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

Yaqian Dong; Guoxiang Jia; Jingwen Hu; Hui Liu; Tingting Wu; Shenshen Yang; Yubo Li; Ting Cai

doi:10.1155/2021/9915027

. 2021 Jul 28;2021:9915027. doi: 10.1155/2021/9915027

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

Yaqian Dong ¹, Guoxiang Jia ¹, Jingwen Hu ¹, Hui Liu ¹, Tingting Wu ¹, Shenshen Yang ^1,^✉, Yubo Li ^1,^✉, Ting Cai ^2,^3,^✉

PMCID: PMC8337118 PMID: 34367714

Abstract

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.

1. Introduction

The 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 [2–4]. The 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. Therefore, 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.

The 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. Methods

2.1. Materials and Instruments

Sophora flavescens (Beijing Tongren Drug Store), matrine, oxymatrine, sophocarpine standard (Chengdu Ruifensi Biotechnology Co., Ltd., China), ethanol (Tianjin Huihang, analytical grade), acetonitrile (Sigma, USA, HPLC grade), formic acid (Sigma, USA, HPLC grade), distilled water (Guangzhou Watsons, China), UPLC-Q-TOF-MS (Waters, Milford, MA, USA), a Waters ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm), and MassLynx V4.1 were used.

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. The 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].

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

2.3. UHPLC and MS Conditions

UHPLC: Waters ACQUITY UHPLC BEH C18 Column, 2.1 × 100 mm, 1.7 μm; column temperature is set to 35°C; mobile phase: the aqueous phase is 0.1% formic acid aqueous solution (A), and the organic phase is acetonitrile solution (B); flow rate: 0.4 mL/min. The gradient elution method is used for chromatographic separation, and the gradient procedure is as follows: 0–10 min, 3–20% B; 10–15 min, 20–30% B; 15–20 min, 30–50% B; 20–25 min, 50–70% B; 25–27 min, 70–100% B; 27–30 min, 100% B; 30–32 min,100–3% B; 32–35 min, 3% B
TOF-MS: electrospray ionization source (ESI), scanning mode: positive and negative ions. The MS parameters are as follows: dry gas temperature: 325°C; dry gas flow rate: 11 ml/min; desolvent gas flow rate: 800 L/h; capillary voltage: 3.0 kV; collision-induced dissociation voltage: 6 kV; collision energy: 20–50 eV; atomizer pressure: 350 psi; auxiliary gas: N₂; positive and negative reference ion calibration ([M+H]⁺ = 556.2771, [M-H]⁻ = 554.2615) to ensure accuracy in spectral acquisition. The range of data acquisition is 50 to 1500.

3. Results and Discussion

3.1. Establishment of the Method

The 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. The 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.

Table 1.

Fragmentation information of Sophora flavescens in the positive and negative ion mode.

No.	Identity	Formula	RT	Theoretical value	Actual value	ppm	Main MS/MS fragments detected (arranged from large to small according to relative intensity)	Ref.
1	Lamprolobine	C₁₅H₂₄N₂O₂	0.72	265.1916	265.1926	3.77	265.1926[M+H]⁺ 263.1771[M+H-H₂]⁺ 150.1293[M+H-H₂O-C₅H₇NO]⁺	[12]

2	Matrine	C₁₅H₂₄N₂O	1.59	249.1967	249.1980	5.22	249.1980[M+H]⁺ 247.1815[M+H-H₂]⁺ 150.1298[M+H-H₂-C₅H₇NO]⁺ 148.1152[M+H-H₂-C₅H₉NO]⁺ 136.1144[M+H-H₂-C₆H₉NO]⁺ 176.1083[M+H-H₂-C₃H₅NO]⁺ 231.1959[M+H-H₂O]⁺	[13, 14]

3	7,11-Dehydromatrine	C₁₅H₂₂N₂O	1.93	247.1810	247.1821	4.45	247.1821[M+H]⁺ 148.1149[M+H-C₅H₉NO]⁺ 176.1066[M+H-C₃H₅NO]⁺	[12]

4	Sophocarpine	C₁₅H₂₂N₂O	1.93	247.1810	247.1821	4.45	247.1821[M+H]⁺ 136.1137[M+H C₄H₄O-C₂H₄NH]⁺ 245.1661[M+H-H₂]⁺ 150.1293[M+H-C₅H₇NO]⁺ 148.1149[M+H-C₅H₉NO]⁺ 179.1542[M+H-C₄H₄O]⁺ 108.0833[M+H-C₄H₄O-C₂H₄NH-C₂H₄]⁺	[14]

