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Qualitative and quantitative analysis of multi-components in Xing-Su-Ning Capsules for quality improvement
⁎Corresponding authors at: Tianjin University of Traditional Chinese Medicine, 10 Poyanghu Road, West Area, Tuanbo New Town, Jinghai District, Tianjin 301617, China. renming2008@126.com (Ming Ren), miaomiaojiang@126.com (Miaomiao Jiang)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Background
Xin-Su-Ning Capsules (XSNC) is an effective prescription for the treatment of arrhythmia composed of eleven Chinese herbs. With the wide application of XSNC in clinic, its quality control issues have also received increasing attention. Based on the multi-components characteristics of Chinese herbal compound, there is an urgent need to establish a quality evaluation system.
Methods
Gas chromatography-mass spectrometry (GC-MS) and ultra high-performance liquid chromatography quadrupole electrostatic orbitrap high resolution mass spectrometry (UHPLC-Q-Exactive-Orbitrap-MS) were performed to identify the preliminary chemical profile of XSNC. Subsequently, a rapid ultra high-performance liquid chromatography coupled with electrospray ionization triple-quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) method was developed to evaluate the quality of XSNC through a simultaneous determination of 16 components.
Results
A total of 21 volatile components and 59 non-volatile compounds were tentatively identified from the XSNC, each identified compound is marked on the corresponding chromatogram. Moreover, sixteen chemical constituents (sophocarpine, matrine, febrifugine, berberine, palmatine, Tangeratin, nobiletin, liensinine, neferine, scopoletin, isoliquiritigenin, liquiritigenin, naringenin, naringin, hesperidin and glycyrrhizic acid) were quantified by the developed UHPLC-QQQ-MS/MS method. The method validation of the sixteen compounds was performed with acceptable linearity (R2, 0.9990-1.0000), precision (RSD, 0.25-2.06%), repeatability (RSD, 0.93-2.90%) and recovery (99.65%-104.03%, RSD≤4.35%).
Conclusions
This qualitative analysis method sensitive and reliable for searching the volatile and non-volatile compounds from XSNC. The linearity, accuracy and precision of the quantitative analysis method were satisfactory. It is proposed that the methods described here can be applied for rapid evaluation, quality control and authenticity establishment of XSNC.
Keywords
Xing-Su-Ning Capsules
Constituent identification
Quantitative analysis
UHPLC-Q-Exactive-Orbitrap-MS
UHPLC-QQQ-MS/MS
- ESI
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electrospray ionization
- GC-MS
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gas chromatography-mass spectrometry
- TCM
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Traditional Chinese medicine
- UHPLC-Q-Exactive-Orbitrap-MS
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ultra performance liquid chromatography tandem quadrupole orbitrap mass spectrometer
- UHPLC-QQQ-MS/MS
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ultra high-performance liquid chromatography coupled with electrospray ionization triple-quadrupole mass spectrometry
- XSNC
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Xin-Su-Ning Capsules
Abbreviations
1 Introduction
Chinese herbal compound has definite curative effect and low side effects. It is the main drug for the clinical treatment of some complex diseases, chronic diseases and other diseases (Li and Du, 2015). Arrhythmia is a common and extremely dangerous cardiovascular diseases, it can not only aggravate the pre-existing heart disease, but also cause sudden death of patients (James and Calkins, 2016; Sossalla and Vollmann, 2018) With the limitation of anti-arrhythmic effect of chemical drugs, Chinese herbal compound represented by Xin-Su-Ning capsules (XSNC) has been more and more recognized in clinical practice because of its remarkable anti-arrhythmic effect in terms of multi-ion channel block and non-ion channel regulation(Li et al., 2019; Sun, 2017; Yao and Fang, 2017). XSNC consists of 11 Chinese herbs, including Coptidis Rhizoma, Pinelliae Rhizoma, Poria, Aurantii fructus Immaturus, Dichroae Radix, Nelumbinis Plumula, Sophorae flavescentis Radix, Artemisiae annuae Herba, Ginseng Radix et Rhizoma, Ophiopogonis Radix, Glycyrrhizae Radix et Rhizoma, it is a good prescription for the treatment of phlegm heat disturbance arrhythmia (Ma et al., 2006). Evidence-based medicine studies carried out from 2014 to 2017 have confirmed that XSNC has a definite clinical effect in treating cardiac arrhythmias caused by phlegm-heat (Zhaiet al., 2017). However, its chemical composition and quality control research are not in-depth, according to the instruction of Committee for the Pharmacopoeia of China in 2015, only berberine was indicated to be a index of XSNC for qualitative identification and quantitative analysis. In view of the complexity of chemical components of traditional Chinese medicine (TCM), its single qualitative and quantitative index is not enough to show the overall quality information of TCM, and it is difficult to fully reflect the effectiveness and safety of TCM. As a result, the comprehensive identification method of chemical composition is of great significance to the research of chemical composition of TCM. A new analytical method is needed to quantitatively determine various active components in XSNC.
In recent years, with the development of analytical technology, ultra high-performance liquid chromatography quadrupole electrostatic orbitrap high resolution mass spectrometry (UHPLC-Q-Exactive-Orbitrap-MS) is widely used in the analysis of TCM and compound prescription due to its fast separation speed, high sensitivity and strong determination accuracy (Eliuk and Makarov, 2015; Yang et al., 2020). Ultra high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) could provide simultaneous quantification of multiple components in the analysis of TCM (Liu et al., 2017, 2017; He et al., 2015).
To achieve the comprehensive chemical characterization of XSNC, we developed methods based on GC-MS and LC-MS to effectively analyze the chemical composition of XSNC. Then, an approach based on UHPLC-QQQ-MS/MS was developed to investigate the content of multi-components in XSNC. The quality of XSNC was comprehensively evaluated by quantifying the content of 16 compounds, which provided a reference for its quality evaluation, and laid a foundation for the later-stage drug-effective material basic research and clinical application.
