的结合度约为9.0。但是, 陈et。探讨了儿茶酚胺-DPBA 络合剂的最佳 ph 值, 发现当 pH 值改变为6.0 和 7.034、35时, CAs 的复合 PS-四氯乙烯纳米纤维的吸附性能是最佳的。刘et . 牵连到, boronate 酸化合物的支撑材料结构, 特别是腔和纳米孔的空间约束, 以及在反应过程中形成的 B N 配体, 也可以显著降低结合 pH 值和增强绑定亲和力38。四氯乙烯-PS 结构和儿茶酚胺结构都配备了-NH 组。因此, 四氯乙烯的性质和纳米孔隙 (如代表结果中所示的数据), 以及形成的 B N 配体, 都可能有助于降低协议中的结合 pH 值。因此, 该协议中的最佳 pH 值可以是中性的, 这大大简化了程序, 避免了分析物的降解。
catcholamine 测定的主要局限性是, 它们在生物样品中的浓度非常低, 其结构不稳定 (易氧化);此外, 很难去除介质中的杂质。因此, 对生物样品中儿茶酚胺的分析, 必须进行预处理, 通常是固相萃取。本文提出的 PFSPE 方法为尿样中的分析物提供了一种简便、方便的预浓度。但是, 在进行实验时, 必须严格控制实验条件, 如避免光暴露, 尽可能缩短预处理时间。
用该方法测定健康婴儿和高危婴儿尿液中三儿茶酚胺和两种代谢物的浓度, 以确定其适用性。两组尿 MHPG 有显著性差异。如前所述, 人体中的 MHPG 量现在主要报告为反映肾上腺素能神经音的指数, 在中枢神经系统和外围神经系统中儿茶酚胺代谢活动 40, 41, 42, 是用于中枢 NE 代谢的有用血浆/尿标记43。对于高危婴儿, 各种因素, 如缺氧缺血性脑病 (HIE) 导致脑外伤和大脑的侮辱, 导致不同程度的儿童神经发育缺陷。这种脑损伤反过来将导致释放神经递质 (MHPG 包括) 到体液中44,45。根据 J.W. Maas 的报告, MHPG46、47的大脑、脑脊液、血浆和尿浓度之间存在显著的相关性。在尿中总 (游离 + 共轭) MHPG 的测量长期被用于评估中心 ne 和周围的 ne 代谢在人的新陈代谢41,48,49。因此, 可以得出结论, 高危婴儿与健康的肾上腺素能神经元的音调损伤相比, 影响中枢和周围神经系统的儿茶酚胺代谢。这一差异表明, 应进一步研究尿 MHPG 水平与神经发育缺损的关系。结果表明, 该方法可用于测定尿中儿茶酚胺的存在, 具有很有前景的临床应用价值, 用于评价与这些神经递质相关的疾病。

