Department of Genetics and Endocrinology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou 510623, China.
Department of Genetics and Endocrinology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou 510623, China.
Clin Chim Acta. 2023 Aug 1;548:117453. doi: 10.1016/j.cca.2023.117453. Epub 2023 Jul 9.
Cerebrospinal fluid (CSF) monoamine neurotransmitters, their precursors and metabolites are essential biomarkers in the diagnosis and follow-up of monoamine neurotransmitter disorders (MNDs). However, their extra low concentrations and potential instability challenge the detection method. Here, we present a method that enables simultaneous quantification of these biomarkers.
With propyl chloroformate /n-propanol, 16 biomarkers in 50 μL of CSF were derivatized in situ within seconds under an ambient temperature. The derivatives were extracted by ethyl acetate and separated by a reverse phase column followed by mass spectrometric detection. The method was fully validated. Optimal conditions for standard solution preparation and storage, as well as CSF sample handling, were investigated. CSF samples from 200 controls and 16 patients were analyzed.
The derivatization reaction stabilized biomarkers and increased sensitivity. Most biomarkers were quantifiable in concentrations between 0.02 and 0.50 nmol/L that were sufficient to measure their endogenous concentrations. The intra- and inter-day imprecision were < 15% for most analytes, and accuracy ranged from 90.3% to 111.6%. The stability study showed that standard stock solutions were stable at -80 °C for six years when prepared in the protection solutions; Analytes in CSF samples were stable for 24 h on wet ice and at least two years at -80 °C; But repeated freeze-thaw should be avoided. With this method, age-dependent reference intervals for each biomarker in the pediatric population were established. Patients with MNDs were successfully identified.
The developed method is valuable for MNDs diagnosis and research, benefiting from its advantages of sensitivity, comprehensiveness, and high throughput.
脑脊液（CSF）单胺神经递质及其前体和代谢物是诊断和随访单胺神经递质疾病（MNDs）的重要生物标志物。然而，其超低浓度和潜在不稳定性对检测方法提出了挑战。在此，我们提出了一种能够同时定量这些生物标志物的方法。
采用丙酰氯/正丙醇，在室温下数秒内将 50 μL CSF 中的 16 种生物标志物原位衍生化。衍生化产物用乙酸乙酯提取，然后通过反相柱分离，再进行质谱检测。该方法经过了全面验证。考察了标准溶液制备和储存的最佳条件以及 CSF 样品处理条件。分析了 200 例对照和 16 例患者的 CSF 样本。
衍生化反应稳定了生物标志物并提高了灵敏度。大多数生物标志物在 0.02-0.50 nmol/L 的浓度范围内可定量，足以测量其内源性浓度。大多数分析物的日内和日间精密度均<15%，准确度范围为 90.3%-111.6%。稳定性研究表明，在保护溶液中制备时，标准储备溶液在-80°C 下可稳定保存 6 年；CSF 样品中的分析物在湿冰上可稳定 24 小时，至少在-80°C 下可稳定两年；但应避免反复冻融。使用该方法，建立了儿科人群中每种生物标志物的年龄相关参考区间。成功识别出 MND 患者。
该方法具有灵敏度高、全面性好、高通量等优点，对 MNDs 的诊断和研究具有重要价值。