2 Materials and methods
2.1 Materials and reagents
Methanol and acetonitrile (chromatographic purity) was purchased from Fisher company (USA), formic acid (MS grade) was purchased from ACS company (USA), and distilled water was purchased from Guangzhou Watsonsfood and beverage company (Guangzhou, China). Reference standards of sophocarpine, matrine, febrifugine, berberine, palmatine, Tangeratin, nobiletin, liensinine, neferine, scopoletin, isoliquiritigenin, liquiritigenin, naringenin, naringin, hesperidin and glycyrrhizic acid, jatrorrhizine were purchased from Sichuan Weikeqi Biotechnology Co., Ltd. or Shanghai Yuanye Biotechnology Co., Ltd. XSNC were supplied by Shanxi Momentum Pharmaceutical Co., Ltd (Shanxi, China).
2.2 Sample solutions preparation
XSNC were completely removed the capsule and weighed 1.77 g of the powder precisely. The powder was placed in a headspace sample bottle and sealed with an aluminum cap. Then, it was injected in the headspace sampler for GC-MS analysis.
The contents of XSNC were extracted by cold leaching with methanol for 24 h and repeated three times, then ultrasonically extracted for three times with methanol, the extracts was combined, concentrated and freeze-dried to obtain freeze-dried powder of non-volatile components. The lyophilized powder was weighed and dissolved in methanol a concentration of 5 mg/mL, then the solution was centrifuged at about 14000 rpm for 10 min. The supernatant was filtered through a 0.22 μm syringe filter and the filtrate was stored at 4 ◦C ready for UHPLC-ESI-Q-Exactive-Orbitrap-MS qualitative analysis.
A total of 0.2 g powder of XSNC was accurately weighed, ultrasound (25 kHz, 35 ◦C, 300 W) for 30 min with 20 mL methanol room temperature. After cooling down, the lost volume of methanol was complemented. Then the extracted solution was centrifuged at 14000 rpm for 10 min and the supernatant was taken. The supernatant 1 mL was accurately measure, diluted it with methanol and constant volume to obtain a test solution diluted 500 times with XSNC extract. The solution was filtered through a 0.22 μm syringe filter and the filtrate was stored at 4 ◦C ready for UHPLC-QQQ-MS/MS quantitative analysis.
2.3 Standard solutions preparation
The standards for matrine, sophocarpine, jatrorrhizine, palmatine, berberine, liquiritigenin, isoliquiritigenin, Tangeratin, nobiletin, and scopoletin were weighed accurately and dissolved in methanol for preparation of 1 mg/mL single reference solutions. Each reference solution was drew 0.1 mL and obtained a 100 µg/mL mixed standard solution. The solutions were filtered with 0.22 μm syringe filters before UHPLC-ESI-Q-Exactive-Orbitrap analysis.
The standards for sophocarpine, matrine, febrifugine, berberine, palmatine, Tangeratin, nobiletin, liensinine, neferine, scopoletin, isoliquiritigenin, liquiritigenin, naringenin, naringin, hesperidin and glycyrrhizic acid were weighed accurately and dissolved in methanol for preparation of single reference substance mother solution. The concentrations of reference substance mother solutions were as follows: sophocarpine 20 µg/mL, matrine 20 µg/mL, febrifugine 1 µg/mL, berberine 20 µg/mL, palmatine 20 µg/mL, Tangeratin 1 µg/mL, nobiletin 1 µg/mL, liensinine 2 µg/mL, neferine 10 µg/mL, scopoletin 5 µg/mL, isoliquiritigenin 2 µg/mL, liquiritigenin 2 µg/mL, naringenin 10 µg/mL, naringin 300 µg/mL, hesperidin 20 µg/mL, and glycyrrhizic acid 40 µg/mL.
Preparation of the standard curve: The above-mentioned reference substance mother solution was taken 50 µL each and the volume was made up to 1 mL with methanol. It contains sophocarpine 1 µg/mL, matrine 1 µg/mL, and febrifugine 50 ng/mL, berberine 1 µg/mL, palmatine 1 µg/mL, Tangeratin 50 ng/mL, nobiletin 50 ng/mL, liensinine 100 ng/mL, neferine 500 ng/ mL, scopoletin 250 ng/mL, isoliquiritigenin 100 ng/mL, liquiritigenin 100 ng/mL, naringenin 500 ng/mL, naringin 15 µg/mL, hesperidin 1 µg/mL and glycyrrhizic acid 2 µg/mL the highest concentration of mixed standard solution. The highest concentration of mixed reference solution was diluted with methanol 1:1 (v: v) by 2, 4, 8, 16, 32, 64 times to obtain a series of mixed reference solution.
2.4 GC-MS analysis
Chromatographic analysis was performed on an Agilent 7890B gas chromatograph (American, Agilent). A HP-5 MS quartz capillary column was used for chromatographic separation. Injections were performed in a split mode (ratio 5:1). High-purity nitrogen was used as a carrier gas and injector temperature was 240 ◦C. The initial column temperature was maintained at 50 ◦C for 2 min, then raised to 200 ◦C at a rate of 4 ◦C/min and held isothermally for 2 min. The column flow was 10 ml/min. Mass spectrometry analysis was performed on an Agilent 5977B mass spectrometer (American, Agilent). EI ionization method was adopted; the ion source temperature and quadrupole temperature were 230 ◦C and 150 ◦C respectively; the full scan mode range m/z 40∼400.
2.5 UHPLC-Q-exactive-orbitrap-MS qualitative analysis
Chromatographic analysis was performed on a Thermo Scientific UltiMate 3000 Ultra Performance Liquid Chromatograph (Thermo Fisher Scientific, USA); A Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1×100 mm) maintained at 35 ◦C was used for chromatographic separation. The mobile phase consisted of water acidified with 0.1 % (v/v) formic acid (A) and acetonitrile (B), was delivered at flow rate of 0.2 mL/min using the following gradient program: 0-2 min, 5-10% B; 2-5 min, 10-15% B; 5-10 min, 15-25% B; 10-15 min, 25-30% B; 15-20 min, 30-45%; 20-25 min, 45-65% B; 25-30 min, 65-95% B.
The Q-Exactive-Orbitrap mass spectrometer (Thermo Fisher Scientific, USA) equipped with an electrospray ion source. The atomizing gas was nitrogen; the spraying voltage was 3.5 KV; the flow rate of the sheath gas and the aux gas was 35 L/h and 10 L/h, respectively; the capillary temperature and auxiliary heating temperature were 350 ◦C; the first level spectrum adopted the positive and negative ion full scan mode, the scan range was 100-1500 m/z, the full scan resolution was 70000 FWHM; the second level fragment spectrum used the target ion detection mode, the resolution was 17500 FWHM; the collision induced dissociation energy gradient was set to 30/40/50 V.
