Elsevier

Journal of Chromatography A

Volume 1598, 2 August 2019, Pages 232-241
Journal of Chromatography A

Development of an on-line analytical platform to screen haptens in shuxuening injection based on high-performance liquid chromatography coupled with ion-trap multistage mass spectrometry and human serum albumin fluorescence detection

https://doi.org/10.1016/j.chroma.2019.04.011Get rights and content

Highlights

  • An on-line HPLC-MSn system was built to screen hapten in SXNI for the first time.

  • The HSA-binding ability of compounds in Shuxuening injection were studied.

  • The sensitization of 15 compounds was studied by RBL-2H3 cells.

Abstract

Shuxuening injection (SXNI), one of the traditional Chinese medicine injections (TCMI), is widely used for the treatment of cardiovascular diseases in the clinic. However, its allergic reactions have impeded the clinical applications of SXNI, such adverse reactions have not been well understood due to the lack of methods for detecting haptens. In this study, a high-performance liquid chromatography-diode–array detector–multi-stage mass spectrometry–human serum albumin–fluorescence detector (HPLC–DAD–MSn–HSA–FLD) system was established to identify and screen haptens for the first time. Flavones, flavonols and their glycosides in SXNI showed strong HSA binding ability in different degrees. Fifteen of these compounds were used to study the association of HSA binding ability and sensitizability using isothermal titration calorimetry (ITC) and fluorescence techniques, furthermore, RBL-2H3 cell experiments were conducted to verify the results. It was found that ginkgolides showed no sensitizability, while flavones and flavonol aglycones showed stronger sensitizability than their glycosides. The system was proven to be precise, stable and reproducible, which lays a foundation for screening haptens in SXNI and relevant samples.

Introduction

Shuxuening injection (SXNI) is a sterile aqueous solution made of Ginkgo biloba leaf [1]. It is famous for its good clinical efficacy as a treatment of ischemic cerebrovascular disease, coronary heart disease, cerebral thrombosis and cerebral vasospasm [2,3]. However, its adverse reactions caused by its haptens have aroused great concern for its expansion on clinical applications. Previous studies have revealed that its major constituents include flavonoid glycosides and ginkgolides [4,5], however, whether these constituents are the haptens causing allergic reactions is still uncertain.

Major adverse reactions of SXNI include anaphylaxis and anaphylactoid reaction. Anaphylaxis occurs when the same substance stimulated the immunized organism again, while anaphylactoid reaction is similar to anaphylaxis except that it occurs when the antigen enters the body for the first time [6]. In anaphylaxis, the hapten can be a small molecule, which after binding to its carrier proteins, forms a hapten-protein complex (complete antigen) to induce allergic reactions [7]. Several known carrier proteins include keyhole limpet hemocyanin, concholepas concholepas hemocyanin, bovine serum albumin, ovalbumin and human serum albumin (HSA) [[8], [9], [10], [11]]. Among them, HSA is the most relevant carrier responsible for human sensitivity. HSA is the most abundant protein accounting for about 60% of total plasma protein in human circulation system. It is composed of 585 amino acid residues, among which tryptophan, lysine and phenylalanine residues confer endogenous fluorescence to HSA [12]. When a compound binds to HSA, its fluorescence decreases [13]. Based on this mechanism, potential HSA binders can be detected by fluorescence detection for further evaluation of their hapten properties. Isothermal titration calorimetry (ITC) was also applied to explore the interaction mode between HSA and flavonoids in Shuxuening injection [14,15].

RBL-2H3 cell is one type of basophilic leukemia cells isolated from basophils, which is commonly used in studies of high-affinity IgE receptors (also known as FcεRI receptors). FcεRI receptor is able to distinguish substances leading to anaphylaxis and anaphylactoid reaction. In anaphylaxis, the allergic model can be established by co-cultured with IgE for cross-linking activation of cell surface receptor [16]. In sensitized state, RBL-2H3 cells will release allergic medium when co-cultured with allergens. Meanwhile, co-culturing compound directly with RBL-2H3 cells can determine whether compound causes class allergic reaction. Histamine and tryptase content then can be detected to determine the degree of allergic reactions and class allergic reactions [16,17].

In this study, a method using high-performance liquid chromatography–diode-array detector–multi-stage mass spectrometry–human serum albumin–fluorescence detector (HPLC–DAD–MSn–HSA–FLD) system was established for the first time. HSA binders were identified in SXNI, and their hapten properties were then validated on RBL-2H3 cells. This system can serve as a high-throughput screening approach for screening and validating haptens from complex samples.

Section snippets

Materials and reagents

SXNI (Lot. No.: 20150809) was obtained from Shi Yao Yin Hu Group Co., Ltd (Shijiazhuang, China). Quercetin (42) was obtained from Sinopharm Group Chemical Reagent Co., Ltd. Quercetin-3-O-rutinoside (8), quercetin-3-O-glucoside (11), apigenin-7-O-glucoside (25), luteolin-7-O-glucoside (30), myricetin (S1) and luteolin (S2) were purchased from China Food and Drug Administration Research Institute (Beijing, Chian). Myricetin-3-O-glucoside (4), kaempferol-3-O-rutinoside (17), isorhamnetin-3-O

Determination of the detection wavelengths of HSA

Fluorescence spectra of off-line detection were performed on a Cary Eclipse fluorescence spectrophotometer (Agilent, Australia). The excitation and emission spectra were set in the scanning ranges of 225–325 nm and 300–600 nm, respectively. The maximum excitation wavelength was set at 285 nm and the maximum emission wavelength was set at 350 nm as shown in Fig. 2A.

Optimization of the on-line HSA binding system

Various conditions including HSA concentration, the pH value, the concentration of NaCl, the reaction temperature, and the mobile

Conclusion

In this study, an on-line HPLC–DAD–MSn–HSA–FLD system was established to screen haptens in SXNI for the first time. Using this system, 63 ingredients were identified in SXNI, flavonoids in which showed different HSA-binding activity. In fluorescence detection and ITC experiments, flavonoid aglycones had stronger HSA binding activity than their glycosides. The aglycones also displayed strong sensitization in RBL-2H3 cell allergic test and class-allergic test. In summary, the workflow of this

Acknowledgments

This work was supported by Nature Science Foundation of Beijing, China (Grant No.7142088).

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