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 (布鲁诺尔, 埃米特和出纳) 表面积, 孔隙体积和孔径大小-分别为2.8297 米2 g-1, 0.009 厘米3 g-1和 12.76 nm。这些数据表明, 该协议所使用的材料在表面上具有纳米尺度的孔隙, 这可能有助于提高吸附效率和降低协议中的结合 pH 值。
此协议使用优化的卷、样本成分、渗滤液、淋洗剂组成、等, 以及在过程中所消耗的 pH 值和时间。在陈et, 淋洗剂组成是一个12.0 米醋酸溶液, 因为酸性条件导致吸附剂成为质子和积极充电, 这是有利于洗脱的CAs34, 35.本研究将淋洗剂组成配方重造为30% 磷酸、15% 乙腈和55% 蒸馏水。淋洗剂组成的最终 pH 值调整为3.0。淋洗剂组成溶剂在洗脱时应保持在酸性环境中, 但要比12.0 米乙酸适中, 这可能会导致系统的峰值出现不良和损坏。
对儿茶酚胺的鉴别, 比较了标准溶液峰样品中峰的色谱保留时间。图 2显示了不同解决方案中的高效液相色谱图谱的例子。如果成功地遵循了该协议, 则应通过高效液相色谱--发育清晰对称、定义良好的峰值和最小背景噪声, 获得三儿茶酚胺及其代谢物的色谱图谱, 如图2所示.为了与常规方法进行比较, 选择了商用 PBA 墨盒作为控制。在图 2(ac)中, 五个目标峰值 (b) 显著高于 thaosein (c), 表明 PFSPE 方法比 PBA 墨盒方法更敏感。此外, DOPAC 峰值未显示在 pba 墨盒提取结果中, 表明 pba 列无法提取 DOPAC。图 2(d) 在没有任何预处理的情况下描述空白尿样的色谱,图 2(e) 显示了用四氯乙烯-PS 复合纳米纤维提取的尿样的图谱。图 2(f) 在提取后使用 PBA 列显示尿样的图谱, 这也表明没有 DOPAC 提取与图 2(c) 中的结果一致。该图表明, PFSPE 方法不仅能有效地提取靶点, 而且能去除尿中的大部分干扰, 为靶化合物提供良好的峰值识别。
统计分析显示, 三儿茶酚胺和两种代谢产物的测量结果可靠地重现 (表 1)。所有目标化合物在1.5 和 100 ng/mL (R2> 0.99) 之间的线性度很好, 每个分析物的标准曲线可以在补充图 1中找到。曲线和 R2值表明, 该分析方法在一定的线性范围内具有良好的线性度和相关性, 适合于尿样中分析物浓度的计算。检测的极限范围从0.25 到 0.54 ng/毫升不等, 定量的限度 (loq 矿用) 分别为0.83 到1.81。信噪比 (S/N) 值等于3。五目标化合物的方法学回收率范围从 97.4% (MHPG) 到 124.2% (DOPAC), 这对于实际样品的应用是令人满意的。日内精度从2.7 到 4.8% (表示为相对标准偏差), 日间精度为 2-8. 1%, 显示良好的精度和重复性。
为检测实际尿样中的靶点, 在苏州市医院儿童保育司招募了28名高危婴儿和22例健康婴儿。这项研究的50名婴儿都是男孩。当他们六月大, 他们被带到例行健康检查在2016年9月。所有的尿样都收集和预处理后的协议步骤3.1 和 3.2, 和样本分析使用其余的步骤3。根据标定曲线计算出浓度。通过方差分析, 分析统计学差异。结果可以在表 2中看到。比较分析了两组间儿茶酚胺和代谢产物的差异。p值表明, 高风险和健康组之间的儿茶酚胺没有显著差异, 而代谢物 MHPG 内容在这些组中不同 (p = 0.001)。高危儿组的 MHPG 比对照组高 (14.8 3.6 ng/毫升 1.4, 0.2 ng/毫升), 这意味着尿 MHPG 水平可能是早期识别高危婴儿的潜在标志。
图 3和表 3描述了用于确定单胺材料的经典量化方法, 并与给出操作过程和兴趣图的其他方法进行比较。与经典方法相比, PFSPE 法具有 timespan (5-10 分钟) 短、操作过程简化、有机溶剂少、环境友好性较好等优点, 具有满意的方法学参数。所需淋洗剂组成量低 (50 µL), 目标浓缩步骤无需蒸发, 大大提高了尿靶化合物的检测灵敏度。与传统的粒子基 SPE 相比, 该方法提高了效率, 简化了制备过程, 降低了分析的时间, 具有可接受的可靠性、选择性和灵敏度。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56445/56445fig1.jpg"> 图 1: PFSPE 过程的示意图流程图及其设备的表示形式.(1) 气密注射器 (2) 吸管尖端和 (3) 填充的纳米纤维.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56445/56445fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56445/56445fig2.jpg"> 图 2: 图谱不同的示例.(a) 将水样本与目标一起加在 (100 ng/毫升) 中而不提取。(b) 由 PFSPE 方法提取的尖水样本, 以及商业苯硼酸 (PBA) 墨盒 (c)。(d) 无提取的真正的空白尿样。(e) PFSPE 方法提取的真实尿样。(f) 由商用 PBA 墨盒提取的真实尿样。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56445/56445fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56445/56445fig3.jpg"> 图 3: 测定方法的分析流程图.(a, b)以前报告的经典提取方法。(a) 由 DPBA 复合体 (b) 和 (c) 通过本文提出的方法提取氧化铝。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56445/56445fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
辅助图 1: 标准曲线和公式.为了利用线方程计算样品中未知的分析物浓度, 构造了五分析物的标准曲线。(a) NE、(b) E、(c) DA、(d) MHPG 和 (E) DOPAC。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56445/supplementary_fig_1.pdf" target="_blank">请单击此处下载此图.</a>
