Method for determining multiple active components in scutellaria baicalensis based on UPLC-QTOF-MS

Abstract

The invention relates to the technical field of chemical analysis. The invention provides a method for determining various active components in scutellaria baicalensis based on UPLC-QTOF-MS. The technical method provided by the invention has the characteristics of rapidness and accuracy, and can qualitatively and quantitatively analyze the types and contents of the active components in the scutellaria baicalensis; the variety and the content of the active ingredients in the roots, the stems and the leaves of the scutellaria baicalensis and in the scutellaria baicalensis obtained at different harvesting times can be compared and analyzed, which is of great significance to the research on the subsequent quality control of the scutellaria baicalensis medicinal material.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis, and in particular to a method for determining multiple active ingredients in scutellaria baicalensis based on UPLC-QTOF-MS. Background Art

[0002] The Chinese Pharmacopoeia records that Scutellaria baicalensis is the dried root of the Lamiaceae plant (Scutellaria baicalensis Georgi). It is cold in nature and bitter in taste. It has the effects of clearing away heat and dampness, purging fire and detoxifying, stopping bleeding, and calming the fetus. Shandong is a province with rich resources of traditional Chinese medicine. Among them, Scutellaria baicalensis is one of the representative authentic Shandong medicinal materials "Ten Shandong Flavors", with a long cultivation history and high market recognition. The main chemical components of Scutellaria baicalensis are flavonoids, phenolic acids, amino acids, phenylethanol, sterols, etc. Among them, flavonoids are the main components, including baicalin, baicalein, wogonin, baicalein, melaleucain, baicalein, etc.

[0003] The traditional medicinal part of Scutellaria baicalensis is the root, and its stems and leaves are often discarded. There is little research on its stems and leaves. The types and contents of compounds in the stems and leaves of Scutellaria baicalensis are unknown, and it also causes a waste of resources. As comprehensive utilization becomes the trend of green development of Chinese medicine resources, whether the stems and leaves of Scutellaria baicalensis have good utilization value and development potential is increasingly worthy of attention. The identification and detection of the types and contents of compounds in the stems and leaves is one of the key issues to be solved. Therefore, it is very necessary to obtain a detection technology solution that is convenient and efficient, has high accuracy, high resolution, and a wide scanning range, and can perform qualitative and quantitative analysis. Summary of the invention

[0004] The object of the present invention is to provide a method for determining the chemical components in Scutellaria baicalensis. The UPLC-QTOF-MS method of the present invention and scientific parameter conditions are fast and accurate, and the types and contents of active components in Scutellaria baicalensis can be qualitatively and quantitatively analyzed. The types and contents of active components in the roots, stems and leaves of Scutellaria baicalensis and Scutellaria baicalensis harvested at different times can also be compared and analyzed, which is of great significance for subsequent research on the quality control of Scutellaria baicalensis medicinal materials.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for determining the chemical components in Scutellaria baicalensis based on UPLC-QTOF-MS, characterized in that it comprises the following steps:

[0007] (1) Grind the Scutellaria baicalensis sample to be tested, mix it with a solvent, perform ultrasonic extraction, and centrifuge, and take the supernatant as the test solution;

[0008] (2) Weigh the standard substances baicalin, wogonin, baicalein, wogonin, apigenin, apigenin 7-O-β-D-glucuronide and chrysin 7-O-β-D-glucuronide respectively, transfer them into a volumetric flask, add methanol to make up to volume, and prepare the reference solution of the corresponding compound;

[0009] (3) The test solution and the reference solution are respectively detected by ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry, and qualitative and quantitative analysis are performed after the component substance peaks are obtained.

[0010] Preferably, in step (1), the mass volume ratio of the crushed Scutellaria baicalensis sample to be tested and the solvent is (0.7-1.5) g: (12-20) mL.

[0011] Preferably, the solvent is ethanol with a volume fraction of 55-75%.