2.6 UHPLC-QQQ-MS/MS quantitative analysis
Chromatographic analysis was performed on an ACQUITY UPLC Ultra Performance Liquid Chromatograph (American, waters company); Chromatographic separation was conducted on a Waters UPLC ACQUITY BEH C18 (1.7 μm, 2.1 mm×100 mm) maintained at 35 ◦C, the mobile phase consisted of 0.1% formic acid solution (A) and acetonitrile (B) using a gradient elution as following: 0-2 min, 5-10% B; 2-5 min, 10-20% B; 5-8 min, 20-25% B; 8-10 min, 25-30% B; 10-15 min, 30-45% B; 15-20 min, 45-95% B; the flow rate was kept at 0.3 mL/min.
MS detection was performed on Waters Xevo TQ-S Triple Quadrupole Mass Spectrometer (American, waterscompany). Quantification was performed using multiple reaction monitoring (MRM) mode. The optimized MS conditions for the positive ion mode were as follows: capillary voltage 3.0 KV, cone voltage 30 V, solvent removal temperature 350 ◦C. The optimized MS conditions for the negative ion mode were as follows: capillary voltage 2.0 KV, cone voltage 37 V, desolvation temperature 350 ◦C. The mass spectrometry analysis conditions of the 16 compounds were optimized and summarized in Table 1.
| Compound | Formula | Parent | Daughters | CV | CE | Detection mode |
|---|---|---|---|---|---|---|
| Sophocarpine | C15H22N2O | 247.14 | 136.13 | 82 | 28 | positive |
| Matrine | C15H24N2O | 249.16 | 148.16 | 80 | 26 | positive |
| Febrifugine | C20H18NO4 | 336.16 | 320.24 | 20 | 28 | positive |
| Berberine | C21H22NO4 | 352.12 | 308.13 | 62 | 28 | positive |
| Palmatine | C16H19N3O3 | 302.11 | 138.13 | 32 | 14 | positive |
| Tangeratin | C20H20O7 | 373.03 | 343.14 | 26 | 26 | positive |
| Nobiletin | C21H22NO8 | 403.10 | 373.15 | 84 | 26 | positive |
| Liensinine | C37H42N2O6 | 611.40 | 206.17 | 100 | 34 | positive |
| Neferine | C38H44N2O6 | 625.42 | 206.16 | 100 | 30 | positive |
| Scopoletin | C10H8O4 | 191.01 | 176.03 | 36 | 16 | negative |
| Isoliquiritigenin | C15H12O4 | 255.11 | 119.15 | 46 | 24 | negative |
| Liquiritigenin | C15H12O4 | 255.17 | 119.15 | 40 | 26 | negative |
| Naringenin | C15H12O5 | 271.04 | 151.07 | 30 | 20 | negative |
| Naringin | C27H32O14 | 579.21 | 151.05 | 80 | 46 | negative |
| Hesperidin | C28H34O15 | 609.22 | 301.18 | 48 | 24 | negative |
| Glycyrrhizic acid | C42H62O16 | 821.56 | 351.09 | 38 | 42 | negative |
2.7 Method validation of UHPLC–QQQ–MS/MS
According to “2.6” analysis conditions and “2.2” extraction conditions, the linearity, limit of detection (LOD), limit of quantification (LOQ), precision, repeatability, stability and recovery rate of 16 compound markers were determined. The standard curve was drew with the concentration x (ng/mL) of the reference substance as the abscissa and the corresponding peak area y of each reference substance as the ordinate. Then linear regression was performed on the standard curve to examine the correlation coefficient and linear range of the resulting linear regression equation. LOD and LOQ were determined based on the standard deviation of response value and the slope of standard curve, calculation formula: LOD=3.3δ/S, LOQ=10δ/S (δ, standard deviation; S, slope). XSNC was accurately weighed to test its precision, repeatability and stability. The test solution was continuously injected 6 times within 24 hours to evaluate the accuracy of the instrument, and 16 identical samples were prepared for repeatability analysis. The stability of the samples was studied after being placed at room temperature for 0, 2, 4, 8, 12 and 24 hours. The test solution and the reference solution were added at a ratio of 1:1, and six parts were measured in parallel to calculate the recovery rate of each component.
3 Results
3.1 Identification of chemical composition of Xin-Su-Ning capsules
3.1.1 Analysis of volatile components
The volatile components in XSNC were analyzed by GC-MS. The total ion flow diagram was shown in Fig. 1. A total of 21 volatile components were identified by searching with NIST mass spectrometry database (Table 2).
