Method for determining chemical components in pseudo-ginseng based on UHPLC-QTOF / MS

Abstract

The invention relates to a method for determining chemical components in pseudo-ginseng based on ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UHPLC-QTOF / MS), and the conditions of the ultra-high performance liquid chromatography comprise a C18 chromatographic column and a C18 guard column. Compared with the prior art, the research identifies more types of chemical components. According to the research, 225 chemical components are identified, 35 of the chemical components are reported for the first time in pseudo-ginseng, including 13 of the chemical components are potential new compounds, 11 of the chemical components are found for the first time in the whole plant of pseudo-ginseng and 11 of the chemical components are found for the first time in the roots of pseudo-ginseng, and the 35 of the chemical components can be detected in a pseudo-ginseng sample to be detected.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and relates to a method for determining chemical components in Panax notoginseng based on UHPLC-QTOF / MS. Background Art

[0002] Panax notoginseng, derived from the dried roots and rhizomes of Panax notoginseng (Burk.) FHChen, a plant of the Araliaceae family, has the effects of dispersing blood stasis and stopping bleeding, reducing swelling and relieving pain. It has a long history of medicinal use and extremely high medicinal value.

[0003] Due to the high medicinal value of Panax notoginseng, many researchers are committed to studying the active ingredients of Panax notoginseng. One of the important research methods is to use plant metabolomics technology. Plant metabolomics technology is a high-throughput analytical technology that uses high-throughput detection and data analysis to simultaneously perform qualitative and quantitative analysis on all small molecule metabolites of plants in a specific physiological period. At present, plant metabolomics technology based on LC-MS, GC-MS, and NMR has been widely used in research fields such as medicinal plant origin, medicinal parts, growth years, and crude drug quality control. Ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UHPLC-QTOF / MS) combines the advantages of UHPLC's fast and efficient chromatographic separation capabilities and Q-TOF's high resolution and high sensitivity. It is one of the sample analysis technologies commonly used in the field of plant metabolomics research.

[0004] Existing literature reports that the number of chemical components identified in Panax notoginseng using UHPLC-QTOF / MS technology is relatively small, for example, 132, 151, and 133, respectively. Therefore, further in-depth research on the chemical components in Panax notoginseng is needed to discover more new chemical components. Summary of the invention

[0005] In order to enrich the types and quantities of chemical components of Panax notoginseng and identify Panax notoginseng from different origins, this application provides a method for determining the chemical components in Panax notoginseng based on UHPLC-QTOF / MS plant metabolomics technology, and 73 Panax notoginseng samples from different origins were analyzed and determined. The quality control sample spectra of the method of this application overlap well, and the retention time and peak height are stable, indicating that the instrument and system are stable during the determination process and the measured results are accurate.

[0006] The present invention adopts the following technical scheme to achieve the above-mentioned purpose.

[0007] The present invention provides a method for determining the chemical components in Panax notoginseng based on ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UHPLC-QTOF / MS), wherein the conditions of the ultra-high performance liquid chromatography include: C 18 Chromatographic columns and C 18Guard column.

[0008] Preferably, the C 18 The chromatographic column was Waters ACQUITY UPLC BEH C 18 (2.1mm×100mm, 1.7μm); the C 18 The guard column was Waters ACQUITY UPLC BEH C 18 (2.1mm×5mm, 1.7μm).

[0009] Preferably, the conditions of the ultra-high performance liquid chromatography also include:

[0010] Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile; the gradient elution program was as follows:

[0011]

[0012] Preferably, the conditions of the ultra-high performance liquid chromatography also include: flow rate: 0.4 mL / min; column temperature: 40° C.; injection volume: 2 μL.

[0013] Preferably, the conditions of the quadrupole time-of-flight mass spectrometry include: ESI ion source, negative ion mode scanning; auxiliary gas (GS1): 55 psi, nebulizer gas (GS2): 55 psi, curtain gas: 35 psi;

[0014] Preferably, the conditions of the quadrupole time-of-flight mass spectrometry further include:

[0015] Ion source temperature: 600°C; ion spray voltage: -4500V; collision energy (CE): 40±20V; declustering voltage (DP): -80V; scanning range: m / z 100~1800MS, m / z 50~1800MS 2 ;

[0016] Preferably, the conditions of the quadrupole time-of-flight mass spectrometry further include:

[0017] The IDA (Information dependent analysis) and DBS (Dynamic background substract) modes were used for data collection.

[0018] Preferably, the method further comprises a sample preparation method, and the sample preparation method comprises:

[0019] Weigh the powder of Panax notoginseng medicinal material accurately, place it in a container, add 70% methanol, perform ultrasonic extraction at room temperature, cool it, and absorb the supernatant through a PTFE microporous filter to obtain the product.