补充图 2: 硼酸与顺二醇组或相邻两羟基的 Boronate 亲和相互作用.(A) 硼酸与多 OH 组之间相互作用的示意图, 形成五-或六-元循环酯。(B) 独联体二醇组或相邻两个红圈羟基组描述了五种物质对硼酸化合物的主要反应部位。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56445/supplementary_fig_2.pdf" target="_blank">请单击此处下载此图.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td>氖</td>
			<td>MHPG</td>
			<td>电子邮件</td>
			<td>是</td>
			<td>DOPAC</td>
			<td>大</td>
		</tr>
<tr>
<td>线性范围 (ng/毫升)</td>
			<td>1.5-400</td>
			<td>1.5-200</td>
			<td>1.5-100</td>
			<td></td>
			<td>1.5-100</td>
			<td>1.5-400</td>
		</tr>
<tr>
<td>R 平方值</td>
			<td>0.9945</td>
			<td>0.995</td>
			<td>0.9976</td>
			<td></td>
			<td>0.9902</td>
			<td>0.9954</td>
		</tr>
<tr>
<td>LOD (ng/毫升)</td>
			<td>0.323</td>
			<td>0.322</td>
			<td>0.313</td>
			<td>0.657</td>
			<td>0.249</td>
			<td>0.543</td>
		</tr>
<tr>
<td>loq 矿用 (ng/毫升)</td>
			<td>1.076</td>
			<td>1.072</td>
			<td>1.043</td>
			<td>2.191</td>
			<td>0.83</td>
			<td>1.809</td>
		</tr>
<tr>
<td>恢复 @ RSD (%) (n=9)</td>
			<td>110.7±2。9</td>
			<td>97.4±9。1</td>
			<td>103.9±5。2</td>
			<td>86.5±7。3</td>
			<td>124.2±3。1</td>
			<td>117.3±5。4</td>
		</tr>
<tr>
<td>精度 (RSD%)(n=9)</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>天内</td>
			<td>4。5</td>
			<td>4</td>
			<td>2。7</td>
			<td>4。8</td>
			<td>3。3</td>
			<td>4。7</td>
		</tr>
<tr>
<td>天际</td>
			<td>4。1</td>
			<td>8。1</td>
			<td>2</td>
			<td>6。3</td>
			<td>3。2</td>
			<td>5。9</td>
		</tr>
<tr>
<td colspan="7">NE, 去甲肾上腺素;MHPG, 3-甲氧基 4-hydroxyphenylglycol;E、肾上腺素;是内部标准;DOPAC, 3, 4-Dihydroxyphenylacetic 酸;DA, 多巴胺;</td>
		</tr>
</tbody></table>表 1: 采用标准溶液测定三个儿茶酚胺和两个代谢物的拟议议定书的分析结果。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>浓度 (ng mL-1)</td>
			<td>控制组 (22)</td>
			<td>高危婴儿组 (28)</td>
			<td>P 值</td>
		</tr>
<tr>
<td>氖</td>
			<td>7.52±1.34</td>
			<td>5.56±1。7</td>
			<td>0.37</td>
		</tr>
<tr>
<td>MHPG</td>
			<td>1.4±0。2</td>
			<td>14.8±3。6</td>
			<td>0.001</td>
		</tr>
<tr>
<td>电子邮件</td>
			<td>24±15。8</td>
			<td>20.9±5.87</td>
			<td>0.841</td>
		</tr>
<tr>
<td>DOPAC</td>
			<td>106.36±30。1</td>
			<td>72.12±18.07</td>
			<td>0.312</td>
		</tr>
<tr>
<td>大</td>
			<td>55.53±11。9</td>
			<td>48.12±20。9</td>
			<td>0.76</td>
		</tr>
<tr>
<td colspan="4">P 0.05 ** 显示两组之间的显著差异</td>
		</tr>
</tbody></table>表 2: 健康婴儿与高危婴儿之间尿中三个儿茶酚胺和两个代谢物的浓度比较.对 MHPG 含量差异进行了统计学分析, 并对其结果进行统计分析, 其意义为p = 0.05。意味着标准偏差显示。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td rowspan="4">示例</td>
			<td>用尽的溶剂 (mL)</td>
			<td>样品容积 (毫升)</td>
			<td>预处理时间 (分钟)</td>
			<td>线性范围</td>
			<td rowspan="4">lod</td>
			<td>相对恢复 (%)</td>
			<td>蒸发和 redissolve</td>
			<td rowspan="4">分析方法</td>
		</tr>
<tr>
<td>溶剂</td>
			<td>卷</td>
			<td>时间 (分钟)</td>
			<td></td>