| No | tR（min） | Compounds | Molecular formula | possibility | Forward match | Reverse match | CAS |
|---|---|---|---|---|---|---|---|
| 1 | 4.249 | 2-Methylbutanal | C5H10O | 85.02 | 910 | 918 | 96-17-3 |
| 2 | 5.382 | Ethylpropenylether | C5H10O | 65.08 | 876 | 888 | 928-55-2 |
| 3 | 7.184 | 2-Ethoxyoxolane | C6H12O2 | 64.13 | 808 | 811 | 13436-46-9 |
| 4 | 10.114 | (Z)-2-Butenoic acid ethyl ester | C6H10O2 | 78.51 | 910 | 911 | 6776-19-8 |
| 5 | 12.116 | Ethyl acetate | C8H16O2 | 90.07 | 868 | 875 | 123-66-0 |
| 6 | 15.183 | Methyl lactate | C4H8O3 | 95.95 | 859 | 883 | 2155-30-8 |
| 7 | 15.716 | Methylheptenone | C8H14O | 73.86 | 778 | 865 | 110-93-0 |
| 8 | 19.984 | Acetoxy-2-acetone | C5H8O3 | 87.60 | 898 | 935 | 592-20-1 |
| 9 | 21.380 | 2-Acetylfuran | C6H6O2 | 74.57 | 943 | 950 | 1192-62-7 |
| 10 | 21.581 | Pyrrole | C4H5N | 81.84 | 939 | 946 | 109-97-7 |
| 11 | 21.781 | 2,4-Dihydroxy-2,5-dimethyl-3 | C6H8O4 | 68.04 | 740 | 797 | 10230-62-3 |
| 12 | 22.113 | Propionic acid | C3H6O2 | 79.50 | 960 | 977 | 137-40-6 |
| 13 | 23.515 | 5-Methylfuran aldehyde | C6H6O2 | 85.14 | 900 | 913 | 620-02-0 |
| 14 | 24.116 | Hotrienol | C10H16O | 73.52 | 789 | 811 | 20053-88-7 |
| 15 | 25.380 | γ-Butyrolactone | C4H6O2 | 60.08 | 961 | 966 | 96-48-0 |
| 16 | 25.849 | Furfuryl alcohol | C5H6O2 | 67.13 | 916 | 916 | 98-00-0 |
| 17 | 27.583 | 5-Methyl-2-furanmethanol | C6H8O2 | 86.83 | 806 | 806 | 3857-25-8 |
| 18 | 29.048 | 2(5H)-Furanone | C4H4O2 | 64.78 | 735 | 934 | 497-23-4 |
| 19 | 34.450 | 2-Acetylpyrrole | C6H7NO | 77.55 | 930 | 935 | 1072-83-9 |
| 20 | 40.646 | Ethyl palmitate | C18H36O2 | 91.76 | 901 | 903 | 628-97-7 |
| 21 | 40.715 | Ethyl hexadecanoate | C18H36O2 | 71.12 | 700 | 721 | 628-97-7 |
3.1.2 Analysis of non-volatile components
UHPLC-MS was used to analyze the non-volatile components in XSNC. The total ion flow diagram of the sample solution and the reference solution were shown in Fig. 2. For the compounds with chemical standards, according to the retention time, as well as accurate and high-resolution mass and tandem mass spectra, as a results, 10 compounds (peak 1, 2, 9, 26, 34, 37, 38, 43, 49 and 50) were identified as matrine, sophocarpine,scopoletin, jatrorrhizine, liquiritigenin, berberine,palmatine, isoliquiritigenin, nobiletin and Tangeratin, respectively. For the compounds without chemical standards, based on the retention time, exact mass data, fragment information, and molecular formula reported in the literatures, a total of 49 compounds were detected. Take hesperidin as an example to illustrate the fragmentation process, peak 20 exhibited the precursor ion [M−H]- ion at m/z 609.1822 in the negative mode and [M+H]+ ion at m/z 611.1976 in the positive mode. It was speculated that the relative molecular weight of the compound was 610 and the predicted molecular formula was C28H34O15. In the first-order mass spectrum of positive ion mode, there were fragments of m/z 449.1439 and m/z 465.1389, and in the second-order mass spectrum, there were fragment ions of m/z 303.0861 [M+H–Rha–Glc]+, m/z 153.0182 [M+H–Rha–Glc–C9H10O2]+, which was consistent with the fragments of hesperidin in the literature, and speculated that the compound was hesperidin (Chen et al., 2012). All in all, a total of 59 chemical constituents were tentatively identified including 18 alkaloids, 33 flavonoids, 5 coumarins, 2 alcoholamines and 1 triterpenoid (Table 3). Among these compounds, sophocarpine, matrine, febrifugine, berberine, palmatine, Tangeratin, nobiletin, liensinine, neferine, scopoletin, isoliquiritigenin, liquiritigenin, naringenin, naringin, hesperidin and glycyrrhizic acid were mainly active constituents with reported bioactivities. As a result, the quantitative analysis of these 16 constituents was performed in XSNC extracts.
| Peak NO. | tR (min) | Formula | Measured (m/z) | Detected (m/z) | Delta (ppm) | Fragments | Identification | class | source |
|---|---|---|---|---|---|---|---|---|---|
| 1# | 2.21 | C15H24N2O | 248.1889 | 249.1959 | –0.642 | 148.1116 150.1271 112.0760 | Matrine | Alkaloids | Sophora flavescens |
| 2# | 2.48 | C15H22N2O | 246.1732 | 247.1804 | –0.283 | 229.1705 179.1541 150.1275 136.1132 | Sophocarpine | Alkaloids | Sophora flavescens |
| 3 | 3.51 | C15H22N2O | 246.1732 | 247.1804 | –0.525 | 148.1121 136.1125 112.0763 | Sophocarpine Isomers | Alkaloids | Sophora flavescens |
| 4 | 3.76 | C15H24N2O2 | 264.1838 | 265.1909 | –0.394 | 150.1278 138.1277 | Hydroxylated matrine | Alkaloids | Sophora flavescens |
| 5 | 4.06 | C16H19N3O3 | 301.1421 | 302.1497 | –0.556 | 284.1398 203.0816 138.0914 | Febrifugine | Alkaloids | Changshan |