5	Sophoridine	C₁₅H₂₄N₂O	1.96	249.1967	249.1983	6.42	249.1983[M+H]⁺ 247.1827[M+H-H₂]⁺ 150.1294[M+H-H₂-C₅H₇NO]⁺ 148.1146[M+H-H₂-C₅H₉NO]⁺ 218.1494[M+H-CH₅N]⁺ 231.1847[M+H-H₂O]⁺	[14]

6	9α-Hydroxysophocarpine	C₁₅H₂₂N₂O₂	2.14	263.1760	263.1776	6.08	263.1776[M+H]⁺ 245.1667[M+H-H₂O]⁺ 150.1299[M+H-H₂O-C₂H₂O-CH₂-C₂HN]⁺ 203.1198[M+H-H₂O-C₂H₂O]⁺ 148.1139[M+H-H₂O-C₂H₂O-CH₂-C₂HN H₂]⁺ 175.1250[M+H-H₂O-C₂H₂O-CH₂-CH₂]⁺ 189.1109[M+H-H₂O-C₂H₂O-CH₂]⁺	[15]

7	Mamanine	C₁₅H₂₂N₂O₂	2.14	263.1760	263.1776	6.08	263.1776[M+H]⁺ 150.1299[M+H-H₂O-C₅H₅NO]⁺ 245.1667[M+H-H₂O]⁺	[12]

8	9α-Hydroxymatrine	C₁₅H₂₄N₂O₂	2.29	265.1916	265.1928	4.53	265.1928[M+H]⁺ 150.1294[M+H-H₂O C₅H₇NO]⁺ 247.1824[M+H-H₂O]⁺	[15]

9	Oxysophoridine	C₁₅H₂₄N₂O₂	2.33	265.1916	265.1933	6.41	265.1933[M+H]⁺ 247.1826[M+H-H₂O]⁺ 148.1142[M+H-H₂O-C₅H₉NO]⁺ 98.0987[M+H-H₂O-C₄H₁₀O₂]⁺ 112.0782[M+H-C₅H₇NO-C₂H₂NO]⁺ 168.1405[M+H-C₅H₇NO]⁺	[12]

10	Oxysophocarpine	C₁₅H₂₂N₂O₂	2.65	263.1760	263.1760	0.00	263.1760[M+H]⁺ 177.1402[M+H-H₂O-C₄H₄O]⁺ 245.1662[M+H-H₂O]⁺ 150.1289[M+H-C₆H₁₁NO]⁺ 148.1143[M+H-C₅H₉NO]⁺ 203.1214[M+H-H₂O-C₃H₆]⁺ 122.1008[M+H-H₂O-C₄H₄O-C₃H₄NO]⁺	[12]

11	Oxymatrine	C₁₅H₂₄N₂O₂	3.24	265.1916	265.1926	3.77	265.1926[M+H]⁺ 247.1823[M+H-H₂O] 205.1355[M+H-H₂O-C₂H₄N]+ 148.1139[M+H-H₂O-C₅H₉NO]+ 175.1254[M+H-C₄H₈O]+ 112.1129[M+H-C₅H₇NO-C₂H₂NO]+	[13, 14]

12	Sophoranol N-oxide	C₁₅H₂₄N₂O₃	4.68	281.1865	281.1879	4.98	281.1879[M+H]⁺ 245.1696[M+H-2H₂O]⁺ 138.1283[M+H-H₂O-C₄H₁₀NO₂]⁺ 263.1678[M+H-H₂O]⁺	[15]

13	Daidzin	C₂₁H₂₀O₉	6.19	417.1186	417.1184	−0.48	417.1184[M+H]⁺ 255.0589[M+H-Glu]⁺ 199.0759[M+H-Glu-2CO]⁺	[12, 16]

14	Baptifoline	C₁₅H₂₀N₂O₂	6.70	261.1603	261.1595	−3.06	243.1435[M+H-H₂O]⁺ 261.1595[M+H]⁺ 146.1015[M+H-C₆H₁₃NO]⁺ 96.0877[M+H-H₂O-C₉H₉NO]⁺ 114.0939[M+H-C₉H₉NO]⁺	[16]

