Dai Zhaolai, Sun Shiqiang, Chen Hui, Liu Moyan, Zhang Lianhua, Wu Zhenlong, Li Ju, Wu Guoyao
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China.
Henan Yinfa Animal Husbandry Co., Xinzheng, Henan, China.
Methods Mol Biol. 2019;2030:131-142. doi: 10.1007/978-1-4939-9639-1_11.
Tryptophan is a nutritionally essential amino acid for both humans and animals. Besides acting as a building block for protein synthesis, tryptophan (Trp) and its metabolites are crucial for maintaining neurological function, immunity, and homeostasis in the body. To uncover the regulatory role of Trp and its metabolites in cell nutrition, metabolism and physiology, various analytical methods, including high-performance liquid chromatography (HPLC), have been developed to determine key Trp metabolites. Here we describe a rapid and sensitive method for the simultaneous analysis of Trp and its metabolites along with other amino acids by HPLC involving in-line pre-column derivatization with o-phthaldialdehyde (OPA) and dual-channel fluorescence detection. OPA reacts very rapidly (within 1 min) with Trp, 5-hydroxytryptophan, 5-hydroxytryptamine, and tryptamine at room temperature (e.g., 20-25 °C) in an autosampler. Their derivatives are immediately injected into the HPLC column without the need for extraction. Trp metabolites that cannot react with OPA but are fluorescent can be detected by setting the excitation and emission wavelengths of the fluorescence detector in another detection channel. The autosampler is programmed to mix Trp and its metabolites with OPA for 1 min to generate highly fluorescent derivatives for HPLC separation and detection (Channel A, excitation = 270 nm and emission = 350 nm; Channel B, excitation = 340 nm and emission = 450 nm). The detection limit for Trp and its metabolites is 30 pmol/mL or 150 fmol/injection. The total time for chromatographic separation (including column regeneration) is 55 min for each sample. Our HPLC method can be used for the analysis of amino acids (including Trp) in alkaline protein hydrolysates and of Trp and its metabolites in biological samples.
色氨酸对人类和动物而言都是营养必需氨基酸。除了作为蛋白质合成的组成部分外，色氨酸（Trp）及其代谢产物对于维持人体神经功能、免疫力和体内平衡至关重要。为了揭示色氨酸及其代谢产物在细胞营养、代谢和生理学中的调节作用，人们开发了包括高效液相色谱法（HPLC）在内的各种分析方法来测定关键的色氨酸代谢产物。在此，我们描述了一种通过HPLC同时分析色氨酸及其代谢产物以及其他氨基酸的快速灵敏方法，该方法采用邻苯二甲醛（OPA）在线柱前衍生化和双通道荧光检测。OPA在室温（例如20 - 25°C）下于自动进样器中与色氨酸、5 - 羟色氨酸、5 - 羟色胺和色胺反应非常迅速（在1分钟内）。它们的衍生物无需萃取即可立即注入HPLC柱。不能与OPA反应但具有荧光的色氨酸代谢产物可通过在另一个检测通道中设置荧光检测器的激发和发射波长来检测。自动进样器被编程为将色氨酸及其代谢产物与OPA混合1分钟，以生成用于HPLC分离和检测的高荧光衍生物（通道A，激发波长 = 270 nm，发射波长 = 350 nm；通道B，激发波长 = 340 nm，发射波长 = 450 nm）。色氨酸及其代谢产物的检测限为30 pmol/mL或150 fmol/进样。每个样品的色谱分离总时间（包括柱再生）为55分钟。我们的HPLC方法可用于分析碱性蛋白质水解物中的氨基酸（包括色氨酸）以及生物样品中的色氨酸及其代谢产物。