| 6 | 4.52 | C19H23NO3 | 313.1678 | 314.1748 | –0.732 | 269.1172 237.0911 107.0495 | Lotusine | Alkaloids | Lotus Seed Heart |
| 7 | 6.48 | C20H24NO4 | 342.1705 | 342.1696 | –1.241 | 265.0858 297.1119 | Magnoflorine | Alkaloids | Coptis |
| 8 | 7.45 | C27H30O14 | 578.1636 | 579.1706 | –0.418 | 271.0599 195.0287 219.0287 153.0181 | Rhoifolin | Flavone | Citrus aurantium |
| 9# | 7.53 | C10H8O4 | 192.0423 | 193.0495 | –0.131 | 178.0261 133.0284 137.0597 145.0958 | Scopoletin | Coumarin | Citrus aurantium |
| 10 | 8 | C27H32O15 | 596.1741 | 595.1671 | 0.347 | 459.1097 287.0555 269.0454 135.0436 | Eriocitrin | Flavone | Citrus aurantium |
| 11 | 8.16 | C26H30O13 | 550.1686 | 549.1609 | –0.754 | 297.0073 255.0655 153.0180 | Glycyrrhizin-4'-apirin | Flavone | Citrus aurantium |
| 12 | 8.36 | C27H30O16 | 610.1484 | 609.1465 | 0.611 | 301.0341 269.0451 201.0556 151.0022 88.9862 | Rutin | Flavone | Citrus aurantium |
| 13 | 8.45 | C27H32O15 | 596.1741 | 595.1669 | 0.146 | 459.1134 339.0707 287.0556 235.0556 | Neoeriocitrin | Flavone | Citrus aurantium |
| 14 | 8.73 | C27H30O15 | 594.1585 | 593.1513 | 0.197 | 447.0923 327.0599 285.0395 | Lonicerin | Flavone | Citrus aurantium |
| 15 | 9.3 | C19H15NO4 | 322.1079 | 322.1071 | –0.914 | 307.0837 294.0759 | Greenland Xanthine | Alkaloids | Coptis |
| 16 | 9.32 | C27H32O14 | 580.1792 | 579.1718 | –0.309 | 339.9276 295.0617 151.0023 | Narirutin | Flavone | Citrus aurantium |
| 17 | 9.75 | C27H32O14 | 580.1792 | 579.1714 | 0.162 | 459.1171 271.0607 151.0022 119.0487 | Naringin | Flavone | Citrus aurantium |
| 18 | 9.8 | C15H12O5 | 272.0685 | 273.0753 | –1.794 | 153.0182 147.0040 171.0287 177.0546 | Naringenin isomers | Flavone | Citrus aurantium |
| 19 | 9.85 | C21H22O10 | 434.1163 | 433.1134 | –1.432 | 363.3987 271.0607 151.0022 83.0123 | Prunin | Flavone | Citrus aurantium |
| 20 | 10.17 | C28H34O15 | 610.0898 | 609.1822 | –0.498 | 325.0722 301.0710 164.0102 151.0022 | Hesperidin | Flavone | Citrus aurantium |
| 21 | 10.41 | C28H32O15 | 608.1691 | 607.167 | 0.34 | 329.1388 299.0554 242.0673 164.0103 125.0228 | Neogeranin | Flavone | Citrus aurantium |
| 22 | 10.64 | C16H14O6 | 302.0785 | 303.0858 | –1.764 | 153.0182 285.0758 322.1056 | Hesperetin | Flavone | Citrus aurantium |
| 23 | 10.7 | C28H34O15 | 610.0898 | 609.18119 | –0.892 | 555.9461 325.0710 301.0711 286.0476 151.0022 | Neohesperidin isomers | Flavone | Citrus aurantium |
| 24 | 10.81 | C22H24O11 | 464.1319 | 463.1247 | 0.227 | 301.0709 286.0476 242.0575 151.0021 | Hesperetin-7-O-β-D-glucoside | Flavone | Citrus aurantium |
| 25 | 10.89 | C28H32O15 | 608.1691 | 607.167 | 0.242 | 489.1394 343.0817 301.0710 267.0657 151.0023 | Geranidin | Flavone | Citrus aurantium |
| 26# | 10.9 | C20H20NO4 | 338.1392 | 338.1383 | –1.108 | 294.1133 97.1016 83.0861 | Jatrorrhizine | Alkaloids | Sophora flavescens |
| 27 | 11.01 | C20H18NO4 | 336.1236 | 336.1226 | –1.232 | 320.0917 292.0962 292.0966 | Dihydroberberine | Alkaloids | Coptis |
| 28 | 11.08 | C26H30O13 | 550.1686 | 549.16156 | 0.357 | 399.1046 255.0655 153.0179 135.0072 | Isoliquiritin glucocelium | Flavone | Licorice |
| 29 | 11.09 | C19H14NO4 | 320.0923 | 320.0913 | –1.232 | 277.0729 262.0871 | Coptisine | Alkaloids | Coptis |
| 30 | 11.17 | C20H20NO4 | 338.1392 | 338.1385 | –2.202 | 323.1144 294.1122 308.0920 | Tetrandrine isomers | Alkaloids | Coptis |
| 31 | 11.48 | C21H22O9 | 418.1264 | 419.1335 | –0.426 | 257.0806 137.0233 239.0702 | Liquiritin | Flavone | Licorice |
| 32 | 11.71 | C20H20NO4 | 338.1392 | 338.1385 | –0.487 | 323.1147 294.1121 | Tetrandrine isomers | Alkaloids | Coptis |
| 33 | 11.96 | C19H15NO4 | 322.1079 | 322.1072 | –0.635 | 307.0838 279.0880 | Berberrubine | Alkaloids | Coptis |
| 34# | 12 | C15H12O4 | 256.0736 | 257.0808 | –0.332 | 211.0753 147.0441 137.0234 119.0494 | Liquiritigenin | Flavone | Licorice |
| 35 | 12.23 | C21H20NO4 | 350.1386 | 350.1387 | –0.042 | 334.1072 322.0706 306.1126 | 13-methylepiberberine | Alkaloids | Coptis |
| 36 | 12.8 | C16H12O5 | 284.0679 | 285.0756 | –0.648 | 270.052 253.0493 225.0546 | Calycosin | Flavone | Sophora flavescens |
| 37# | 12.96 | C20H18NO4 | 336.1236 | 336.1227 | –1.055 | 321.0984 292.0966 306.0757 | Berberine | Alkaloids | Coptis |
| 38# | 13.27 | C21H22NO4 | 352.1549 | 352.1539 | –1.149 | 337.1296 322.1074 308.1280 | Palmatine | Alkaloids | Coptis |
| 39 | 13.91 | C28H34O14 | 594.1943 | 595.2021 | 0.03 | 287.0911 153.0182 | Poncirin | Flavone | Citrus aurantium |
| 40 | 14.93 | C15H12O5 | 272.0685 | 273.0756 | –0.476 | 153.0182 147.0440 119.0494 | Naringenin | Flavone | Citrus aurantium |
| 41 | 15.08 | C21H20NO4 | 350.1387 | 350.1386 | –1.358 | 335.1142 320.0918 306.1123 292.0971 254.0569 | 13-methylberberine | Alkaloids | Coptis |