15	Daidzein	C₁₅H₁₀O₄	11.03	255.0657	255.0690	12.94	255.0690[M+H-Glu]⁺ 137.0274[M+H-C₂O₃]⁺ 199.0759[M+H-Glu-2CO]⁺	[16]

16	Kurarinone	C₂₆H₃₀O₆	19.43	439.2121	439.2128	1.59	439.2128[M+H]⁺ 179.0361[^1,3A⁺-C₉H₁₆]⁺ 303.1609^1,3A⁺ 136.0179^1,3B⁺ (136.0179^{1, 3}B⁺ is the fragment ion produced by RDA fragmentation reaction) 421.2132[M+H-H₂O]⁺	[17, 18]

17	Stamens isoflavones	C₁₆H₁₂O₅	12.26	283.0606	283.0618	4.24	283.0616[M-H]⁻ 268.0373[M-H-CH₃]⁻ 211.0400[M-H-C₂O₃]⁻ 253.0472[M-H-3H₂O]⁻ 271.1014[M-H-H₂O]⁻	[12, 19]

18	(2R,3R)-8-Isopentenyl-7,4-dihydroxy-5-methoxy dihydroflavonol	C₂₁H₂₂O₆	16.04	369.1338	369.1349	2.98	369.1349[M-H]⁻ 313.0851[M-H-C₃H₄O]⁻ 353.9778[M-H-CH₃]⁻ 351.1122[M-H-H₂O]⁻	[12]

19	Formononetin	C₁₆H₁₂O₄	16.29	267.0657	267.0669	4.49	267.0669[M-H]⁻ 252.0430[M-H-CH₃]⁻ 223.0404[M-H-CO₂]⁻	[20]

20	2′-Hydroxy-isoxanthohumol	C₂₁H₂₂O₆	16.58	369.1338	369.1346	2.17	369.1346[M-H]⁻ 207.1024[M-H-H₂O-C₈H₁₆O₂]⁻ 341.1375[M-H-CO]⁻ 351.1252[M-H-H₂O]⁻ 354.4461[M-H-CH₃]⁻	[12]

21	Kuraridinol	C₂₆H₃₂O₇	16.68	455.2070	455.2076	1.32	455.2076[M-H]⁻ 161.0246[C₉H₅O₃]⁻ 293.1761^1,4A⁻ 437.1793[M-H-H₂O]⁻	[19, 21]

22	Leachianone G	C₂₀H₂₀O₆	17.37	355.1182	355.1202	5.63	355.1202[M-H]⁻ 161.0214[C₉H₅O₃]⁻ 337.1068[M-H-H₂O]⁻ 235.1366^1,3A⁻	[17]

23	Norkurarinol	C₂₅H₃₀O₇	18.24	441.1913	441.1928	3.40	279.1612^1,3A⁻ 441.1928[M-H]⁻ 161.0251[C₉H₅O₃]⁻ 211.1704[^1,3A⁻-C₃O₂]⁻ 162.0280^1,3B⁻ 423.1707[M-H-H₂O]⁻	[21]

24	Kushenol Q	C₂₅H₂₈O₁₁	18.39	441.1913	441.1935	4.99	279.1617^1,3A⁻441.1935[M-H]⁻ 161.0255[C₉H₅O₃]⁻ 211.1747[^1,3A⁻-C₃O₂]⁻ 331.1563[M-H-C₈H₁₄]⁻	[22]

25	Maackiain	C₁₆H₁₂O₅	18.48	283.0606	283.0598	−2.83	283.0598[M-H]⁻ 268.0368[M-H-CH₃]⁻ 211.0394[M-H-C₂O₃]⁻ 227.0754[M-H-2CO]⁻ 255.0628[M-H-CO]⁻	[16]

26	Kushenol N	C₂₆H₃₀O₇	18.76	453.1913	453.1913	0.00	453.1913[M-H]⁻ 177.0195[C₉H₅O₄]^−1,4B⁻ 275.1653[C₁₇H₂₃O₃]^−1,4A⁻ 149.0249[^1,4B⁻-CO]⁻ 137.0254[^1,4A−-C₉H₁₅-CH₃]⁻	[23]

27	Kushenol I	C₂₆H₃₀O₇	19.04	453.1913	453.1908	−1.10	453.1908[M-H]⁻ 177.0191[C₉H₅O₄]^−1,4B⁻ 149.0256[^1,4B⁻-CO]⁻ 435.1866[M-H-H₂O]⁻ 425.2036[M-H-CO]⁻	[16]