			<td>恢复 (%)</td>
			<td>溶剂和</td>
		</tr>
<tr>
<td>毫升)</td>
			<td>毫升)</td>
			<td></td>
			<td></td>
			<td></td>
			<td>重组</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>残留</td>
		</tr>
<tr>
<td>尿</td>
			<td>0。5</td>
			<td>0.05</td>
			<td>10</td>
			<td>2.0–200/毫升 (NE, E, DA)</td>
			<td>0.2-0.5 ng/毫升 (NE, E, DA)</td>
			<td>88.5-94.5 (NE, E, DA)</td>
			<td>不</td>
			<td>HPLC-幼儿发展 (陈等, 2016)</td>
		</tr>
<tr>
<td>尿</td>
			<td>19</td>
			<td>0。7</td>
			<td>—</td>
			<td>47–167µg/升 (DA)</td>
			<td>166-500 毫微米 (DA)</td>
			<td>98.3-101.1 (DA)</td>
			<td>不</td>
			<td>HPLC-紫外线 (彼得等, 2016)</td>
		</tr>
<tr>
<td>尿</td>
			<td>1。3</td>
			<td>0.01</td>
			<td>—</td>
			<td>0.5–1250 (NE, E, DA, NMN, 锰)</td>
			<td>0.5-2.5 ng/毫升 (NE, E, DA, NMN, 锰)</td>
			<td>74.1–97.3 (NE, E, DA, NMN, 锰)</td>
			<td>不</td>
			<td>LC-ms (李等, 2016)</td>
		</tr>
<tr>
<td>尿液和血浆</td>
			<td>20</td>
			<td>0.05</td>
			<td>10</td>
			<td>0.04-2. 5 毫升 (NE, E, DA)</td>
			<td>0.01-0. 02 毫升 (NE, E, DA)</td>
			<td>87.0-97.5 (NE, E, DA)</td>
			<td>不</td>
			<td>HPLC-紫外线 (穆罕默德等, 2016)</td>
		</tr>
<tr>
<td>尿</td>
			<td>< 0。5</td>
			<td>0。1</td>
			<td>5</td>
			<td>1.5-400 ng/毫升 (NE, MHPG, E, DOPAC, DA)</td>
			<td>0.249-0. 543 毫升 (NE, MHPG, E, DOPAC, DA)</td>
			<td>97.4-124.2 (NE, MHPG, E, DOPAC, DA)</td>
			<td>不</td>
			<td>这项工作</td>
		</tr>
<tr>
<td colspan="9">-, 未定义。 PBA, 苯硼酸</td>
		</tr>
</tbody></table>表 3: 本工作与近年来单胺类的预处理研究或其他相关课题的比较。
辅助表 1: 用 HPLC 法测定和定量分析纸中分析物的仪器参数.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56445/supplementary_table_1_RM.xlsx" target="_blank">请单击此处下载此表.</a>
补充表 2: 为五个分析物准备标准曲线.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56445/supplementary_table_2_RM.xlsx" target="_blank">请单击此处下载此表.</a>

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

Nanyang Technological University

Research

JoVE Journal

Chemistry

14.5K 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

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

植物材料的快速高通量物种鉴定实时高分辨质谱法直接分析

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

科研

化学

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

<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 ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

高效液相色谱分析和触多巴胺摄取对小鼠多巴胺能稳态的评价

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

自由运动大鼠脑电图记录过程中兴奋氨基酸的微透析

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

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

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此视频中的章节

植物材料的快速高通量物种鉴定实时高分辨质谱法直接分析

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

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

高效液相色谱分析和触多巴胺摄取对小鼠多巴胺能稳态的评价

自由运动大鼠脑电图记录过程中兴奋氨基酸的微透析