[0012] Preferably, the power of the ultrasonic extraction in step (1) is 150-250 W, the frequency of the ultrasonic extraction is 25-35 kHz, the time of the ultrasonic extraction is 15-25 min / time, and the number of ultrasonic extractions is 1-2 times.

[0013] Preferably, the centrifugal speed in step (1) is 14000-18000 r / min, and the centrifugal time is 6-13 min.

[0014] Preferably, the volume fraction of methanol in step (2) is 65-75%.

[0015] Preferably, the chromatographic conditions in the detection process of step (3) are: the chromatographic column is ACQUITY UPLC BEH C 18 The chromatographic column was (2.1 mm × 100 mm, 1.7 μm), the mobile phase A was 0.1% formic acid in acetonitrile, the mobile phase B was 0.1% formic acid in water, the column temperature was 40°C, the injection volume was 2 μL, and the flow rate was 0.4 mL / min.

[0016] Preferably, the gradient elution program in the detection process of step (3) is 0-5 min, 31%→24% A, 69%→76% B; 5-9 min, 24%→30% A, 76%→70% B; 9-15 min, 30%→32% A, 70%→68% B; 15-25 min, 32%→45% A, 68%→55% B; 25-40 min, 45%→47% A, 55%→53% B.

[0017] Preferably, the mass spectrometry conditions in the detection process of step (3) are electrospray ionization source (ESI), positive and negative ion detection modes (ESI+, ESI-), and full scan mode; MS parameters: mass range m / z 50-1500Da, scan time 0.2s.

[0018] Preferably, the Scutellaria baicalensis sample to be tested includes the root part and the stem and leaf part of Scutellaria baicalensis.

[0019] By adopting the above technical solution, the present invention has the following beneficial effects:

[0020] 1. The present invention can accurately determine the types and content changes of different compounds in Scutellaria baicalensis by combining the determination method with optimized operating conditions, and the operation is simple, the analysis sensitivity is higher, the resolution is higher, the accuracy is good, and it has good repeatability and reproducibility.

[0021] 2. The chemical components and contents of different parts of Scutellaria baicalensis and different harvesting times were determined by the technical solution of the present invention. The experimental results show that the contents of baicalin, wogonin, baicalein, and wogonin in the root of Scutellaria baicalensis are relatively high, and the contents of compounds such as baicalein, apigenin, apigenin 7-O-β-D-glucuronide, and chrysin 7-O-β-D-glucuronide in the stems and leaves of Scutellaria baicalensis are relatively high; the contents of compounds in the roots and stems and leaves of Scutellaria baicalensis harvested in March and November are relatively high, while the contents of compounds in different parts of Scutellaria baicalensis harvested in other months are relatively low. The quality of traditional Chinese medicine depends on the content of effective chemical components, which also provides a scientific basis for the quality research of Scutellaria baicalensis medicinal materials. DETAILED DESCRIPTION

[0022] The present invention provides a method for determining the chemical components in Scutellaria baicalensis based on UPLC-QTOF-MS, comprising the following steps:

[0023] (1) Grind the Scutellaria baicalensis sample to be tested, mix it with a solvent, perform ultrasonic extraction, and centrifuge, and take the supernatant as the test solution;

[0024] (2) Weigh the standard substances baicalin, wogonin, baicalein, wogonin, apigenin, apigenin 7-O-β-D-glucuronide and chrysin 7-O-β-D-glucuronide respectively, transfer them into a volumetric flask, add methanol to make up to volume, and prepare the reference solution of the corresponding compound;

[0025] (3) The test solution and the reference solution are respectively detected by ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry, and qualitative and quantitative analysis are performed after the component substance peaks are obtained.

[0026] In the present invention, the Scutellaria baicalensis sample to be tested includes the root part and the stem and leaf part of Scutellaria baicalensis.