28	Sophoraflavanone B	C₂₀H₂₀O₅	19.08	339.1232	339.1231	−0.29	219.0664^1,4A⁻ 339.1231[M-H]⁻ 119.0504[^1,4A-C₇H₁₆]⁻ 275.1648[M-H-C₄H₄O]⁻ 321.9993[M-H-H₂O]⁻	[16]

29	Noranhydroicaritin	C₂₀H₁₈O₆	19.17	353.1025	353.1034	2.55	353.1034[M-H]⁻ 298.0487[M-H-C₄H₇]⁻ 136.0176[M-H-C₄H₇-C₉H₂₂O₂]⁻ 338.0812[M-H-CH₃]⁻ 161.0250[C₉H₅O₃]⁻	[24]

30	Kushenol L	C₂₅H₂₈O₇	19.43	439.1757	439.1760	0.68	439.1760[M-H]⁻ 275.1653[C₁₇H₂₃O₃]⁻ 177.0201[C₉H₅O₄]⁻ 149.0247[M-H-C₉H₁₄O]⁻ 421.1667[M-H-H₂O]⁻	[24]

31	Sophoraisoflavanone A	C₂₁H₂₂O₆	20.18	369.1338	369.1360	5.96	161.0255^1,4B⁻ 369.1360[M-H]⁻ 135.0452[^1,4B⁻-CH₂]⁻ 275.1674[M-H-C₈H₁₀]⁻ 208.1087^1,4A⁻	[12]

32	8-Lavandulylkaempferol	C₂₆H₃₀O₅	20.39	421.2015	421.2027	2.85	421.2027[M-H]⁻ 301.1454^1,4A⁻ 119.0511[^1,4A⁻-C₉H₁₄O-C₃H₈]⁻ 163.0040[^1,4A⁻-C₉H₁₄O]⁻	[12, 16]

33	Kushenol D	C₂₇H₃₂O₆	20.56	451.2121	451.2118	−0.66	451.2118[M-H]⁻ 301.1442^1,4A⁻ 149.0617^1,4B⁻ 217.0517[^1,4A⁻-C₆H₁₂]⁻ 419.1871[M-H-C₂O₂]⁻	[24]

34	Norkurarinone	C₂₅H₂₈O₆	20.62	423.1808	423.1805	−0.71	423.1805[M-H]⁻ 161.0249^1,4B⁻ 262.1535^1,4A⁻ 138.0323[^1,4A⁻-C₉H₁₅]⁻ 193.1601[M-H-H₂O-C₁₅H₁₇O]⁻ 405.1707[M-H-H₂O]⁻	[17]

35	Kushenol X	C₂₅H₂₈O₇	21.15	439.1757	439.1755	−0.46	261.1497[M-H-H₂O-ringB-C₁₀H₁₅]⁻ 177.0191[M-H-H₂O-ringB-C₁₀H₁₅]⁻ 149.0251[M-H-C₉H₁₄O]⁻ 439.1755[M-H]⁻ 421.1631[M-H-H₂O]⁻ 287.1290^1,3A⁻ 109.0298[M-H-H₂O-ringB-C₁₀H₁₅-C₄H₇]⁻ 152.0812^1,3B⁻	[12, 22]

36	Kuraridin	C₂₆H₃₀O₆	23.04	437.1964	437.1984	4.57	161.0258^1,4B⁻ 275.1667^1,4A⁻ 437.1984[M-H]⁻ 151.0412[^1,4A⁻-C₉H₁₆]⁻	[23]

37	5-Methylkushenol C	C₂₇H₃₂O₆	24.61	451.2121	451.2141	4.43	451.2141[M-H]⁻ 301.1473^1,3A⁻ 192.0471[^1,3A⁻-C₈H₁₃]⁻ 313.0871[M-H-C₆H₁₂-C₄H₆]⁻ 367.1223[M-H-C₆H₁₂]⁻	[12, 21]

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Characteristic fragments and neutral loss of the chemical composition of *Sophora flavescens*.

The base peak ion (BPI) flow diagram of *Sophora flavescens* in the (a) positive and (b) negative mode.