| 42 | 16.18 | C16H14O6 | 302.079 | 301.0718 | 0.261 | 286.0479 257.0841 242.0574 233.0796 | Hesperetin | Flavone | Sophora flavescens |
| 43 | 17.95 | C15H12O4 | 256.0736 | 255.0661 | –0.162 | 211.0753 135.0072 119.0487 | Isoliquiritigenin | Flavone | Licorice |
| 44 | 18.33 | C16H12O4 | 268.073 | 269.0805 | –1.358 | 237.0542 137.0233 118.0414 | Formononetin | Coumarin | Citrus aurantium |
| 45 | 18.53 | C15H16O4 | 260.1043 | 261.1114 | –2.74 | 189.0545 243.1012 159.0440 131.0492 | Hesperitone | Coumarin | Citrus aurantium |
| 46 | 19.67 | C26H30O8 | 470.1935 | 471.2009 | –0.414 | 425.1963 339.1952 213.0911 161.0598 95.0132 | Limonin | Flavone | Citrus aurantium |
| 47 | 19.64 | C19H18O6 | 342.1098 | 343.1172 | –0.335 | 313.0704 285.0755 181.0129 373.0918 | 4',5,7,8-tetramethoxyflavonoid | Flavone | Citrus aurantium |
| 48 | 19.97 | C42H62O16 | 822.4038 | 821.3964 | –0.108 | 683.7745 513.6673 443.4119 351.0563 175.0234 | Glycyrrhizic acid | Flavone | Citrus aurantium |
| 49# | 20.7 | C21H22O8 | 402.1315 | 403.1383 | –0.404 | 388.1163 373.0916 355.0822 | Nobiletin | Flavone | Citrus aurantium |
| 50# | 22.17 | C20H20O7 | 372.1209 | 373.1277 | –1.397 | 343.0811 358.1042 325.0703 | Tangeratin | Flavone | Citrus aurantium |
| 51 | 22.09 | C26H30O6 | 438.2042 | 437.1969 | –0.142 | 301.1429 151.0386 91.0539 | Kurarinone | Flavone | Sophora flavescens |
| 52 | 22.21 | C16H35NO2 | 273.2662 | 274.2737 | –1.261 | 256.2634 | Cetyl-Dihydrosphingosine | Alcoholamines | Pinellia |
| 53 | 23.67 | C27H32O6 | 452.2193 | 453.2268 | –0.85 | 329.1025 303.1590 197.0440 | 2’-Methoxymatrine | Flavone | Sophora flavescens |
| 54 | 23.84 | C25H30O6 | 424.1886 | 423.1816 | 0.634 | 261.1491 161.0231 109.0281 | Kushenol E isomers | Flavone | Sophora flavescens |
| 55 | 25.97 | C25H30O6 | 424.1886 | 423.1967 | –0.553 | 261.1491 161.0231 109.0281 | Kushenol E isomers | Flavone | Sophora flavescens |
| 56 | 26.54 | C26H30O6 | 438.2042 | 437.1967 | –0.553 | 275.1648 161.0230 109.0277 | Kuraridin | Flavone | Sophora flavescens |
| 57 | 27.18 | C30H46O4 | 470.3391 | 469.3312 | –0.326 | 425.3423 409.3109 | Glycyrrhetinic acid | Triterpene | Licorice |
| 58 | 27.47 | C20H43NO2 | 329.3288 | 330.3361 | –1.623 | 312.3257 106.0866 88.0762 | 2-amino-1,3-eicosanediol | Alcoholamines | Pinellia |
| 59 | 28.21 | C19H22O3 | 298.1563 | 299.1633 | –0.871 | 189.0542 163.0389 119.0494 | Grapefruit lactone | Coumarin | Citrus aurantium |
Note: # stands for comparison with standard products.
3.2 Quantitative analysis
Through LC-MS multiple reaction detection mode, the test solution was prepared according to the method under “2.2”, and the prepared test solution was determined under the detection conditions “2.6”. Aiming at the problem of large difference in mass spectrum response and content of various types of compounds in the complex system of XSNC, through the multiple dilution method, the same sample was prepared by preparing a low dilution ratio test solution to detect components with low mass spectrometry response and low content (including febrifugine, liensinine, neferine, scopoletin, isoliquiritigenin, liquiritigenin, naringenin, naringin, hesperidin, and glycyrrhizic acid); then low dilution ratio sample was diluted by times to obtain the test solution with high dilution ratio to detect the components with high mass response and high content (including sophocarpine, matrine, berberine, palmatine, Tangeratin and nobiletin). The rapid detection of various components in the sample was realized by different dilution methods.
3.2.1 Methodology validation
LC-MS was used for the quantitative analysis of sophocarpine, matrine, febrifugine, berberine, palmatine, Tangeratin, nobiletin, liensinine, neferine, scopoletin, isoliquiritigenin, liquiritigenin, naringenin, naringin, hesperidin and glycyrrhizic acid .The 16 index components had a good linear relationship within the corresponding concentration range, and their R2 were all greater than 0.999, the LOD and LOQ were 0.146-60.074 ng/mL and 0.442-182.043 ng/mL, the results were shown in Table 4. The relative standard deviation (RSD) values of accuracy, repeatability, and stability were all less than 2.90 %, indicating that the instrument had good precision, the method had high repeatability, and the sample solution was stable for 24 h at room temperature. The sample recovery rate was between 99.65%-104.03%, and the RSD value was less than 4.35 %, indicating that the recovery rates of the 16 compounds in XSNC were good, and the established method had sufficient reliability and accuracy, the results were summarized in Table 5 (Supporting information Table S1-S5).