[0027] In the present invention, the scutellaria sample to be tested is crushed, preferably crushed to a mesh size of 50-80 mesh, further preferably 55-70 mesh, and further preferably 60 mesh. The mass volume ratio of the scutellaria sample to be tested after crushing and the solvent mixed in the present invention is preferably (0.7-1.5) g: (12-20) mL, further preferably (0.8-1.2) g: (15-19) mL, and further preferably 1 g: 18 mL. The solvent in the present invention is ethanol, and the volume fraction is preferably 55-75%, further preferably 60-72%, and further preferably 70%.

[0028] In the present invention, the power of the ultrasonic extraction is preferably 150-250W, further preferably 180-230W, and further preferably 200W; the frequency of the ultrasonic extraction is preferably 25-35kHz, further preferably 28-32kHz, and further preferably 30kHz; the time of the ultrasonic extraction is preferably 15-25min / time, further preferably 18-23min / time, and further preferably 22min / time; the number of ultrasonic extractions is preferably 1-2 times, and further preferably 2 times.

[0029] In the present invention, the centrifugal speed is preferably 14000-18000 r / min, further preferably 15000-17000 r / min, further preferably 16000 r / min; the centrifugal time is preferably 6-13 min, further preferably 7-11 min, further preferably 8 min.

[0030] In the present invention, the volume fraction of methanol is preferably 65-75%, further preferably 68-72%, and further preferably 70%.

[0031] In the present invention, the chromatographic conditions in the detection process are: the chromatographic column is an ACQUITYUPLC BEH C18 chromatographic column (2.1 mm×100 mm, 1.7 μm), the mobile phase A is 0.1% formic acid acetonitrile, the mobile phase B is 0.1% formic acid aqueous solution, the column temperature is 40° C., the injection volume is 2 μL, and the flow rate is 0.4 mL / min.

[0032] In the present invention, the gradient elution program in the detection process is 0-5 min, 31%→24% A, 69%→76% B; 5-9 min, 24%→30% A, 76%→70% B; 9-15 min, 30%→32% A, 70%→68% B; 15-25 min, 32%→45% A, 68%→55% B; 25-40 min, 45%→47% A, 55%→53% B.

[0033] In the present invention, the mass spectrometry conditions in the detection process are electrospray ionization source (ESI), positive and negative ion detection modes (ESI+, ESI-), and full scan mode; MS parameters: mass range m / z 50-1500Da, scan time 0.2s.

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] Example 1

[0036] (I) Preparation of test samples and reference substances

[0037] The root and stem and leaf medicinal materials of Radix Scutellariae were respectively placed in a 60°C oven to dry to constant weight, and then crushed to 50 meshes by a grinder. 1 g of Radix Scutellariae powder was mixed with 18 mL of 70% ethanol, and ultrasonically extracted twice at 200 W, 30 kHz, and 22 min / time. Then, centrifuged at 16000 r / min for 8 min, and the supernatant was taken as the test solution. The information of the root and stem and leaf medicinal materials of Radix Scutellariae is shown in Table 1.

[0038] Table 1 Information on samples of Radix Scutellariae

[0039]

[0040]

[0041] Accurately weigh 0.2 g each of baicalin, wogonin, baicalein, wogonin, apigenin, apigenin 7-O-β-D-glucuronide and chrysin 7-O-β-D-glucuronide (purchased from Shanghai Yuanye Biotechnology Co., Ltd.), add 70% methanol to dissolve and make up to 10 mL, shake well to obtain a mixed reference solution with a concentration of 20 μg / mL. (two)

[0043] The test solution and the reference solution were detected by ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry, respectively, and the chromatograms of the test solution and the reference solution were obtained for analysis. At the same time, the structure of the compound was further identified in combination with relevant literature reports.

[0044] The chromatographic conditions were as follows: the chromatographic column was ACQUITY UPLC BEH C 18 The chromatographic column was (2.1 mm × 100 mm, 1.7 μm), the mobile phase A was 0.1% formic acid in acetonitrile, the mobile phase B was 0.1% formic acid in water, the column temperature was 40°C, the injection volume was 2 μL, and the flow rate was 0.4 mL / min.