3.2. 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 H₂O (18), C₅H₇NO (97), and C₅H₉NO (99) in the collision process, resulting in characteristic fragments of m/z 150 and m/z 148. Nitrogen oxides of matrine alkaloids easily lose H₂O (18) and OH (17), resulting in high-abundance fragments [M+H-H₂O]⁺ and [M+H-OH]⁺ [13, 14]. The cleavage of C7-C13/C9-C11 and C6-C7/C1-C10 of broom alkaloid bonds will produce characteristic fragments such as 146[M+H-C₃H₉N]⁺ and 148[M+H-C₂H₅N]⁺ which are related to the methyl substituents at position 12 [12]. The characteristic fragments of daidzein alkaloids include 243[M+H-H₂O]⁺, 205 [M+H-H₂O-C₃H₄]⁺, 123[M+H-C₈H₁₄N₂]⁺, and 114[M+H-C₉H₈NO]⁺ [17].

The molecular formula of compound 2 is C₁₅H₂₄N₂O, and the retention time is 1.59 min. The main secondary fragments are 247.1815, 231.1959, 176.1083, 150.1298, 148.1152, and 136.1144. In the positive ion mode, the molecular ion peak is m/z 249.1980[M+H]⁺, and its parent ion removes a molecule of H₂ and H₂O, respectively, resulting in an ion peak of m/z 247.1815[M+H-H₂]⁺and a dehydration peak of m/z 231.1959[M+H-H₂O]⁺. Then, the compound undergoes RDA cleavage. On this basis, characteristic matrine-type ion fragments are m/z 150.1298[M+H-H₂-C₅H₇NO]⁺, m/z 148.1152[M+H-H₂-C₅H₉NO]⁺, and m/z 136.1144 [M+H-H₂-C₆H₉NO]⁺. On the basis of losing a molecule of H₂, the compound can continue to lose a molecule of C₃H₅NO and form an ion peak of m/z 176.1083[M+H-H₂-C₃H₅NO]⁺. According to fragment information, standard reference substance retention time, relative molecular mass, and MS and MS information, the compound is identified as matrine. The fragmentation rules are shown in Figure 3.

The fragmentation process of matrine in the positive ion mode.

The molecular formula of compound 4 is C₁₅H₂₂N₂O, and the retention time is 1.93 min. In the positive ion mode, the molecular ion peak is 247.1821[M+H]⁺, and the main secondary fragments are 245.1661, 179.1542, 150.1293, 148.1149, 136.1137, and 108.0833. It is conjectured that the fragmentation process of compound 4 is as follows. Firstly, the parent ions lose a molecule of C₅H₇NO (97) and C₅H₉NO (99) to produce the characteristic ion fragments of matrine type: m/z 150.1293[M+H-H₂-C₅H₇NO]⁺ and m/z 148.1149[M+H-H₂-C₅H₉NO]⁺. Secondly, the parent ion can lose a molecule of C₄H₄O resulting in the fragment 179.1542[M+H-C₄H₄O]⁺ and then directly lose a molecule of C₂H₄NO or lose C₂H₄ to yield 136.1137[M+H-C₄H₄O-C₂H₄NO]⁺ and m/z 108.0833[M+H-C₄H₄O-C₂H₄NO-C₂H₄]⁺ ion fragments. The fragment ion 245.1661 is obtained by direct loss of a molecule of H₂ by the parent ion. Based on the fragmentation rules and standard information, it can be inferred that the compound is sophocarpine. The fragmentation process is shown in Figure 4.

The fragmentation process of sophocarpine in the positive ion mode.

3.3. 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. Therefore, mass spectrometry can well distinguish the two [26]. It is easy to remove neutral molecules from flavonoids such as H₂O, CH₃, CO, CO₂, C₂H₂O, and C₂O₃ in the negative ion mode. Most of the dihydroflavonoids in Sophora flavescens have fragment information such as [M-H]⁻, [M-H-H₂O]⁻, [M-H-CO]⁻, and [M-H-CH₃]⁻, and these compounds are prone to RDA cleavage at positions 1,2 and 3,4, resulting in ^1,3A⁻fragment ions. Chalcone compounds form 261[C₁₆H₂₁O₃]⁻ and 161[C₉H₅O₃]⁻ ion fragments under the anion mode B ring, and the 1,4 cleavage occurs in different positions of dihydroflavonoids, resulting in ^1,4A⁻ and B^1,4− characteristic fragment ions [12, 22]. The main fragment of dihydroflavonols is that the C ring is rearranged by RDA to produce characteristic fragments such as 177[C₉H₅O₄]⁻, 275[C₁₇H₂₃O₃]⁻, ^1,3A⁻, and ^1,3B⁻, and the hydroxyl group at position 3 is unstable, so it is easy to eliminate the reaction and lose H₂O to form a double bond. Flavonol compounds undergo RDA cleavage to produce characteristic fragments ^1,3A⁻ and ^1,3B⁻ and continue to lose neutral molecules such as CO (28) and CO₂ (44). Isoflavones easily lose neutral molecules such as CO, 2CO, CO₂, and C₂O₃ [23]. Based on the above mass spectrometry information combined with retention time, the chemical constituents of flavonoids in Sophora flavescens were identified quickly.

