Laboratory co-established by the province and the state, School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 110016, China.
J Mass Spectrom. 2013 Aug;48(8):969-78. doi: 10.1002/jms.3243.
Neurotransmitters and their metabolites in central nervous system were known to play a significant role in sedation and hypnosis. A rapid and sensitive UFLC-MS/MS method for simultaneous determination of serotonin, 5-hydroxyindole acetic acid (5-HIAA), tryptophan (Try), dopamine (DA), norepinephrine (NE), γ-aminobutyric acid (GABA), glutamic acid (Glu) and acetylcholine (Ach) in rat brain without derivatization, ion-pairing reagent or pre-concentration was developed. Analytes and IS were separated on a Inertsil ODS-EP column (150 mm × 4.6 mm, 5 µm particles) and analyzed in a single chromatographic run in less than 9.0 min, using gradient elution with the mobile phase consisting of methanol and 0.01% acetic acid in water at a flow rate of 1.2 ml min(-1) . The detection of the analytes was performed on 4000Q UFLC-MS/MS system with turbo ion spray source in positive ion and multiple reaction monitoring mode. The developed method provided excellent linear calibration curves for the assay of analytes (R(2) ≥ 0.9915). Limits of quantification were in the range of 1.0 ng ml(-1) to 1.0 µg ml(-1) for the analytes in rat brain. Intra- and inter-day precision and accuracy of analytes were well within acceptance criteria (15%). Mean extraction recoveries of analytes and IS from rat brain were all more than 80.0%. Furthermore, the validated method was successfully applied to comparing profiles of analytes in normal and insomnic rat brains. Results indicated that there were statistically significant differences for serotonin, 5-HIAA, DA, NE, Glu and Ach, but no significant difference for Try and GABA between two groups.
中枢神经系统中的神经递质及其代谢物被认为在镇静和催眠中发挥着重要作用。建立了一种无需衍生化、离子对试剂或预浓缩，即可同时测定大鼠脑组织中 5-羟色胺（5-HT）、5-羟吲哚乙酸（5-HIAA）、色氨酸（Trp）、多巴胺（DA）、去甲肾上腺素（NE）、γ-氨基丁酸（GABA）、谷氨酸（Glu）和乙酰胆碱（Ach）的超快速液相色谱-串联质谱（UFLC-MS/MS）方法。分析物和内标在 Inertsil ODS-EP 柱（150mm×4.6mm，5μm 颗粒）上分离，在不到 9.0min 内以甲醇和 0.01%乙酸水溶液为流动相进行梯度洗脱，流速为 1.2ml·min-1，在单一次色谱运行中进行分析。采用正离子和多反应监测模式的 4000Q UFLC-MS/MS 系统，以涡轮离子喷雾源进行检测。该方法为大鼠脑组织中分析物的测定提供了优异的线性校准曲线（R2≥0.9915）。分析物的定量下限范围为 1.0ng·ml-1至 1.0μg·ml-1。分析物的日内和日间精密度和准确度均符合可接受标准（15%）。分析物和内标的大鼠脑组织平均提取回收率均大于 80.0%。此外，该验证方法成功应用于比较正常和失眠大鼠脑组织中分析物的谱图。结果表明，两组间 5-HT、5-HIAA、DA、NE、Glu 和 Ach 有统计学差异，但 Trp 和 GABA 无统计学差异。