[0045] The gradient elution program was as follows: 0-5 min, 31%→24% A, 69%→76% B; 5-9 min, 24%→30% A, 76%→70% B; 9-15 min, 30%→32% A, 70%→68% B; 15-25 min, 32%→45% A, 68%→55% B; 25-40 min, 45%→47% A, 55%→53% B.

[0046] The mass spectrometry conditions were electrospray ionization source (ESI), positive and negative ion detection modes (ESI+, ESI-), and full scan mode; MS parameters: mass range m / z 50-1500Da, scan time 0.2s; spray voltage: -4500V; nebulizer gas pressure (GS1): 0.55kPa; auxiliary gas pressure (GS2): 0.5kPa.

[0047] According to the analysis of the test spectra of the test solution and the reference solution, a total of 53 compounds were separated and identified, including 45 flavonoids, 5 organic acid compounds, and 3 other compounds, including protocatechuic acid, malic acid, D-glucaric acid, baicalein-7-O-glucoside, scutellaria baicalin, baicalin, wogonin, naringenin-7-O-glucuronide, baicalein, wogonin, plantago asiatica, luteolin-7-O-glucuronide, chrysin-7-O-glucuronide and chrysin, etc.

[0048] (III) Methodological investigation

[0049] 1. Precision experiment

[0050] The injection was repeated 6 times on the same day, the peak areas of the 7 target components were recorded, and the RSD values ​​of their peak areas were calculated. The injection was repeated for 3 days, and the peak areas of the 7 target components were measured for 3 days according to the above chromatographic conditions, and the RSD values ​​of their peak areas were calculated. The results showed that the RSD values ​​of the 7 target components, baicalin, wogonin, baicalein, wogonin, apigenin, apigenin 7-O-β-D-glucuronide, and chrysin 7-O-β-D-glucuronide, were 1.11%, 0.98%, and 0.99%, respectively.

[0051] 1.11%, 1.75%, 1.64%, 2.34% and 0.87%, all less than 15%, indicating that the precision of the instrument is good.

[0052] 2. Stability test

[0053] 1 mL of the same test solution was accurately aspirated and injected at 0, 2, 4, 8, 12, 16 and 24 h under the above-mentioned chromatographic conditions, and the peak area RSDs of the six target components were calculated. The results showed that the peak area RSD values ​​of the seven target components, including baicalin, wogonin, baicalein, wogonin, apigenin, apigenin 7-O-β-D-glucuronide and chrysin 7-O-β-D-glucuronide, were 1.68%, 2.05%, 1.59%, 3.86%, 3.52%, 5.14% and 4.14%, respectively, which were all less than 15%. The test solution had good stability within 24 h.

[0054] 3. Repeatability

[0055] The same test solution was taken and measured according to the above chromatographic conditions, and the peak area RSDs of the six target components were calculated. The results showed that the peak area RSD values ​​of the seven target components, including baicalin, wogonin, baicalein, wogonin, apigenin, apigenin 7-O-β-D-glucuronide and chrysin 7-O-β-D-glucuronide, were 2.78%, 2.51%, 3.68%, 3.45%, 3.75%, 2.87% and 4.39%, respectively, which were all less than 15%. The method of the present invention has good repeatability.

[0056] (IV) Principal component analysis

[0057] The principal component analysis of the spectra obtained from the Scutellaria baicalensis samples numbered 1-8 was performed using SIMCA-P software to analyze the differences in the types of compounds in different parts of Scutellaria baicalensis. The contents of baicalin, wogonin, baicalein, wogonin, apigenin, apigenin 7-O-β-D-glucuronide and chrysin 7-O-β-D-glucuronide in the Scutellaria baicalensis samples with different numbers were calculated according to the external standard method.