The molecular formula of compound 34 is C₂₆H₂₈O₆, and the retention time is 20.62 min. The main secondary fragment ions are 405.1707, 262.15351, 193.1601, 161.0249, and 138.0323. In the negative ion mode, the parent ion peak is m/z 423.1805[M-H]⁻. The parent ion 2′-OH is chemically active, which means that it easily loses a molecule of H₂O and produces dehydrated fragments m/z 405.1707[M-H-H₂O]⁻. On this basis, the neutral losing fragment C₁₅H₁₇O is lost, and the ion fragment m/z 193.1601[M-H-H₂O-C₁₅H₁₇O]⁻ is obtained. Due to the presence of 2′-OH, the compound does not easily produce RDA cleavage, but breaks at the 1′4 position of the C ring, resulting in ion fragments ^1,4A m/z 261.1501 and ^1,4B m/z 162.0281, and then the ion fragments m/z 138.0323[^1,4A-C₉H₁₅]⁻ are obtained when C₉H₁₅ is lost in ^1,4A. Based on the above law, it is inferred that the compound is norkurarinone. The fragmentation process is shown in Figure 5.

The fragmentation process of norkurarinone in the negative ion mode.

The molecular formula of compound 35 is C₂₅H₂₈O₇, the retention time is 21.15, and the main secondary fragment ions are 421.1631, 287.12901, 261.1497, 177.0191, 152.08121, 149.0251, and 109.0298. In the negative ion mode, the precursor ion peak is m/z 439.1761[M-H]⁻, and the precursor ion removes one molecule of H₂O to obtain m/z 421.1631[M-H-H₂O]⁻ ion fragment. After that, the 1,4 positions of the C ring break off one molecule of C₉H₆O₃ to obtain the ion fragment 261.1497[M-H-H₂O-C₉H₆O₃]⁻. In addition, the parent ion of the compound can also directly lose one molecule of C₁₆H₂₀O₂, generating ion fragments of m/z 177.0197[M-H-H₂O-ringB-C₁₀H₁₅]⁻, on the basis of which another molecule of C₄H₇ is lost, and m/z 109.0298[M-H-H₂O-ringB-C₁₀H₁₅-C₄H₇]⁻. In the case of RDA rearrangement of this compound, m/z 287.1290 ^1,3A⁻ and m/z 152.0812 ^1,3B⁻ ion fragments can be generated, and then the characteristic fragment ^1,3A⁻ loses C₉H₁₄O to produce m/z 149.0251[^1,3A⁻-C₉H₁₄O]⁻. Based on the above fragmentation rules, it can be inferred that this compound is kushenol X. The fragmentation process is shown in Figure 6.

The fragmentation process of kushenol X in the negative ion mode.

4. Conclusion

The 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.

Acknowledgments

This work was funded by the National Natural Science Foundation of China (no. 81903933), the Tianjin Science and Technology Development Fund Project (2018KJ002), and the Tianjin Talent Development Special Support Project for High-Level Innovation and Entrepreneurship.

Contributor Information

Shenshen Yang, Email: shine2099@163.com.

Yubo Li, Email: yaowufenxi001@sina.com.

Ting Cai, Email: caiting@ucas.ac.cn.

Data Availability

No data were used to support this study.

Conflicts of Interest

The authors declare no conflicts of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

No data were used to support this study.