| Compounds | Regression equation | R2 | Linearity range (ng/mL) | LOD (ng/mL) | LOQ (ng/mL) |
|---|---|---|---|---|---|
| Sophocarpine | y =8033.50x + 112717.00 | R2 = 0.9992 | 15.625-1000 | 4.910 | 14.880 |
| Matrine | y = 3834.20x- 25.18 | R2 = 0.9997 | 15.625-1000 | 3.362 | 10.187 |
| Febrifugine | y =2782.70x- 1136.30 | R2 = 0.9994 | 0.781-50 | 0.146 | 0.442 |
| Berberine | y = 13757.00x + 16757.00 | R2 = 0.9999 | 15.625-1000 | 3.722 | 11.280 |
| Palmatine | y = 16658.00x - 3566.10 | R2 = 1.0000 | 15.625-1000 | 3.236 | 9.805 |
| Tangeratin | y = 92881.00x + 5240.10 | R2 = 0.9994 | 0.781-50 | 0.320 | 0.971 |
| Nobiletin | y = 73488.00x + 16786.00 | R2 = 0.9998 | 0.781-50 | 0.498 | 1.509 |
| Liensinine | y = 721.52x - 757.60 | R2 = 0.9990 | 1.563-100 | 0.429 | 1.299 |
| Neferine | y = 1079.90x - 3426.70 | R2 = 0.9993 | 7.813-500 | 2.296 | 6.959 |
| Scopoletin | y = 148.42x - 293.17 | R2 = 0.9995 | 3.906-250 | 1.230 | 3.729 |
| Isoliquiritigenin | y = 418.49x - 279.76 | R2 = 0.9994 | 1.563-100 | 0.461 | 1.395 |
| Liquiritigenin | y = 233.48x - 115.91 | R2 = 0.9992 | 1.563-100 | 0.158 | 0.480 |
| Naringenin | y =295.30x - 599.14 | R2 = 0.9996 | 7.813-500 | 2.435 | 7.378 |
| Naringin | y = 156.15x + 1022.20 | R2 = 1.0000 | 234.375-15000 | 60.074 | 182.043 |
| Hesperidin | y = 433.93x + 109.30 | R2 = 0.9992 | 15.625-1000 | 4.831 | 14.641 |
| Glycyrrhizic acid | y = 213.75x - 2960.30 | R2 = 0.9993 | 31.250-2000 | 5.541 | 16.792 |
| Compounds | Precision RSD (%) | Repeatability RSD (%) | Stability RSD (%) | Recovery | |
|---|---|---|---|---|---|
| Mean | RSD (%) | ||||
| Sophocarpine | 0.77 | 0.93 | 0.44 | 100.43 | 4.35 |
| Matrine | 0.28 | 0.93 | 0.30 | 101.14 | 2.15 |
| Febrifugine | 1.94 | 2.04 | 2.17 | 101.58 | 1.32 |
| Berberine | 0.82 | 1.26 | 0.81 | 99.92 | 0.43 |
| Palmatine | 1.65 | 1.18 | 1.02 | 100.35 | 1.25 |
| Tangeratin | 0.77 | 1.23 | 0.54 | 101.65 | 0.25 |
| Nobiletin | 0.49 | 1.25 | 0.51 | 101.19 | 1.03 |
| Liensinine | 1.90 | 2.34 | 1.29 | 101.98 | 2.79 |
| Neferine | 2.06 | 1.22 | 1.05 | 99.65 | 1.92 |
| Scopoletin | 0.45 | 2.61 | 0.49 | 101.81 | 1.47 |
| Isoliquiritigenin | 0.68 | 2.29 | 0.73 | 99.71 | 0.77 |
| Liquiritigenin | 1.98 | 2.90 | 1.82 | 101.50 | 2.09 |
| Naringenin | 1.11 | 1.90 | 2.42 | 99.71 | 2.50 |
| Naringin | 0.37 | 2.73 | 0.80 | 104.03 | 0.60 |
| Hesperidin | 0.25 | 2.47 | 0.84 | 99.89 | 1.27 |
| Glycyrrhizic acid | 0.47 | 1.90 | 1.14 | 101.06 | 1.27 |
3.2.2 Determination of sample content
Multiple reaction monitoring (MRM) is a highly specific and sensitive mass spectrometry technique for quantifying predefined compounds of interest. The UHPLC–MS/MS analysis method described above was subsequently used to simultaneously quantify 16 compounds in 10 collected batches. Every sample was analyzed in triplicates to acquire the average contents of the constituents. The results were shown in Table 6.
| Sample batch | Sophocarpine (mg/g) | Matrine (mg/g) | Febrifugine (mg/g) | Berberine (mg/g) | Palmatine (mg/g) | Tangeratin (mg/g) | Nobiletin (mg/g) | Liensinine (mg/g) | Neferine (mg/g) | Scopoletin (mg/g) | Isoliquiritigenin (mg/g) | Liquiritigenin (mg/g) | Naringenin (mg/g) | Naringin (mg/g) | Hesperidin (mg/g) | Glycyrrhizic acid (mg/g) |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 220201 | 3.06 ± 0.27 | 11.62 ± 0.75 | 0.05 ± 0.00 | 31.05 ± 1.31 | 7.56 ± 0.32 | 0.46 ± 0.01 | 0.55 ± 0.02 | 0.06 ± 0.01 | 0.38 ± 0.01 | 0.36 ± 0.03 | 0.11 ± 0.01 | 0.15 ± 0.01 | 0.43 ± 0.04 | 28.01 ± 0.01 | 9.86 ± 0.21 | 1.66 ± 0.03 |
| 220202 | 2.65 ± 0.10 | 11.65 ± 0.21 | 0.06 ± 0.01 | 31.66 ± 0.64 | 7.79 ± 0.06 | 0.46 ± 0.00 | 0.55 ± 0.01 | 0.05 ± 0.00 | 0.37 ± 0.01 | 0.40 ± 0.01 | 0.12 ± 0.00 | 0.17 ± 0.00 | 0.52 ± 0.01 | 32.64 ± 0.05 | 10.99 ± 0.23 | 1.59 ± 0.04 |
| 210701 | 4.39 ± 0.20 | 15.90 ± 1.62 | 0.06 ± 0.00 | 34.25 ± 1.44 | 8.77 ± 0.21 | 0.45 ± 0.01 | 0.55 ± 0.01 | 0.07 ± 0.00 | 0.45 ± 0.11 | 0.69 ± 0.02 | 0.12 ± 0.00 | 0.24 ± 0.01 | 0.77 ± 0.02 | 47.02 ± 0.94 | 13.24 ± 0.13 | 1.60 ± 0.04 |
| 220101 | 2.52 ± 0.29 | 10.38 ± 0.99 | 0.17 ± 0.02 | 34.96 ± 0.93 | 8.82 ± 0.03 | 0.36 ± 0.00 | 0.43 ± 0.01 | 0.06 ± 0.01 | 0.41 ± 0.03 | 0.42 ± 0.02 | 0.20 ± 0.01 | 0.23 ± 0.01 | 0.44 ± 0.01 | 34.38 ± 0.36 | 13.46 ± 0.08 | 1.25 ± 0.04 |
| 210901 | 3.15 ± 0.05 | 12.16 ± 0.07 | 0.15 ± 0.01 | 25.35 ± 1.21 | 6.31 ± 0.26 | 0.27 ± 0.00 | 0.30 ± 0.01 | 0.05 ± 0.00 | 0.33 ± 0.03 | 0.53 ± 0.02 | 0.10 ± 0.00 | 0.16 ± 0.01 | 0.37 ± 0.02 | 24.66 ± 0.49 | 6.77 ± 0.10 | 1.66 ± 0.04 |
| 210502 | 4.05 ± 0.03 | 14.39 ± 0.77 | 0.10 ± 0.01 | 37.07 ± 0.54 | 8.79 ± 0.13 | 0.59 ± 0.02 | 0.58 ± 0.01 | 0.07 ± 0.01 | 0.55 ± 0.02 | 0.45 ± 0.01 | 0.19 ± 0.00 | 0.20 ± 0.01 | 0.42 ± 0.03 | 26.34 ± 0.84 | 7.57 ± 0.21 | 1.59 ± 0.04 |
| 210301 | 1.72 ± 0.08 | 9.20 ± 0.39 | 0.04 ± 0.01 | 20.60 ± 0.62 | 5.18 ± 0.17 | 0.37 ± 0.01 | 0.41 ± 0.01 | 0.05 ± 0.00 | 0.40 ± 0.01 | 0.32 ± 0.01 | 0.12 ± 0.01 | 0.15 ± 0.01 | 0.36 ± 0.01 | 19.03 ± 0.01 | 5.11 ± 0.05 | 1.72 ± 0.03 |
