A combined analysis method for analyzing the chemical components of abelmoschus gel capsules

Abstract

The present application relates to the technical field of analysis, in particular to a combined analysis method for analyzing the chemical components of Huangkui capsules, which is a fast, efficient and accurate method for analyzing the chemical components of Huangkui capsules based on UHPLC-Q-TOF / MS combined with MS-DIAL and GNPS strategy. By setting conditions such as positive and negative ion mode full scan, molecular ion peak pairing, accurate molecular weight mass error and isotope peak abundance, the compounds can be accurately identified, the molecular formula is generated, and the identification of the compounds is completed based on the self-built compound database. Further combined with MS-DIAL and GNPS, two molecular cages related to flavonoid structure are successfully identified according to MS / MS similarity, and a total of 13 compounds other than the self-built compound database are identified. It provides a useful reference for further development and utilization of Huangkui capsules.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical techniques, in particular, a combined analysis method for analyzing the chemical components of Huangkui capsules. BACKGROUND

[0002] The component analysis of traditional Chinese medicines and their compound compounds has always been a hot and difficult point in the field of analysis, and is also a key point for elucidating their effective substances. In recent years, due to the high sensitivity and high resolution characteristics of UHPLC-Q-TOF / MS technology, it has been widely used in the component analysis of traditional Chinese medicines and their compound compounds. However, based on the high separation degree of high performance liquid chromatography (UHPLC) and the high resolution ability of quadrupole time-of-flight tandem mass spectrometry (Q-TOF / MS) in MS and MS / MS modes, the high-resolution accurate mass spectrometry of mass spectrometry is used for qualitative analysis of compounds. Generally, only the chemical components in the self-built library are identified; and due to the large number of compounds contained in traditional Chinese medicines, similar structures, and the existence of most isomers, it is impossible to avoid the false positive results based on software automatic identification.

[0003] Huangkui capsules are traditional Chinese medicines made of Abelmoschus manihot (L.) ethanol extract, which are widely used in the treatment of chronic glomerulonephritis, nephrotic syndrome, diabetic nephropathy and other kidney diseases. At present, more than 100 compounds have been extracted, separated and identified from Abelmoschus manihot (L.), mainly flavonoids, amino acids, nucleosides, polysaccharides and organic acids. Among them, flavonoids are the main chemical components and pharmacodynamic active ingredients of Huangkui capsules and Abelmoschus manihot (L.). At present, many literatures based on UHPLC-Q-TOF / MS have carried out qualitative analysis on flavonoids contained in Abelmoschus manihot (L.) extract, and continuously increase the structural information of flavonoids (Chi Yu-mei, Zhu Hua-yun, Ju Ling, et al. Analysis of flavonols in Abelmoschus manihot (L.) by high performance liquid chromatography-quadrupole time-of-flight mass spectrometry [J]. Analysis Chemistry, 2009, 37 (2): 227-231. Gao Xin, Wan Yao-yao, Li Chang-yin, et al. Systematic analysis of flavonoids in Huangkui based on liquid chromatography-time-of-flight mass spectrometry and mass loss filtering technology [J]. Analysis Chemistry, 2020, 48 (2): 262-268.).

[0004] Comprehensive understanding of the chemical structure information of flavonoids in Huangkui capsules and its in vivo components is the premise and basis for elucidating its pharmacodynamic material basis, evaluating the quality and safety of related preparations. Therefore, it is essential to establish an analysis method with high selectivity, high sensitivity and high accuracy. SUMMARY

[0005] The purpose of the present application is to provide a combined analysis method for analyzing the chemical components of Huangkui capsules.

[0006] In recent years, many scholars have begun to use other methods to analyze the mass spectrometry data collected by UHPLC-Q-TOF / MS directly or further, to improve the accuracy of compound identification. The specific process is as follows Figure 1 .