[B1] 1.He X.-R., Li C.-G., Zhu X.-S., et al. High-performance liquid chromatography coupled with tandem mass spectrometry technology in the analysis of Chinese medicine formulas: a bibliometric analysis (1997–2015) Journal of Separation Science. 2017;40(1):81–92. doi: 10.1002/jssc.201600784. [DOI] [PubMed] [Google Scholar]

[B2] 2.Yang S., Shan L., Luo H., Sheng X., Du J., Li Y. Rapid classification and identification of chemical components of schisandra chinensis by UPLC-Q-TOF/MS combined with data post-processing. Molecules. 2017;22(10):p. 1778. doi: 10.3390/molecules22101778. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3.Jones E. E., Zhang W., Zhao X., et al. Tissue localization of glycosphingolipid accumulation in a gaucher disease mouse brain by LC-ESI-MS/MS and high-resolution MALDI imaging mass spectrometry. SLAS DISCOVERY: Advancing the Science of Drug Discovery. 2017;22(10):1218–1228. doi: 10.1177/2472555217719372. [DOI] [PubMed] [Google Scholar]

[B4] 4.Gu W.-Y., Li N., Leung E. L.-H., et al. Rapid identification of new minor chemical constituents from smilacis glabrae rhizoma by combined use of UHPLC-Q-TOF-MS, preparative HPLC and UHPLC-SPE-NMR-MS techniques. Phytochemical Analysis. 2015;26(6):428–435. doi: 10.1002/pca.2577. [DOI] [PubMed] [Google Scholar]

[B5] 5.Liu P., Zhao P., Cooks R. G., Chen H. Atmospheric pressure neutral reionization mass spectrometry for structural analysis. Chemical Science. 2017;8(9):6499–6507. doi: 10.1039/c7sc01999h. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6.Yu C., Xu Y., Wang M., Xie Z., Gao X. Application of characteristic fragment filtering with ultra high performance liquid chromatography coupled with high‐resolution mass spectrometry for comprehensive identification of components in Schisandrae chinensis fructus. Journal of Separation Science. 2019;42(7):1323–1331. doi: 10.1002/jssc.201801203. [DOI] [PubMed] [Google Scholar]

[B7] 7.Fang Y. G., Zhu H. D., Liu X. Q., et al. Revision research on the quality standard of matrine and decoction pieces. Chinese Journal of Traditional Chinese Medicine. 2020;8(1):1–9. in Chinese. [Google Scholar]

[B8] 8.Wang X., Zheng R., Huang X., et al. Effects of alkaloids from Sophora flavescens on osteoblasts infected with Staphylococcus aureus and osteoclasts. Phytotherapy Research. 2018;32(4):1354–1363. doi: 10.1002/ptr.6069. [DOI] [PubMed] [Google Scholar]

[B9] 9.Kwon M., Ko S.-K., Jang M., et al. Inhibitory effects of flavonoids isolated from Sophora flavescens on indoleamine 2,3-dioxygenase 1 activity. Journal of Enzyme Inhibition and Medicinal Chemistry. 2019;34(1):1481–1488. doi: 10.1080/14756366.2019.1640218. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10.Yu Q., Cheng N., Ni X. Identifying 2 prenylflavanones as potential hepatotoxic compounds in the ethanol extract of Sophora flavescens. Journal of Food Science. 2013;78(11):T1830–T1834. doi: 10.1111/1750-3841.12275. [DOI] [PubMed] [Google Scholar]

[B11] 11.Wu G. Optimization of the Extraction Process of a Compound Chinese Medicine and Preparation of Granules. Mishan, China: Heilongjiang Bayi Land Reclamation University; 2014. [Google Scholar]

[B12] 12.Li X. N., Dong X., Bao B. Q., et al. Quick analysis and identification of the chemical constituents of Sophora flavescens based on Q-exactive high-resolution mass spectrometry. Chinese Medicinal Materials. 2019;42(1):103–109. (in Chinese) [Google Scholar]

[B13] 13.Sabatino L., Scarangella M., Lazzaro F., et al. Matrine and oxymatrine in corroborant plant extracts and fertilizers: HPLC/MS-MS method development and single-laboratory validation. Journal of Environmental Science and Health, Part B. 2015;50(12):862–870. doi: 10.1080/03601234.2015.1062656. [DOI] [PubMed] [Google Scholar]

[B14] 14.Wu Z. J., Sun D.-M., Fang D.-M., Chen J.-Z., Cheng P., Zhang G.-L. Analysis of matrine-type alkaloids using ESI-QTOF. International Journal of Mass Spectrometry. 2013;341-342:28–33. doi: 10.1016/j.ijms.2013.03.002. [DOI] [Google Scholar]