| 210501 | 3.96 ± 0.05 | 11.79 ± 0.03 | 0.07 ± 0.01 | 30.90 ± 1.30 | 7.50 ± 0.22 | 0.46 ± 0.01 | 0.53 ± 0.01 | 0.08 ± 0.01 | 0.68 ± 0.01 | 0.29 ± 0.01 | 0.12 ± 0.01 | 0.18 ± 0.00 | 0.61 ± 0.02 | 35.63 ± 0.40 | 9.46 ± 0.03 | 2.97 ± 0.02 |
| 201001 | 2.86 ± 0.06 | 10.56 ± 0.76 | 0.02 ± 0.00 | 17.13 ± 0.78 | 4.46 ± 0.15 | 0.35 ± 0.01 | 0.39 ± 0.01 | 0.04 ± 0.01 | 0.32 ± 0.01 | 0.35 ± 0.02 | 0.05 ± 0.00 | 0.09 ± 0.00 | 0.32 ± 0.02 | 17.33 ± 0.33 | 3.31 ± 0.05 | 0.55 ± 0.01 |
| 211101 | 2.16 ± 0.03 | 9.40 ± 0.03 | 0.05 ± 0.00 | 26.73 ± 0.49 | 6.34 ± 0.26 | 0.34 ± 0.01 | 0.37 ± 0.00 | 0.07 ± 0.01 | 0.66 ± 0.01 | 0.37 ± 0.00 | 0.09 ± 0.00 | 0.16 ± 0.01 | 0.40 ± 0.02 | 23.38 ± 0.34 | 6.40 ± 0.14 | 1.89 ± 0.09 |
Note: values are expressed as the mean ± SD of three parallel samples.
4 Discussion
How to combine the basic requirements of “safe, effective and controllable quality” with the characteristics of TCM is the key problem of TCM quality research and control. In this context, Academician Liu Changxiao and his team put forward the concept of Chinese medicine quality marker (Q-Marker) (Liu, 2019; Liu et al., 2016). In view of this, a method for simultaneous quantitative analysis of 16 chemical components in XSNC was established. Berberine and palmatine are derived from the prince drug Coptidis Rhizoma. A large number of studies have shown that berberine mainly exerts anti-arrhythmic effects by affecting potassium ion channels (Chen et al., 2018); Palmatine and berberine have similar structures and have higher content in Coptidis Rhizoma, it also has better anti-arrhythmic activity (Liu et al., 2017, 2017). Sophocarpine and matrine are derived from the official medicine Sophorae flavescentis Radix, and liensinine, neferine come from the central medicine Nelumbinis Plumula. These alkaloids can exert their anti-arrhythmic effects by influencing myocardial cell ion channels and prolonging APD (Jain and Parmar, 2011). Hesperidin is derived from the adjuvant Aurantii Fructus Immaturus, and isoliquiritin is derived from the drug Glycyrrhizae Radix et Rhizoma. These ingredients are all antiarrhythmic active ingredients (Ojha et al., 2013). Another research report glycyrrhizic acid has a protective effect on the heart (Ding et al., 2018). Dichroae Radix has small poison and febrifugine is the active component of Dichroae Radix, the content of febrifugine is determined to ensure the safety of TCM compound preparation. The active ingredients and characteristic ingredients contained in each component of the TCM are selected as indicators for quantitative analysis, which can provide a better reference for the quality evaluation and the material basis of the medicinal effect of the traditional Chinese medicine compound.
The chemical components of XSNC are complex and it is difficult to achieve baseline separation by liquid chromatography. In this experiment, multi reaction monitoring technology (MRM) in LC-MS technology was selected for quantitative analysis of the selected 16 chemical components. MRM monitoring mode can detect and analyze specific compounds with strong specificity, high sensitivity and high accuracy. Different compounds have different mass spectrometric responses, and the content of each component is completely different. To solve this problem, a multiple dilution method was constructed to realize the simultaneous quantitative analysis of 16 components in XSNC.
In this study, qualitative analysis of the chemical components of XSNC was carried out by GC-MS and LC-MS techniques. A total of 21 volatile components and 59 non-volatile components were identified, which further clarified the chemical composition of XSNC. It provides a reference for the characterization of chemical components of other TCM preparations; on the basis of LC-MS technology, considering the active components and characteristic components contained in various components of XSNC, the quantitative analysis of 16 chemical components with great content difference in XSNC was realized by constructing the double ratio dilution method. The method has high sensitivity and good selectivity, which can provide experimental basis for formulating a comprehensive quality control method of XSNC.
Acknowledgements
This work was supported by the National Key Research and Development Plan of China (No.2018YFC1707403); the Science and Technology Program of Tianjin (No.20ZYJDJC00120 and 21ZYJDJC00080).
Funding information
National Key Research and Development Plan of China, Grant Number: 2018YFC1707403; Science and Technology Program of Tianjin, Grant Numbers: 20ZYJDJC00120 and 21ZYJDJC00080.
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.
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Appendix A
Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2022.103825.
Appendix A
Supplementary data
The following are the Supplementary data to this article:Supplementary Data 1
Supplementary Data 1