[0007] In general, there are currently three methods for analyzing traditional Chinese medicine and its compound analysis process based on UHPLC-Q-TOF / MS analysis Figure 1 ): Method I, as follows steps 1, 2, 3 and 4, the conventional process, that is, the analysis software of the instrument is used for analysis; Method II, as follows steps 1, 2, 3, 4 and 5, the analysis software of the instrument is combined with MS-DIAL analysis; Method III, as follows steps 1, 2, 3, 4 and 6, the analysis software of the instrument is combined with GNPS analysis. The advantage of MS-DIAL is that it can further identify and supplement the results of Mass Hunter, and identify the structure of unknown compounds combined with MS-FINDER. The disadvantage is that it takes a long time to confirm each compound due to the large number of automatically identified compounds; The advantage of GNPS is that it can identify compounds of the same type, and the disadvantage is that it may not match the chemical components of traditional Chinese medicine in the constructed network, resulting in false positive results.

[0008] Step 1: Establish a compound database based on the instrument supplier's software platform (such as Agilent Mass Hunter, Water UNIFI, etc.), which includes information such as compound name, molecular formula, molecular weight, chemical structure, etc., referred to as "self-built library".

[0009] Step 2: Optimize the pretreatment and UHPLC-Q-TOF / MS analysis method, analyze the traditional Chinese medicine and its chemical profile in positive and / or negative ion mode; optimize different collision energies (such as 10, 20, 30 V) and acquisition modes to collect the secondary mass spectrum of the sample.

[0010] Step 3: Import the self-built library into the supplier's software platform such as Agilent MassHunter software, optimize the extraction parameters, calculate and screen, and then use the Find by Formula function to extract and match the target compound according to the accurate mass (m / z), isotopic abundance and retention time characteristics (Rt), to obtain the preliminary identification results.

[0011] Step 4: Analyze the secondary mass spectrum of the compound by comparing the retention time (Rt), multi-stage mass spectrum (MS / MS fragmentation), ultraviolet absorption spectrum, etc., combined with the mass spectrum fragment characteristics of the compound type and literature retrieval, to obtain the final identification results.

[0012] Step 5: MS-DIAL supports most instrument acquisition data formats, including Agilent Technologies (.D), AB Sciex (.Wiff), Thermo Fisher Scientific (.RAW), Bruker Daltonics (.D), and Waters (.RAW); based on MS-DIAL, the raw mass spectrometry data collected is filtered, peak-aligned, peak-extracted, peak-identified, and normalized, combined with MS-FINDER to extract characteristic peaks and mark them, and compared with the sample in the mass spectrometry database for fingerprint area and fragmentation, listing a series of possible compounds, and then confirming the structure of the compound according to the self-built library and the information of the reference substance (Tsugawa H, Cajka T, Kind T, et al. MS-DIAL: data-independent MS / MS deconvolution for comprehensive metabolome analysis [J]. Nat Methods. 2015, 12(6): 523-526. Tu Yan, Sun Lian-na, Dong Zhi-ying, et al. Analysis of the chemical components of Danshen medicinal materials from different regions based on UPLC-QTOF-MS technology [J]. Chinese Herbal Medicines. 2021, 44(6) 1335-1340.).

[0013] Step 6: To make up for the deficiency of manually establishing a local database and identifying compounds, a molecular network based on the Global Natural Products Social Molecular Networking (GNPS) database platform can be used to integrate similar compounds with similar secondary mass spectrometry fragments into a visual network through computer algorithms. Known compounds in the network can be identified in combination with the diagnostic ion identification results, and the structures of unknown compounds can be inferred according to the similarities and differences of the mass spectrometry data of related nodes (Fan Q, Chen XB, Wang YM, et al. Diterpenoid alkaloid components in Cistanche tubulosa based on UPLC-Q-TOF-MS / MS and GNPS technology [J]. Journal of Sun Yat-Sen University: Natural Science Edition, 2021, 60(3) 30-44. Lei HB, Zhang YH, Zu XiP, et al. Comprehensive profiling of the chemical components and potential markers in raw and processed Cistanche tubulosa by combining ultra-high-performance liquid chromatography coupled with tandem mass spectrometry and MS / MS-based molecular networking [J]. Anal Bioanal Chem. 2021, 413(1): 129-139.).

[0014] Therefore, the present application comprehensively analyzes the advantages of GNPS and MS-DIAL (Method IV, i.e. Steps 1, 2, 3, 4, 5 and 6) to improve the efficiency and accuracy of compound identification, and takes Huangkui capsules as an example to carry out identification and analysis of the chemical components contained therein, especially flavonoid components.