[0058] Table 2 Content of target components in different numbers of Scutellaria baicalensis samples

[0059]

[0060]

[0061] The results showed that the overall chemical composition of the root and stem and leaf of Scutellaria baicalensis was quite different, while the chemical composition of the stem and leaf of Scutellaria baicalensis was similar. The compound that contributed the most to the difference between different parts of Scutellaria baicalensis was mainly baicalin. The content of baicalin in the root of Scutellaria baicalensis was very high, while the content in the stem and leaf was relatively low. However, compounds such as baicalein, apigenin, apigenin 7-O-β-D-glucuronide, and chrysin 7-O-β-D-glucuronide existed in small amounts in the root of Scutellaria baicalensis, and their contents were relatively high in the stem and leaf of Scutellaria baicalensis, so they still had good effects of clearing away heat and dampness, purging fire and detoxifying, and anti-inflammatory. The contents of baicalin, wogonin, baicalein, and wogonin were relatively high in the root of Scutellaria baicalensis, and their contents were relatively low in the stem and leaf of Scutellaria baicalensis, so the bitter and cold properties of Scutellaria baicalensis were relatively weakened.

[0062] The results also showed that there were obvious differences in the content of compounds in the roots and stems and leaves of Scutellaria baicalensis harvested at different times. The content of compounds in the roots and stems and leaves of Scutellaria baicalensis harvested in March and November was relatively high, while the content of compounds in different parts of Scutellaria baicalensis harvested in other months was relatively low.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for determining the chemical components in Scutellaria baicalensis based on UPLC-QTOF-MS, characterized in that: The steps include: (1) Grind the Scutellaria baicalensis sample to be tested, mix it with a solvent, perform ultrasonic extraction, and centrifuge, and take the supernatant as the test solution; (2) Weigh the standard substances baicalin, wogonin, baicalein, wogonin, apigenin, apigenin 7-O-β-D-glucuronide and chrysin 7-O-β-D-glucuronide respectively, transfer them into a volumetric flask, add methanol to make up to volume, and prepare the reference solution of the corresponding compound; (3) The test solution and the reference solution are respectively detected by ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry, and qualitative and quantitative analysis are performed after the component substance peaks are obtained.

2. The method according to claim 1, characterized in that: The mass volume ratio of the crushed Scutellaria baicalensis sample to be tested and the solvent mixed in step (1) is (0.7-1.5) g: (12-20) mL; The solvent is ethanol with a volume fraction of 55-75%.

3. The method according to claim 1, characterized in that: The power of the ultrasonic extraction in step (1) is 150-250W, the frequency of the ultrasonic extraction is 25-35kHz, the time of the ultrasonic extraction is 15-25min / time, and the number of ultrasonic extractions is 1-2 times.

4. The method according to claim 1, characterized in that: The centrifugal speed in step (1) is 14000-18000 r / min, and the centrifugal time is 6-13 min.

5. The method according to claim 1, characterized in that The volume fraction of methanol in step (2) is 65-75%.

6. The method according to claim 1, characterized in that The chromatographic conditions in the detection process of step (3) are as follows: the chromatographic column is ACQUITY UPLC BEH C 18 The chromatographic column was (2.1 mm × 100 mm, 1.7 μm), the mobile phase A was 0.1% formic acid in acetonitrile, the mobile phase B was 0.1% formic acid in water, the column temperature was 40°C, the injection volume was 2 μL, and the flow rate was 0.4 mL / min.

7. The method according to claim 1, characterized in that The gradient elution program in the detection process of step (3) is as follows: 0-5 min, 31%→24% A, 69%→76% B; 5-9 min, 24%→30% A, 76%→70% B; 9-15 min, 30%→32% A, 70%→68% B; 15-25 min, 32%→45% A, 68%→55% B; 25-40 min, 45%→47% A, 55%→53% B.

8. The method according to claim 1, characterized in that The mass spectrometry conditions in the detection process of step (3) are electrospray ionization source (ESI), positive and negative ion detection modes (ESI+, ESI-), and full scan mode; MS parameters: mass range m / z 50-1500 Da, scan time 0.2 s.

9. The method according to claim 1, characterized in that: The scutellaria baicalensis sample to be tested includes the root of scutellaria baicalensis and the stem and leaf of scutellaria baicalensis.

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