An effective method for the determination of 20 free amino acids in tea samples using 4-(carbazole-9-yl)-benzyl chloroformate (CBBC-Cl) as a derivatization reagent by high-performance liquid chromatography combined with fluorescence detection (HPLC-FLD) has been established. CBBC-Cl quickly derivatized amino acids within 6 min in basic solution (pH 9.0) at room temperature (ca. 25 °C). The maximum excitation and emission wavelengths of the stable derivatives were 290 nm and 370 nm, respectively. The separation of 20 amino acids, including the theanine (THE) derivative, was optimized on a Hypersil BDS-C18 column. Identification of these derivatives was performed via liquid chromatography/electrospray ionization mass spectrometry (LC/ESI/MS) in positive-ion detection mode. Under optimal conditions, excellent linearity for all the amino acids was obtained, with determination coefficients of r 2 > 0.9986. The detection limits (LODs) and quantitation limits (LOQs) ranged from 0.13 to 1.5 μg·kg −1 and 0.43 to 4.95 μg·kg −1 , respectively. Satisfactory recoveries, ranging from 86.7 to 106.4%, were obtained for all the amino acids in the tea samples. The developed method has been successfully applied to the quantification of 20 amino acids in tea samples.

中文翻译：

---

使用 4-(咔唑-9-基)-苄基氯甲酸酯作为衍生化试剂，通过荧光检测的高效液相色谱法测定茶叶样品中的氨基酸

以4-(咔唑-9-基)-氯甲酸苄酯(CBBC-Cl)为衍生试剂的高效液相色谱结合荧光检测(HPLC-FLD)测定茶叶样品中20种游离氨基酸的有效方法） 已经建立。CBBC-Cl 在室温（约 25 °C）的碱性溶液（pH 9.0）中在 6 分钟内快速衍生氨基酸。稳定衍生物的最大激发和发射波长分别为 290 nm 和 370 nm。在 Hypersil BDS-C18 色谱柱上优化了 20 种氨基酸的分离，包括茶氨酸 (THE) 衍生物。这些衍生物的鉴定通过液相色谱/电喷雾电离质谱 (LC/ESI/MS) 在正离子检测模式下进行。在最佳条件下，获得了所有氨基酸的优异线性，决定系数 r 2 > 0.9986。检测限(LOD) 和定量限(LOQ) 的范围分别为0.13 至1.5 μg·kg -1 和0.43 至4.95 μg·kg -1 。茶叶样品中的所有氨基酸均获得了令人满意的回收率，范围为 86.7% 至 106.4%。所开发的方法已成功应用于茶叶样品中 20 种氨基酸的定量分析。