[0015] Based on this, the present application provides a combined analysis method for analyzing the chemical components of Huangkui capsules, comprising the following steps:

[0016] Step A: Establish a compound database based on the instrument supplier software platform (such as Agilent Mass Hunter, Water UNIFI, etc.), which includes information such as compound name, molecular formula, molecular weight, chemical structure, etc., referred to as "self-built library";

[0017] Step B: Optimize the pretreatment and UHPLC-Q-TOF / MS analysis method, analyze the traditional Chinese medicine and its chemical profile in positive and / or negative ion mode; optimize different collision energies (such as 10, 20, 30V) and acquisition modes to collect the secondary mass spectrum of the sample;

[0018] Step C: Import the self-built library into the vendor software platform such as Agilent MassHunter software, optimize the extraction parameters, calculate and screen, and then find the target compounds according to the accurate mass (m / z), isotopic abundance and retention time characteristics (Rt) by the function of Find by Formula, perform peak extraction and matching, and obtain the preliminary identification results;

[0019] Step D: By comparing the retention time (Rt), MS / MS fragmentation, ultraviolet absorption spectrum and other information, combining the mass spectrum fragment characteristics of the compound type and literature retrieval, the secondary mass spectrum of the compound is analyzed, and the final identification result is obtained;

[0020] Step E: MS-DIAL supports most instrument acquisition data formats, including Agilent Technologies (.D), AB Sciex (.Wiff), Thermo Fisher Scientific (.RAW), Bruker Daltonics (.D) and Waters (.RAW); based on MS-DIAL, the original mass spectrum data collected is filtered, peak alignment, peak extraction, peak identification and normalization, MS-FINDER is used to extract characteristic peaks and mark, and the fingerprint area and fragmentation of the sample in the mass spectrum database are compared, a series of compounds with high possibility are listed, and the structure of the compound is confirmed according to the self-built library and reference substance information;

[0021] Step F: In order to make up for the deficiency of manually establishing a local database and identifying compounds, the molecular network established based on the Global Natural Products Social Molecular Networking (GNPS) database platform can generate similar secondary mass spectrum fragments according to similar structural compounds, and the similarity size is integrated in the visual network through computer algorithm. The known compounds in the network can be identified in combination with the diagnostic ion identification results, and the structure of the unknown compounds can be inferred according to the similarities and differences of the related node mass spectrum data.

[0022] The advantages of the present application are:

[0023] The application is based on a rapid, efficient and accurate method for analyzing chemical components in Huangkui capsules established by UHPLC-Q-TOF / MS combined with MS-DIAL and GNPS strategy. The method can accurately identify compounds, generate their molecular formula, and complete the identification of compounds based on the self-built compound database by setting conditions such as positive and negative ion mode full scan, molecular ion peak pairing, accurate molecular weight mass error and isotope peak abundance, and 58 compounds are identified. Further combined with MS-DIAL and GNPS, two molecular cages related to flavonoid structure are successfully identified based on MS / MS similarity, and 13 compounds other than the self-built compound database are identified. The above results help to expand the understanding of the chemical substance basis of Huangkui capsules, and provide a useful reference for further elucidation of the efficacy of Huangkui capsules and further development and utilization. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 . Based on UHPLC-Q-TOF / MS analysis of traditional Chinese medicine and its compound analysis flowchart; method I (purple arrow): conventional process, i.e. analysis by instrument self-analysis software; method II (blue arrow): instrument self-analysis software combined with MS-DIAL analysis; method III (green arrow): instrument self-analysis software combined with GNPS analysis; method IV (red arrow): instrument self-analysis software combined with GNPS and MS-DIAL analysis.

[0025] Figure 2 . UHPLC-Q-TOF / MS analysis of Huangkui capsule extract TIC graph (A-positive ion mode, B-negative ion mode).

[0026] Figure 3 . Analysis of compound structure based on MS-DIAL (taking quercetin as an example); (A) compound primary mass spectrum information, (B) compound secondary mass spectrum information, (C) MS-DIAL and MS-FINDER matching results, (D) compound cleavage rule.

[0027] Figure 4 . Two flavonoid compounds molecular cages (I and II) in Huangkui capsule extract. DETAILED DESCRIPTION

[0028] The specific embodiments provided by the application will be described in detail below in combination with examples.