[B15] 15.Zeng Z., Guo Z., Peng B., et al. Comparative study on the alkaloids of Sophora flavescens and Sophora flavescens by UPLC/Q-TOF MS∼E. Natural Product Research and Development. 2015;27(5):804–808. [Google Scholar]

[B16] 16.Li X., Dong X., Li N., et al. Quick analysis and identification of chemical constituents of siwei tumuxiang powder by HPLC-Q-exactive-MS/MS high resolution mass spectrometry. Chinese Journal of Experimental Traditional Medical Formulae. 2020;26(6):121–131. [Google Scholar]

[B17] 17.Yang Z., Zhang W., Li X., Shan B., Liu J., Deng W. Determination of sophoraflavanone G and kurarinone in rat plasma by UHPLC-MS/MS and its application to a pharmacokinetic study. Journal of Separation Science. 2016;39(22):4344–4353. doi: 10.1002/jssc.201600681. [DOI] [PubMed] [Google Scholar]

[B18] 18.Zhang L., Xu L., Xiao S.-S., et al. Characterization of flavonoids in the extract of Sophora flavescens ait. by high-performance liquid chromatography coupled with diode-array detector and electrospray ionization mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis. 2007;44(5):1019–1028. doi: 10.1016/j.jpba.2007.04.019. [DOI] [PubMed] [Google Scholar]

[B19] 19.Yang W.-Z., Ye G., Meng A.-H., et al. Rapid characterisation of flavonoids from Sophora alopecuroides L. by HPLC/DAD/ESI-MSn. Natural Product Research. 2013;27(4-5):323–330. doi: 10.1080/14786419.2012.688052. [DOI] [PubMed] [Google Scholar]

[B20] 20.Guo P., Dong L., Yan W., Wei J., Wang C., Zhang Z. Simultaneous determination of linarin, naringenin and formononetin in rat plasma by LC-MS/MS and its application to a pharmacokinetic study after oral administration of bushen guchi pill. Biomedical Chromatography. 2015;29(2):246–253. doi: 10.1002/bmc.3267. [DOI] [PubMed] [Google Scholar]

[B21] 21.Fabre N., Rustan I., Hoffmann E., Quetin-Leclercq J. Determination of flavone, flavonol, and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass spectrometry. Journal of the American Society for Mass Spectrometry. 2001;12(6):707–715. doi: 10.1016/S1044-0305(01)00226-4. [DOI] [PubMed] [Google Scholar]

[B22] 22.Qi J., Xu D., Zhou Y.-F., Qin M.-J., Yu B.-Y. New features on the fragmentation patterns of homoisoflavonoids in Ophiopogon japonicus by high-performance liquid chromatography/diode-array detection/electrospray ionization with multi-stage tandem mass spectrometry. Rapid Communications in Mass Spectrometry. 2010;24(15):2193–2206. doi: 10.1002/rcm.4608. [DOI] [PubMed] [Google Scholar]

[B23] 23.Liu Y., Chen L., Cai W., Zhao L.-L., Mo Z.-X. Use of an UHPLC-MS/MS method for determination of kuraridin and characterization of its metabolites in rat plasma after oral administration. Molecules. 2018;23(2):p. 132. doi: 10.3390/molecules23020132. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24.Zhao F. Study on the Difference of Chemical Composition and Content in Different Parts of Sophora flavescens. Beijing, China: China Academy of Chinese Medical Sciences; 2015. [Google Scholar]

[B25] 25.Rashid H. U., Xu Y., Muhammad Y., Wang L., Jiang J. Research advances on anticancer activities of matrine and its derivatives: an updated overview. European Journal of Medicinal Chemistry. 2019;161:205–238. doi: 10.1016/j.ejmech.2018.10.037. [DOI] [PubMed] [Google Scholar]

[B26] 26.Weng Z., Zeng F., Zhu Z., et al. Comparative analysis of sixteen flavonoids from different parts of Sophora flavescens ait. by ultra high-performance liquid chromatography-tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis. 2018;156:214–220. doi: 10.1016/j.jpba.2018.04.046. [DOI] [PubMed] [Google Scholar]

PERMALINK

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

Yaqian Dong

Guoxiang Jia

Jingwen Hu

Hui Liu

Tingting Wu

Shenshen Yang

Yubo Li

Ting Cai

Abstract

1. Introduction