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Using 4-(Carbazole-9-yl)-benzyl Chloroformate as a Derivatization Reagent for the Measurement of Amino Acids in Tea Samples by High-Performance Liquid Chromatography with Fluorescence Detection

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Abstract

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 r2 > 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.

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Acknowledgements

This work was supported by National Natural Science Foundation of China (No. 21475075, 21677085 and 31801624) and the National Natural Science Foundation of Shandong Province (No. ZR2015CM040, ZR2017JL012, and ZR2018MC031).

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Authors and Affiliations

  1. Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal University, Qufu, 273165, China

    Xu Wang, Jinmao You, Zhiwei Sun, Zhongyin Ji, Shijuan Zhang, Jiangkun Tan & Xianzhu Luo

  2. Key Laboratory of Tibetan Medicine Research and Qinghai Key Laboratory of Qinghai-Tibet Plateau Biological Resources, Northwest Institute of Plateau Biology, Chinese Academy of Science, Xining, 810001, China

    Honglun Wang

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  1. Xu Wang
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Wang, X., You, J., Sun, Z. et al. Using 4-(Carbazole-9-yl)-benzyl Chloroformate as a Derivatization Reagent for the Measurement of Amino Acids in Tea Samples by High-Performance Liquid Chromatography with Fluorescence Detection. Chromatographia 83, 487–496 (2020). https://doi.org/10.1007/s10337-020-03864-5

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