The presence of monoamines and their cofactors (the pterins and vitamin B₆ (pyridoxal phosphate (PLP))) in human cerebrospinal fluid (CSF) can be used as indicators of the biosynthesis and turnover of dopamine and serotonin in the brain. In addition, abnormalities in the CSF levels of these molecules are associated with various neurological diseases, including genetic diseases leading to dopamine and serotonin deficiency. Here, we provide a set of quantitative high-performance liquid-chromatography (HPLC) approaches to determine CSF levels of monoamines and their cofactors. This protocol describes step-by-step procedures for CSF sample preparation for the analysis of different molecules, HPLC calibration and analysis, and data quantification and interpretation. Unlike plasma/tissue/blood samples, CSF requires minimal sample preparation: in this protocol, only the analysis of PLP requires mixing with trichloroacetic acid to release the protein-bound vitamin, centrifugation, and mixing of the supernatant with phosphate buffer and sodium cyanide for derivatization in alkaline conditions. Monoamines are analyzed by HPLC with coulometric electrochemical detection (ED), pterins are analyzed by HPLC with coupled coulometric electrochemical and fluorescence detection, and PLP is analyzed by HPLC with fluorescence detection. The quantification of all compounds is achieved by external calibration procedures, and internal quality control and standards are analyzed in each run. We anticipate that investigation of dopamine and serotonin disturbances will be facilitated by measurements of these compounds in human CSF and other biological samples. The estimated time for the different procedures primarily depends on the electrochemical detector stabilization. Overnight stabilization of this detector is advised, and, after that step, preanalytical equilibration rarely exceeds 3 h.

中文翻译：

---

HPLC分析人脑脊髓液中的单胺及其辅助因子。

单胺及其辅因子的存在（蝶呤和维生素B ₆人脑脊液（CSF）中的（磷酸吡rid醛（PLP））可以用作大脑中多巴胺和5-羟色胺的生物合成和更新的指标。另外，这些分子的CSF水平异常与多种神经系统疾病有关，包括导致多巴胺和5-羟色胺缺乏的遗传疾病。在这里，我们提供了一套定量高效液相色谱（HPLC）方法来确定单胺及其辅助因子的脑脊液水平。该协议描述了CSF样品制备的分步程序，用于分析不同的分子，HPLC校准和分析以及数据定量和解释。与血浆/组织/血液样品不同，CSF需要的样品制备量最少：在此方案中，只有对PLP的分析需要与三氯乙酸混合以释放蛋白质结合的维生素，然后进行离心分离，然后将上清液与磷酸盐缓冲液和氰化钠混合以在碱性条件下衍生化。单胺通过库仑电化学检测（ED）进行HPLC分析，蝶呤通过库仑电化学和荧光检测相结合的HPLC分析，PLP通过萤光检测进行HPLC分析。通过外部校准程序对所有化合物进行定量，并在每次运行中分析内部质量控制和标准品。我们预计，通过测量人脑脊液和其他生物样品中的这些化合物，可以促进对多巴胺和5-羟色胺紊乱的研究。不同程序的估计时间主要取决于电化学检测器的稳定性。建议该检测器通宵稳定，并且在此步骤之后，分析前的平衡很少超过3小时。