[0020] In a specific embodiment, the method of the present invention for determining the chemical components in Panax notoginseng based on ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UHPLC-QTOF / MS) comprises:

[0021] (1) Sample preparation

[0022] Weigh 0.5 g of Panax notoginseng powder, weigh accurately, place in a 100 mL conical flask, add 25 mL of 70% methanol accurately with a pipette, extract at room temperature by ultrasonic (500 W) for 60 min, cool to room temperature for 10 min, and draw the supernatant through a 0.22 μm PTFE microporous filter to obtain;

[0023] (2) The samples prepared above were tested under the following chromatographic and mass spectrometric conditions:

[0024] Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile; flow rate: 0.4 mL / min; column temperature: 40°C; injection volume: 2 μL; gradient elution program was as shown in the following table; ESI ion source, negative ion mode scanning; auxiliary gas (GS1): 55 psi, nebulizer gas (GS2): 55 psi, curtain gas: 35 psi; ion source temperature: 600°C; ion spray voltage: -4500 V; collision energy (CE): 40±20 V; declustering voltage (DP): -80 V; scanning range: m / z 100-1800 MS, m / z 50-1800 MS 2 ; Data collection was performed using IDA (Information dependent analysis) and DBS (Dynamic background substract) modes;

[0025] Gradient elution procedure for UHPLC-QTOF / MS analysis

[0026]

[0027] Compared with the prior art, this study identified a larger number of chemical components. A total of 225 chemical components were identified in this study, of which 35 compounds were reported for the first time in Panax notoginseng, including 13 potential new compounds (see Table 2), 11 were first discovered in the plant Panax notoginseng (the whole plant of Panax notoginseng) (see Table 3), and 11 were first discovered in the roots of Panax notoginseng (see Table 4). The above 35 compounds were detected in all 73 Panax notoginseng samples measured in the present invention. Therefore, the results of this study have enriched the types and quantities of chemical components of Panax notoginseng medicinal materials to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Overlay of TIC graphs in negative ion mode for all QC samples (eight injections in total). DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described below in conjunction with specific implementation methods.

[0030] Embodiment 1:

[0031] In this example, a total of 73 three-year-old Panax notoginseng samples collected from the authentic production area of ​​Wenshan, Yunnan and other non-authentic production areas outside Wenshan in Yunnan Province, Sichuan and Chongqing, and Guizhou were crushed and sieved to obtain 73 portions of Panax notoginseng medicinal material powder, completing the sample preparation work, and UHPLC-QTOF / MS was used to measure the 73 Panax notoginseng samples.

[0032] 1. Experimental Materials

[0033] (I) Sample information

[0034] The Panax notoginseng samples were 73 three-year-old Panax notoginseng collected from 12 production areas including the authentic production area of ​​Wenshan, Yunnan and the non-authentic production areas of Kunming, Yuxi, Qujing, Honghe, Lijiang, Pu'er, Tengchong, Funing, Chuxiong in Yunnan and Xingyi in Guizhou, and the Sichuan-Chongqing region (including Suining, Sichuan, Langzhong, Sichuan, Tongnan, Chongqing, and Rongchang, Chongqing) from September to November 2019 and September to November 2020. All samples were identified by the Pharmacognosy Laboratory, School of Pharmacy, Peking University as the underground parts of Panax notoginseng (Burk.) FHChen of the Araliaceae family, and the voucher specimens are stored in the Pharmacognosy Laboratory, School of Pharmacy, Peking University.

[0035] After cleaning the underground samples while they are still fresh, place them on newspapers and dry them in the laboratory (room temperature is about 20°C, relative humidity is about 30%). From the time the herbs are dry to the touch, weigh them once every 24 hours. If the weight change is within 1% within 48 hours, it is considered to be air-dried. The drying process takes about 20 to 35 days. After about 7 days of drying, separate the main root, rhizome, branch root, and fibrous root. All samples are experimental materials with the main root.

[0036] (II) Instruments and Equipment

[0037] AB SCIEX Triple TOF 6600+ LC / MS (SCIEX, USA): including SCIEX Exion LC ADSystem; chromatographic column: Waters ACQUITY UPLC BEH C 18 (2.1 mm × 100 mm, 1.7 μm); Guard column: Waters ACQUITY UPLC BEH C 18 (2.1mm×5mm, 1.7μm); one-tenth balance (OHAUS, AR1140, USA); KQ-500DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Milli-Q ultrapure water preparation instrument (Millipore, USA); grinder (Xiaoxiong Electric Co., Ltd., FSJ-A05N6); Pharmacopoeia No. 4 sieve (R40 / 3 series, Shaoxing Shangyu Huafeng Hardware Instrument Co., Ltd.).

