Xie Li, Chen Liqin, Gu Pan, Wei Lanlan, Kang Xuejun
School of Public Health of Southeast University, Laboratory of Environment and Biosafety Research Institute of Southeast University in Suzhou;
School of Public Health, Tianjin Medical University.
J Vis Exp. 2018 Mar 1(133):56445. doi: 10.3791/56445.
The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.
生物体液中儿茶酚胺神经递质的提取与分析对于评估神经系统功能及相关疾病具有重要意义，但其精确测量仍是一项挑战。已有多种通过各种仪器测量神经递质的方法被描述，包括高压液相色谱法（HPLC）。然而，这些方法存在缺点，如操作复杂或难以检测多个目标，这些缺点无法避免，目前，由于其高灵敏度和良好的选择性，主要的分析技术仍是HPLC结合灵敏的电化学或荧光检测。在此，描述了一种详细的方法，使用由聚合物冠醚与聚苯乙烯组成的电纺复合纳米纤维作为吸附剂（也称为填充纤维固相萃取（PFSPE）方法），对婴儿实际尿液样本中的儿茶酚胺进行高压液相色谱电化学检测（HPLC - ECD）的预处理和检测。我们展示了尿液样本如何通过纳米纤维填充的固相柱轻松进行预净化，以及样本中的分析物如何在ECD系统上快速富集、解吸和检测。PFSPE极大地简化了生物样本的预处理程序，减少了时间、费用，并降低了目标物的损失。总体而言，这项工作阐述了一种简单便捷的固相萃取与HPLC - ECD系统联用的方法，用于同时测定婴儿尿液中的三种单胺神经递质（去甲肾上腺素（NE）、肾上腺素（E）、多巴胺（DA））及其两种代谢物（3 - 甲氧基 - 4 - 羟基苯乙二醇（MHPG）和3,4 - 二羟基苯乙酸（DOPAC））。所建立的方法被应用于评估围产期脑损伤高危婴儿与健康对照之间尿儿茶酚胺及其代谢物的差异。比较分析显示两组之间尿MHPG存在显著差异，表明儿茶酚胺代谢物可能是婴儿脑损伤风险病例早期诊断的重要候选标志物。