[0029] Example:

[0030] 1. Experiment and materials

[0031] 1.1 Materials and equipment

[0032] Ultra-high performance liquid chromatograph (Agilent, USA), equipped with an Agilent 1290 Infinity binary pump, autosampler, column oven, and diode array detector (DAD); quadrupole-time-of-flight tandem mass spectrometer (Agilent 6530, USA), equipped with an Agilent jet stream ion focusing (AJS) ion source and a MassHunter Qualitative Analysis 10.0 workstation; ultrasonic cleaner (SK7200H, 350W, 53kHz, Shanghai Keda Ultrasonic Instrument Co., Ltd.); timed adjustable speed vortex mixer (Iabnet VX-200, USA); 1 / 100,000 electronic balance (Sartorius CPA255D, Germany); freeze dryer (BUCHI Lyovapor L-200, Switzerland); Eppendorf mini spin centrifuge (Eppendorf, Germany); Eppendorf 5430r centrifuge (Eppendorf, Germany).

[0033] Reference standards included rutin, hyperoside, isoquercitrin, myricetin, quercetin, myricetin-3-O-glucoside, and gossypol, all purchased from Dalian Meilun Pharmaceutical Co., Ltd. (purity > 98%). Abelmoschus manihot capsules (batch number 21102305) were purchased from Jiangsu Suzhong Pharmaceutical Group Co., Ltd. Physiological saline was provided by the preparation room of Changzheng Hospital. Water was deionized water; methanol, acetonitrile, and formic acid were mass spectrometry grade (E. Merck, Germany); all other reagents were analytical grade.

[0034] 1.2 Preparation of the test solution

[0035] Accurately weigh 0.12 g of the contents of the okra capsules and place them in a stoppered conical flask. Add 10 mL of pure methanol, weigh the flask, and extract using ultrasound for 30 min. Allow the flask to cool to room temperature and weigh it again. Make up the lost mass with pure methanol. Centrifuge the extract at 3000 rpm for 10 min. Transfer the supernatant to a 1.5 mL centrifuge tube and centrifuge at 15000 rpm for 10 min. Transfer 200 μL of the supernatant to a brown vial for analysis by UHPLC-Q-TOF / MS.

[0036] 1.3 Preparation of reference solution

[0037] The reference substance (seven kinds of quercetin, myricetin, hyperoside, isoquercitrin, rutin, myricetin 3-O-β-D-glucoside, gossypetins-8-O-β-D-glucuronide) was precisely weighed, and methanol was added to prepare a 1 mg / mL stock solution. 10 μL of the above stock solution was precisely taken and mixed in a 1.5 mL centrifuge tube, vortexed, and methanol was added to prepare a mixed standard solution with a final concentration of 10 μg / mL.

[0038] 1.4 UHPLC-Q-TOF / MS analysis conditions

[0039] UHPLC conditions: Waters ACQUITY UPLC HSS T3 column (100 mm x 2.1 mm, 1.7 μm) was used, 0.05% formic acid water (A) - 0.05% formic acid acetonitrile (B) was used as the mobile phase, the elution gradient was set as: 0-2 min, 5% B; 2-15 min, 5%-16% B; 15-20 min, 16%-50% B; 20-28 min, 50%-80% B; 28-33 min, 80%-100% B; 33-35 min, 100% B; 35.1-40 min, 5% B, flow rate 0.3 ml / min; column temperature 30°C, injection volume 2 μl. Detection wavelength: 254 nm.

[0040] Q-TOF / MS conditions: ESI ion source, data was collected in positive and negative ion modes (m / z 100-1700) respectively. The detection mode was Full MSMS2, ion source temperature 350°C, capillary voltage 3.5 kV (positive ion), 4.0 kV (negative ion), atomization gas pressure 45 Psi, dry gas flow rate 11 L / min, sheath gas flow rate 11 L / min, sheath gas temperature 350°C, fragment voltage 140 V, collision energy 40 eV.

[0041] 1.5 Establishment of compound analysis library and data analysis

[0042] Pubchem, Web of Science, CNKI and Wanfang databases were used as retrieval sources to search for the separation and analysis of compound information contained in Abrus precatorius. Based on the defult document provided by Agilent, an Abrus precatorius compound database was established. Data analysis was performed using Agilent MassHunter Qualitative Analysis 10.0 software.

