Rapid stacking of amino acids in soybean and Dendrobium officinale by on-capillary sandwich derivatization in capillary electrophoresis
Graphical abstract
Introduction
Capillary electrophoresis (CE) with its powerful separation function has been widely used in the separation and detection of many substances, such as DNA (Przybylski et al., 2018), enzymes (Hu et al., 2018), inorganic ions (Opekar, Hraníček, & Tůma, 2020), and small organic molecules in complex samples (Gogolashvili et al., 2021). However, the small sample injection volumes and short optical path length compromise the detection limits of CE. To address these issues, various methods have been established to improve the detection efficiency of CE. Among these, stacking techniques have received a lot of attention and interest as an online preconcentration method. Various stacking approaches, including field-amplified sample stacking (FASS) (Zayed & Belal, 2020), field-enhanced sample injection (FESI) (Thang, See, & Quirino, 2016) large volume sample stacking (LVSS) (Medrano et al., 2019), and micelle to solvent stacking (MSS) (Song et al., 2020) are devoted to enhancing the detection sensitivity of multiple substances in CE. During the stacking process, the compounds are stacked at the boundary between the background solution (BGS) and the sample zone to form a narrow band for enrichment purposes. Thus, better separation efficiency and detection sensitivity are obtained by stacking (Breadmore et al., 2019, Malá et al., 2007). Recently, combination technology is the current hotspot, CE coupled with mass spectrometry (CE-MS) (Pero-Gascon et al., 2020) and CE with laser-induced fluorescence detection (CE-LIF) (Szilágyi et al., 2018) dramatically improve the detection sensitivity. However, the unstable detection process and the expensive instruments make the study of these coupling techniques somewhat difficult. UV–vis detector, as the most universal detector in CE, is economical compared with other methods (Xu et al., 2015), but there is a difficulty in detection for some analytes without UV absorption. Thus, it is an interesting study to develop a new CE enrichment technique to improve the detection sensitivity of analytes in complex sample matrices, especially for compounds without UV absorption.
Amino acids (AAs) are one of the most essential small molecules in the body, not only as a material basis but also as important for our physiological regulation (Dullius et al., 2020). AAs have been the focus of academic research due to their essential role in life and health, in which the establishment of a faster and more effective detection method is an important research direction. Currently, CE has been applied to determine AAs in human cells and tissues, foods, and medicinal plants (Castro-Puyana et al., 2007, Ta et al., 2021). Since most AAs do not have chromophores, label derivatization is important to enhance their optical properties and enhance detection. In conventional pre-column derivatization techniques, o-phthaldialdehyde (OPA), fluorescein isothiocyanate (FITC), and 9-fluoroenylmethylchloroformate (FMOC) are common derivatization reagents in AAs detection (Rufian-Henares et al., 2002, Lalljie and Sandra, 1995, Chan et al., 1993). Besides, aphthalene −2,3-dicarboxaldehyde has the disadvantage of requiring the involvement of a toxic catalyst, NaCN, when used for the derivatization of AAs (Celá, Mádr, & Glatz, 2017). Carboxytetramethylrhodamine succinimidyl ester and 7-amino-1,3-naphthalenedisulfonic acid monopotassium salt were successfully employed as a pre-column derivatization reagent for AAs analysis (Liu and Wang, 2013, Song et al., 2011). Additionally, diverse indirect CE techniques for AAs detection have been developed successively, such as solid phase extraction coupled with CE and capillary coating techniques (Li et al., 2019, Vitali et al., 2014). Moreover, the technique of capillary and chip electrophoresis in combination with contactless conductivity detection is also a promising method for detecting AAs (Tůma, 2021, Tůma et al., 2022). These methods for the indirect detection of AAs by CE have their advantages, but they also have shortcomings, such as cumbersome operation and high reagent consumption. In-capillary derivatization has currently attracted broad attention due to its advantages of simplicity, environmental protection, and high efficiency (Xie et al., 2017). Online derivatization makes up for these adverse effects in analyzing AAs in samples to make the whole process simpler and more efficient, directly injecting the sample and derivatization reagent into the capillary. For CE on-capillary derivatization, there are some related reports concerning the online complexation of AAs with copper ions (Luo et al., 2017), microdialysis (Harstad & Bowser, 2016), and electrophoretically mediated microanalysis (Celá, Mádr, & Glatz, 2017). These techniques have contributed to improving the efficiency of AAs detection, but most of these techniques employed only the single online derivatization method that did not involve an enrichment step and had relatively low sensitivity. Therefore, it is of great significance to develop a more secure and efficient method that can achieve simultaneous online derivatization and enrichment of AAs.
Since soybean and Dendrobium officinale are functional food for their high content of the AAs and other nutrients, qualitative and quantitative detection is necessary for their quality identification (Wang et al., 2020). In this study, a novel online sandwich derivatization and stacking approach by CE was established to preconcentrate multi-AAs in two functional food, and it was more convenient and efficient for the online sandwich derivatization and enrichment by CE. The online sandwich derivatization procedure was performed by the injection sequence of AAs, 4-Chloro-7-nitro-1, 2, 3-benzoxadiazole (NBD-Cl), and AAs. Furthermore, through a series of optimization, including running buffer concentration, injection pressure, derivatization reagent injection time, reaction waiting time, sample matrix concentration, and sample injection time, the optimal conditions were obtained and applied to the preconcentration of 9 AAs in soybean and Dendrobium officinale.
Section snippets
Chemicals
Borax was purchased from Sinopharm Chemical Reagent Co., ltd. (Shanghai, China). Sodium hydroxide (NaOH) was supplied by Hangzhou Xiaoshan Chemical Reagent Factory Co., ltd. (Hangzhou, China). Hydrochloric acid (HCl) was from Sinopharm Group Chemical Reagent Co., ltd. (Shanghai, China). Distilled water was employed throughout the experiment, which was obtained from Wahaha Group Co. ltd. (Hangzhou, China). Ethanol and acetonitrile of HPLC grade were got from Fisher Chemical (Pittsburg, USA) and
Mechanism
This study was devoted to developing a convenient technique of online sandwich derivatization and stacking to fulfill the online derivatization of AAs to simplify the operation process and reduce time consumption. A schematic depiction of processes occurring in online sandwich derivatization and stacking is presented in Fig. 1. Before working, the borax buffer was filled in the capillary. For the online derivatization process, the regents of AAs, NBD-Cl, and AAs were injected hydrodynamically
Conclusions
The novel online sandwich derivatization and stacking technique have been successfully developed for the preconcentration of nine AAs in the complexsamples. Excellent stability, high sensitivity, reasonable recovery, satisfactory linearity, favorable LOD, and LOQ have been obtained for all AAs due to the employment of the online derivatization and stacking procedure. The SEFs were in range of 8–62 for nine AAs, compared to those for typical injections in CE. Furthermore, the validated method
CRediT authorship contribution statement
Ya-Ling Yu: Visualization, Investigation, Software, Writing – original draft. Min-Zhen Shi: Investigation, Software. Si-Chen Zhu: Investigation. Jun Cao: Conceptualization, Supervision, Methodology, Project administration.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This study was supported by Hangzhou Social Development of Scientific Research Project (20191203B13), Zhejiang Province Basic Public Welfare Research Program (LGF18H280006), Hangzhou 131 Middle aged and Young Talent Training Plan (China, 2017).
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