[0038] (III) Reagents and consumables

[0039] Acetonitrile (LC-MS grade, Fisher Scientific, lot No: 204142); methanol (LC-MS grade, Fisher Scientific, lot No: 158531); formic acid (LC-MS grade, Fisher Scientific, lot No: 205178); water: Milli-Q ultrapure water; 0.22 μm PTFE microporous filter (Beijing Dima Technology Co., Ltd., lot No: F201201).

[0040] 2. Experimental Methods

[0041] (I) Sample preparation Each sample was crushed in a grinder and passed through the pharmacopoeia No. 4 sieve. The particles that could not pass through the sieve were ground in a mortar and then sieved until all the samples were crushed and sieved to obtain 73 portions of Panax notoginseng medicinal powder. In addition, 0.2 g of each of the 73 portions of medicinal powder was weighed accurately, placed in the same 50 mL centrifuge tube, and fully mixed as quality control (QC) samples.

[0042] Weigh 0.5g of each of 73 portions of Panax notoginseng powder, weigh accurately, place in 100mL conical flasks, add 25mL of 70% methanol accurately with a pipette, and seal with a sealing film. Ultrasonic extraction (500W) at room temperature for 60min (the actual ultrasonic water temperature varies between 18 and 23℃, the same below), cool to room temperature for 10min, and draw the supernatant through a 0.22μm PTFE microporous filter to obtain the sample solution to be tested.

[0043] Weigh 0.5 g of QC sample powder, accurately weigh it, place it in a 100 mL conical flask, accurately add 25 mL of 70% methanol with a pipette, and seal it with a sealing film. Ultrasonic extraction (500 W) at room temperature for 60 minutes, cool it to room temperature for 10 minutes, and draw the supernatant through a 0.22 μm PTFE microporous filter to obtain the QC sample solution.

[0044] (II) Chromatographic and mass spectrometry conditions

[0045] Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile; flow rate: 0.4 mL / min; column temperature: 40°C; injection volume: 2 μL; gradient elution program as shown in Table 1; ESI ion source, negative ion mode scanning; auxiliary gas (GS1): 55 psi, nebulizer gas (GS2): 55 psi, curtain gas: 35 psi; ion source temperature: 600°C; ion spray voltage: -4500 V; collision energy (CE): 40 ± 20 V; declustering voltage (DP): -80 V; scanning range: m / z 100-1800 MS, m / z 50-1800 MS 2 ; The IDA (Information dependent analysis) and DBS (Dynamic background substract) modes were used for data collection.

[0046] Table 1 Gradient elution program for UHPLC-QTOF / MS analysis

[0047]

[0048] (III) Experimental steps

[0049] Before officially running the sequence, blank solvent (70% methanol) and three QC samples were first injected to ensure that there were no interfering peaks in the system and blank solvent and the instrument status was stable. Then the sequence was officially run. During the sequence running process, one blank solvent and one QC sample were inserted for every seven samples (there were no interfering peaks in the blank solvent). All samples were injected and analyzed in random order, and the sequence was run continuously until all samples were analyzed.

[0050] In this experiment, the QC samples were prepared by mixing all the samples to be tested in equal amounts using the same preparation method as the samples to be tested. Its function is to monitor whether the instrument and system are stable during the sequence operation. After the sequence operation is completed, all the spectra of the QC samples are superimposed to observe whether the retention time and peak height overlap of the ion peaks in the graphs are good, and the clustering effect of all QC samples is evaluated using multivariate statistical analysis methods to comprehensively judge whether the sample analysis method and the determination process are stable and accurate.

[0051] 3. Experimental Results

[0052] The sequence was run continuously for about 63 hours, and eight QC samples were injected. The overlay of the TIC graphs of the eight QC samples in negative ion mode is shown in the figure below. Figure 1 shown.

[0053] 4. Mass spectrometry data preprocessing and multivariate statistical analysis

[0054] The applicant first used XCMS plus online software to preprocess the large amount of data obtained by the previous UHPLC-QTOF / MS measurement, and then used multivariate statistical analysis methods (PCA, PLS-DA and OPLS-DA) to analyze the preprocessed LC-MS data sets.

[0055] 1. Data analysis software

[0056] AB SCIEX Triple TOF 6600+ LC / MS software: XCMS plus online software; SIMCA14.1 software (Umetrics, Umea, Sweden); Microsoft Excel 2016.

