Determination of Alkaloids and Flavonoids in Sophora flavescens by UHPLC-Q-TOF/MS

This study is based on UHPLC-Q-TOF/MS and fragment ions to achieve classification and identification of alkaloids and flavonoids in Sophora flavescens. By reviewing the available and relevant literature, the mass fragmentation rules of alkaloids and flavonoids were summarized. 0.1% formic acid water (A) and acetonitrile (B) were used as mobile phases. 37 chemical constituents were identified, including 13 alkaloids and 24 flavonoids. This research method offers a complete strategy based on the fragmentation information of characteristic fragment ions and neutral loss obtained by MS/MS to characterize the chemical composition of Sophora flavescens.


Introduction
e analytical methods of traditional Chinese medicine (TCM) are not sufficient for the separation and identification of many complex chemical components, which brings challenges in terms of the quality control and clinical application of TCM [1]. Ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) has become the main means of component analysis of modern traditional Chinese medicine because of its high speed, high efficiency, and high resolution. It can overcome the limitation of ultraviolet detectors, so it is suitable for component analysis in the complex traditional Chinese medicine system [2][3][4]. e chemical components in TCM can be classified and quickly identified on the basis of secondary fragments [5,6]. In the process of treating diseases, traditional Chinese medicine often has multiple components and multiple targets, which often lead to the problem of unclear components. erefore, the classification and identification of chemical components in traditional Chinese medicine are very meaningful. According to differences in the chemical structure, the compounds can be divided into different parent nuclear structure types. Compounds with the same parent nuclear type will produce some ion fragments which are the same in the process of mass spectrometry collision. e traditional Chinese medicinal herb Sophora flavescens comes from dried roots of Sophora flavescens Ait., a leguminous plant which is listed as middle grade in Shennong Materia Medica, and is bitter and cold in taste. Alkaloids and flavonoids are considered to be the main active components of Sophora flavescens [7]. Studies have shown that alkaloids in Sophora flavescens can reduce the secretion of inflammatory factor TNF-α by regulating the expression of BMP2, Runx2, and other proteins, so as to increase the activity of alkaline phosphatase to treat chronic osteomyelitis caused by Staphylococcus aureus infection [8]. Indoleamine 2-dioxygenase-1 (IDO1), a tumor cell survival factor, can lead to the escape of many kinds of cancer cells. As inhibitors of IDO1, many flavonoids in Sophora flavescens have potential uses in cancer immunotherapy [9]. In view of the good clinical efficacy and research prospects of Sophora flavescens, it is of great significance to establish a technique that can quickly classify and identify the chemical composition of Sophora flavescens.
Based on the UHPLC-Q-TOF/MS technology, this study summarized the characteristic fragments and neutral losses during the cleavage process of compounds, classified and identified the chemical components in Sophora flavescens, and identified 37 alkaloids and flavonoids in Sophora flavescens. 2.2. Preparation of Samples. 5.0 g of Sophora flavescens was precisely weighed, refluxed, and extracted twice with 8 times and 6 times of 70% ethanol for 2 hours each time. e combined extract was evaporated and concentrated to 0.1 g/ mL and then filtered by a 0.22 μm microporous membrane, which was the sample solution to be injected [10,11].

Materials and
1 mg of matrine, oxymatrine, and sophocarpine was precisely weighed. en, 1 ml of 70% ethanol was added to dissolve and passed through a 0.22 μm microporous filter membrane.

Establishment of the Method.
e mass spectrometry experimental data reported in the literature were used to summarize the fragments missing from the fragment ion peaks of known chemical components in Sophora flavescens and summarize the fragmentation rules of different fragment ions. Subsequently, MassLynx software was used for peak matching, and the chemical composition of Sophora flavescens was deduced based on the retention time of its components and the fragmentation rules. Finally, 13 alkaloids and 24 flavonoids were identified, as shown in Table 1.
e fragmentation rules of the chemical components in Sophora flavescens are shown in Figure 1, and the base peak ion (BPI) chromatogram of the Sophora flavescens extract in positive and negative ions is shown in Figure 2.

Fragmentation Rules of Alkaloid Compounds.
According to the structure type of the mother nucleus, the alkaloids in Sophora flavescens are mainly divided into matrine type, broom alkali type, anagyrine type, and lupine type [25]. Among them, matrine-type compounds easily lose H 2 O (18), C 5 H 7 NO (97), and C 5 H 9 NO (99) in the collision process, resulting in characteristic fragments of m/z 150 and m/z 148. Nitrogen oxides of matrine alkaloids easily lose H 2 O (18) and OH (17), resulting in high-abundance fragments [M+H-H 2 O] + and [M+H-OH] + [13,14]. e cleavage of C7-C13/C9-C11 and C6-C7/C1-C10 of broom alkaloid bonds will produce characteristic fragments such as 146 [M+H-C 3 H 9 N] + and 148[M+H-C 2 H 5 N] + which are related to the methyl substituents at position 12 [12].   [12] Journal of Analytical Methods in Chemistry 3  Journal of Analytical Methods in Chemistry   Based on the fragmentation rules and standard information, it can be inferred that the compound is sophocarpine. e fragmentation process is shown in Figure 4.

Fragmentation Rules of Flavonoid Compounds.
Flavonoids in Sophora flavescens mainly include dihydroflavonoids, chalcones, dihydroflavonols, flavonols, and isoflavones, in which dihydroflavonoids and chalcones are easy to change. erefore, mass spectrometry can well distinguish the two [26]. It is easy to remove neutral molecules from flavonoids such as H 2 O, CH 3 [12,22]. e main fragment of dihydroflavonols is that the C ring is rearranged by RDA to produce characteristic fragments such as 177[C 9 H 5 O 4 ] − , 275[C 17 H 23 O 3 ] − , 1,3 A − , and 1,3 B − , and the hydroxyl group at position 3 is unstable, so it is easy to eliminate the reaction and lose H 2 O to form a double bond. Flavonol compounds undergo RDA cleavage to produce characteristic fragments 1,3 A − and 1,3 B − and continue to lose neutral molecules such as CO (28) and          Figure 6: e fragmentation process of kushenol X in the negative ion mode. inferred that the compound is norkurarinone. e fragmentation process is shown in Figure 5. e molecular formula of compound 35 is C 25

Conclusion
e UHPLC-Q-TOF/MS technique combined with characteristic fragments and neutral loss was applied to the tracking and identification of alkaloids and flavonoids in Sophora flavescens, and the fragmentation rules of different parent ions were inferred. A total of 13 alkaloids and 24 flavonoids were identified. Analytical strategies for characterizing the structure of compounds by obtaining diagnostic fragment ions based on excimer ion peaks and MS/ MS were summarized.

Data Availability
No data were used to support this study.

Conflicts of Interest
e authors declare no conflicts of interest.