[0043] 2 RESULTS

[0044] 2.1 Identification of chemical components

[0045] The UHPLC-Q-TOF / MS analysis TIC chromatogram of the optimized extract of Huangqi capsule is shown in Figure 2 The identification results are shown in Table 1. According to the steps of method IV, the results of Mass Hunter were first combined with MS-DIAL analysis, and a total of 58 compounds were obtained. Among them, 34 compounds were obtained in positive ion mode, including 24 flavonoids, 5 nucleosides, 1 nucleoside, 1 organic acid, 1 sugar, 1 sterol and 1 other compound; 50 compounds were obtained in negative ion mode, including 27 flavonoids, 11 organic acids, 6 amino acids, 3 nucleosides and 3 sugars. Eight compounds were identified in positive ion mode alone, 24 compounds were identified in negative ion alone, and 26 compounds were identified in both modes. As shown in Figure 2 Compound 48 (Rt = 28.82 min) lost a CO to produce a fragment ion of 273.0, which belongs to a typical flavonoid component. Its C ring undergoes cross-ring cleavage to gradually produce fragment ions of m / z 178.9, 151.0 and 107.0; it can also undergo another one-cross-ring cleavage to produce a fragment ion of m / z 150.8, increasing the intensity at m / z 150.8; in addition, the compound can lose a neutral fragment B ring to produce an ion of m / z 192.8. Based on the above information (Du LY, Tao JH, Jiang S, et al. Metabolic profiles of the Flos Abelmoschus manihot extract by intestinal bacteria from the normal and CKD model rats based on UPLC-Q-TOF / MS [J]. Biomedical chromatography, 2017, 31(2), e3795.), the compound was identified as quercetin; further comparative analysis with the control confirmed the result.

[0046] Secondly, based on the similarity of MS / MS spectra, a visual molecular cage network of Huangqi capsule extract was established, and two obvious flavonoid molecular cages were obtained Figure 4). Among them, molecular cage I is flavonoid aglycone class component, and molecular cage II is flavonoid glycoside class component. Through GNPS network and deduplication analysis of known compounds, 10 flavonoids included in the self-built library were determined, and a total of 13 flavonoids not included in the self-built library were preliminarily identified (Table 2). For example, in this network diagram, the node m / z 301.035 presents m / z 245, 179, 151, 121 and 107, etc. Fragment ions, consistent with the quercetin reference substance, which is determined to be quercetin, which also proves the rationality of GNPS for compound structure analysis from another angle. The node m / z 463.087 presents m / z 301, 300, 273, 245, 193 and 167, etc. Fragment ions, according to the fragmentation rule of flavonoids and the structure predicted by GNPS database, it is determined to be quercetin-7-O-β-D-glucopyranoside structure; The adjacent node m / z 495.075 shows the secondary fragment ions consistent with quercetin-7-O-β-D-glucopyranoside, but the self-built library does not contain the compound, therefore, we speculate that it is myricetin 3'-xyloside structure through MS-DIAL software and the secondary mass spectrum information of the compound.

[0047] Method II, i.e. steps 1, 2, 3, 4 and 5, instrument self-analysis software combined with MS-DIAL analysis; Method III, i.e. steps 1, 2, 3, 4 and 6, instrument self-analysis software combined with GNPS analysis. The advantage of MS-DIAL is that it can further check and supplement the results of MassHunter and identify the structure of unknown compounds combined with MS-FINDER, and the disadvantage is that it takes a long time to confirm each compound due to the large number of automatically identified compounds; The advantage of GNPS is that it can identify compounds of the same type, and the disadvantage is that it may not match traditional Chinese medicine chemical components in the constructed network, and there are false positive results. Therefore, the present application comprehensively analyzes according to the advantages of GNPS and MS-DIAL (Method IV, i.e. steps 1, 2, 3, 4, 5 and 6), improves the efficiency and accuracy of compound identification, and takes Huangkui capsule as an example to carry out identification and analysis of chemical components contained therein, especially flavonoids.