[0057] 2. Data preprocessing and multivariate statistical analysis methods

[0058] The parameters of XCMS plus software were set as follows: characteristic peak detection (centWave): m / z=30 (maximaltolerated m / z deviation in consecutive scans), minimum peak width: 10s, maximumpeak width: 60s, Signal / Noise threshold: 6. Retention time correction (obiwarp): profStep=1. Peak alignment parameters: bw=5, minfrac=0.5, mzwid=0.025. The remaining parameters were set to default. The XCMS multigroup method was used to group the LC-MS data of Panax notoginseng taproot samples from 12 production areas (Wenshan, Kunming, Yuxi, Qujing, Honghe, Lijiang, Pu'er, Tengchong, Funing, Chuxiong in Yunnan and Xingyi, Guizhou, and Sichuan and Chongqing) to obtain the retention time (t R ), mass-to-charge ratio (m / z), and peak area, with a total of 27,280 ion information.

[0059] 5. Mass spectrometry analysis

[0060] A total of 225 chemical components were found by analyzing the mass spectrometry results, of which 35 compounds were reported for the first time in Panax notoginseng, including 13 potential new compounds (see Table 2), 11 first discovered in the whole plant of Panax notoginseng (see Table 3), and 11 first discovered in the root of Panax notoginseng (see Table 4). The above 35 compounds were detected in all 73 Panax notoginseng samples determined by the present invention.

[0061] Table 2 Summary of information on potential new compounds

[0062]

[0063] Table 3 Summary of compounds first discovered in Panax notoginseng

[0064]

[0065] Table 4 Summary of compounds first discovered in the roots of Panax notoginseng

[0066]

[0067]

Claims

1. A method for determining the chemical components of Panax notoginseng based on ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UHPLC-QTOF / MS), wherein: The conditions of the ultra-high performance liquid chromatography include: C 18 Chromatographic columns and C 18 Guard column.

2. The method according to claim 1, wherein: The C 18 The chromatographic column was Waters ACQUITY UPLC BEH C 18 (2.1mm×100mm, 1.7μm); the C 18 The guard column was Waters ACQUITY UPLC BEH C 18 (2.1mm×5mm, 1.7μm).

3. The method according to claim 1 or 2, wherein: The conditions of the ultra-high performance liquid chromatography also include: Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile; the gradient elution program was as follows:

4. The method according to any one of claims 1 to 3, wherein: The conditions of the ultra-high performance liquid chromatography also include: flow rate: 0.4 mL / min; column temperature: 40° C.; injection volume: 2 μL.

5. The method according to any one of claims 1 to 4, wherein: The conditions of the quadrupole time-of-flight mass spectrometry include: ESI ion source, negative ion mode scanning; auxiliary gas (GS1): 55 psi, nebulizer gas (GS2): 55 psi, curtain gas: 35 psi.

6. The method according to any one of claims 1 to 5, wherein: The conditions of the quadrupole time-of-flight mass spectrometry also include: Ion source temperature: 600°C; ion spray voltage: -4500V; collision energy (CE): 40±20V; declustering voltage (DP): -80V; scanning range: m / z 100~1800MS, m / z 50~1800MS 2 .

7. The method according to any one of claims 1 to 6, wherein: The conditions of the quadrupole time-of-flight mass spectrometry also include: The IDA (Information dependent analysis) and DBS (Dynamic background substract) modes were used for data collection.

8. The method according to any one of claims 1 to 7, wherein: The method also includes a sample preparation method, and the sample preparation method includes: Weigh the powder of Panax notoginseng medicinal material accurately, place it in a container, add 70% methanol, perform ultrasonic extraction at room temperature, cool it, and absorb the supernatant through a PTFE microporous filter to obtain the product.

9. The method according to any one of claims 1 to 8, wherein: The method comprises: (1) Sample preparation Weigh 0.5 g of Panax notoginseng powder, weigh accurately, place in a 100 mL conical flask, add 25 mL of 70% methanol accurately with a pipette, extract at room temperature by ultrasonic (500 W) for 60 min, cool to room temperature for 10 min, and draw the supernatant through a 0.22 μm PTFE microporous filter to obtain; (2) The samples prepared above were tested under the following chromatographic and mass spectrometric conditions: Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile; flow rate: 0.4 mL / min; column temperature: 40°C; injection volume: 2 μL; gradient elution program was as shown in the following table; ESI ion source, negative ion mode scanning; auxiliary gas (GS1): 55 psi, nebulizer gas (GS2): 55 psi, curtain gas: 35 psi; ion source temperature: 600°C; ion spray voltage: -4500 V; collision energy (CE): 40±20 V; declustering voltage (DP): -80 V; scanning range: m / z 100-1800 MS, m / z 50-1800 MS 2 ; Data collection was performed using IDA (Information dependent analysis) and DBS (Dynamic background substract) modes; Gradient elution procedure for UHPLC-QTOF / MS analysis

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