[0048] 3CONCLUSION

[0049] The application is based on a kind of fast, efficient and accurate method for analyzing chemical components in Huangqi Capsules established by UHPLC-Q-TOF / MS combined with MS-DIAL and GNPS strategy, which can accurately identify compounds, generate their molecular formula, complete the identification of compounds based on the self-built compound database by setting conditions such as positive and negative ion mode full scan, molecular ion peak pairing, accurate molecular weight mass error and isotope peak abundance, and 58 compounds are identified; further combined with MS-DIAL and GNPS, 2 molecular cages related to flavonoid structure are successfully identified based on MS / MS similarity, and 13 compounds other than the self-built compound database are identified. The above results help to expand the understanding of the chemical substance basis of Huangqi Capsules, and provide a useful reference for further elucidation of the efficacy of Huangqi Capsules and further development and utilization.

[0050] The preferred embodiments of the application have been specifically described above, but the application is not limited to the described embodiments, and those skilled in the art can make various equivalent modifications or replacements without departing from the spirit of the application. These equivalent modifications or replacements are all included in the scope defined by the claims of the present application.

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Claims

1. A combined analytical method for analyzing the chemical components of okra capsules, characterized in that, Includes the following steps: Step A: Establish a compound database based on the instrument vendor's software platform, Agilent Mass Hunter or Water UNIFI, which includes information such as compound name, molecular formula, molecular weight, and chemical structure. This is called a "self-built library". Step B: Optimize the pretreatment and UHPLC-Q-TOF / MS analysis methods to analyze the traditional Chinese medicine and its chemical profile in positive and / or negative ion modes; optimize different collision energies and acquisition modes to acquire secondary mass spectra of the samples; UHPLC conditions: A Waters ACQUITY UPLC HSS T3 column was used. Mobile phase A was 0.05% formic acid in water, and mobile phase B was 0.05% formic acid in acetonitrile. The elution gradient was set as follows: 0–2 min, 5% B; 2–15 min, 5%–16% B; 15–20 min, 16%–50% B; 20–28 min, 50%–80% B; 28–33 min, 80%–100% B; 33–35 min, 100% B; 35.1–40 min, 5% B. The flow rate was 0.3 ml / min; the column temperature was 30℃; the injection volume was 2 μl; and the detection wavelength was 254 nm. Q-TOF / MS conditions: ESI ion source, data acquisition in both positive and negative ion modes, m / z 100~1700; detection mode: Full MSMS 2 The ion source temperature was 350 ℃, the capillary voltage was 3.5 kV for positive ions and 4.0 kV for negative ions, the nebulizing gas pressure was 45 Psi, the drying gas flow rate was 11 L / min, the sheath gas flow rate was 11 L / min, the sheath gas temperature was 350 ℃, the fragment voltage was 140 V, and the collision energy was 40 eV. Step C: Import the self-built library into the supplier's software platform, Agilent Mass Hunter or Water UNIFI software. After optimizing the extraction parameters and calculating the screening, use the chemical formula search function to extract and match the target compounds based on the precise mass m / z, isotopic abundance, and retention time characteristics Rt to obtain preliminary identification results. Step D: By comparing information such as retention time Rt, multi-stage mass spectrometry (MS / MS) fragmentation, and ultraviolet absorption spectrum, combined with the mass spectrometry fragmentation characteristics of the compound type and literature search, the secondary mass spectrometry of the compound is analyzed to obtain the final identification result; Step E: Based on MS-DIAL, the acquired raw mass spectrometry data is filtered, peak aligned, peak extracted, peak identified and normalized. MS-FINDER is used in conjunction to extract characteristic peaks and mark them by searching the mass spectra of unknown samples. The fingerprint region and fragmentation are compared with the samples in the mass spectrometry database to list a series of likely compounds. The structure of the compounds is then confirmed based on the self-built library and reference information. Step F: Based on the Global Natural Products Social Molecular Network (GNPS) database platform, a molecular network is established. According to the characteristic that similar structural compounds produce similar secondary mass spectrometry fragments, the similarity is integrated into the visualized network through computer algorithms. The known compounds in the network are identified by combining the diagnostic ion identification results. Then, the structure of unknown compounds is inferred based on the similarities and differences of the mass spectrometry data of relevant nodes.

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