A method for determining the content of chemical components in Panax notoginseng based on UPLC-MS / MS

Abstract

The present invention belongs to the technical field of quality control of Panax ginseng. The present invention discloses a method for determining the content of chemical components in Panax ginseng medicinal materials based on UPLC-MS / MS. The present invention establishes a method for simultaneously determining the content of 12 chemical components in Panax ginseng medicinal materials based on UPLC-MS / MS, and applies the method to the determination of the content of Panax ginseng medicinal materials from different batches. The detection method of the present invention has high sensitivity and strong specificity, and has good precision, repeatability, and stability. It can reflect the differences in the content of the measured chemical components in different batches of Panax ginseng medicinal materials and can provide a reference for the quality evaluation of Panax ginseng medicinal materials.

Description

Technical Field

[0001] The present invention relates to the technical field of quality control of Panax notoginseng, and in particular to a method for determining the contents of chemical components in Panax notoginseng medicinal materials based on UPLC-MS / MS. Background Art

[0002] Chinese ginseng, also known as Huashan ginseng, is the dried root of PhysochlainainfundibularisKuang, a plant of the Solanaceae family. It is named after Huashan Mountain in the Qinling Mountains where it is mainly produced. The "Chinese Pharmacopoeia" (2020 edition) records that Chinese ginseng is warm in nature, sweet and slightly bitter in taste, enters the lung and heart meridians, and is poisonous; it has the effects of warming the lungs and removing phlegm, relieving asthma and cough, and calming the nerves; clinically, it is mainly used for symptoms such as palpitations, insomnia, easy fright, and cough with cold phlegm. Chinese ginseng has a rich variety of chemical components, mainly including alkaloids, coumarins, organic acids, and volatile oils.

[0003] Due to factors such as geographical conditions, climate, and environment, the quality of Chinese ginseng produced in different production areas is inevitably different. Therefore, there is an urgent need to establish an efficient and accurate method for determining the content of multiple components in Chinese ginseng to provide a basis for quality evaluation of Chinese ginseng. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for determining the content of chemical components in Panax notoginseng based on UPLC-MS / MS, so as to solve the problem that there is a gap in the research on the determination method of the content of multiple components in Panax notoginseng in the art.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for determining the content of chemical components in Panax notoginseng based on UPLC-MS / MS, wherein the chemical components are scopolamine, anisodamine, hyoscyamine, protocatechuic acid, hyoscyamine, scopolamine, p-hydroxybenzoic acid, caffeic acid, tropic acid, scopoletin, isoquercetin and scopolamine.

[0007] The method comprises the following steps:

[0008] (1) Scopolamine, anisodamine, hyoscyamine, protocatechuic acid, hyoscyamine, scopolamine, p-hydroxybenzoic acid, caffeic acid, tropic acid, scopolamine, isoquercetin, and scopolamine were mixed with methanol to obtain standard reference solutions of the chemical components;

[0009] (2) mixing each standard reference solution obtained in step (1) with methanol to obtain a mixed reference solution; measuring the mixed reference solution of different concentrations using UPLC-MS / MS, establishing a linear regression equation with the concentration of the chemical component as the abscissa and the peak area of ​​the chemical component as the ordinate;

[0010] (3) Extracting Panax notoginseng with methanol to obtain a Panax notoginseng medicinal material solution, filtering the extracted solution to obtain a test solution; measuring the test solution using UPLC-MS / MS to obtain the peak area of ​​the chemical components of the test solution, and combining the linear regression equation obtained in step (2) to obtain the concentration of the chemical components of the test solution.

[0011] Preferably, in steps (1), (2) and (3), the volume fraction of methanol is independently 40 to 60%.

[0012] Preferably, in step (1), the concentration of the standard reference solution of each chemical component is independently 0.5 to 2 mg / mL.

[0013] Preferably, in the mixed reference solution of step (2), the concentrations of anisodamine, scopolamine, scopolamine lactone, and hyoscyamine are 40-60 μg / mL, the concentration of scopolamine is 180-220 μg / mL, the concentration of scopolamine lactone is 1-3 μg / mL, the concentration of protocatechuic acid is 7-9 μg / mL, the concentrations of scopolamine, tropic acid, caffeic acid, and p-hydroxybenzoic acid are 15-25 μg / mL, and the concentration of isoquercetin is 0.5-1.5 μg / mL.

[0014] Preferably, the mixed reference solution of different concentrations in step (2) is obtained by diluting the mixed reference solution in sequence by 2 times, 2.5 times, 2 times, 2 times, 2.5 times, 2 times, 2.5 times, 2 times, and 2 times; the volume fraction of methanol used for dilution is 40-60%.

[0015] Preferably, in step (3), the particle size of the Panax notoginseng medicinal material is ≥50 mesh; the extraction is ultrasonic extraction, the extraction time is 50 to 80 minutes, the ultrasonic power is 280 to 320 W, and the ultrasonic frequency is 30 to 50 kHz; the concentration of the Panax notoginseng medicinal material solution is 18 to 22 mg / mL; and the pore size of the filter membrane used for filtration is 0.22 μm.

[0016] Preferably, in steps (2) and (3), the UPLC-MS / MS chromatographic conditions are:

[0017] The chromatographic column is ACQUITY UPLC C SHC 18 Column; in the mobile phase, phase A is a formic acid aqueous solution with a volume fraction of 0.05-0.2%, and phase B is acetonitrile; the elution method is gradient elution; the flow rate is 0.2-0.4 mL / min; the column temperature is 28-32° C.; and the injection volume is 1.9-2.2 μL.

[0018] Preferably, the gradient elution procedure is: 0 min, 10% volume fraction of phase B, 90% volume fraction of phase A; 7 min, 55% volume fraction of phase B, 45% volume fraction of phase A; 8 min, 61% volume fraction of phase B, 39% volume fraction of phase A.

[0019] Preferably, in steps (2) and (3), the mass spectrometry conditions of the UPLC-MS / MS are:

[0020] The ion source is an electrospray ion source; the detection mode is multiple reaction ion monitoring; the scanning mode is positive and negative ion scanning mode; the gas temperature is 280-320°C; the gas flow rate is 10-12 L / min; and the nebulizer pressure is 32-38 Psig.

[0021] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention establishes a UPLC-MS / MS method for simultaneously determining the contents of 12 chemical components in Panax notoginseng, including hyoscyamine, anisodamine, hyoscyamine, protocatechuic acid, hyoscyamine, scopolamine, p-hydroxybenzoic acid, caffeic acid, tropic acid, scopoletin, isoquercetin and scopolamine, in Panax notoginseng medicinal materials. The contents of the components in different batches of medicinal materials are compared, providing a reference for the quality evaluation of Panax notoginseng and the research on the basis of pharmacological effects. The detection method of the present invention has high sensitivity and strong specificity, and has good precision, repeatability and stability. It can reflect the content differences of the measured chemical components in different batches of Panax notoginseng medicinal materials, and the above can provide a reference for the quality evaluation of Panax notoginseng medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0024] Figure 1 The MRM diagram of the mixed control sample solution and the test solution described in Example 1, wherein A is the mixed control sample solution, and B is the test solution; 1-scopolamine, 2-anisodamine, 3-hyoscyamine, 4-protocatechuic acid, 5-hyoscyamine, 6-scopolamine, 7-p-hydroxybenzoic acid, 8-caffeic acid, 9-tropic acid, 10-scopoletin, 11-isoquercetin, and 12-scopolamine. DETAILED DESCRIPTION

[0025] The present invention provides a method for determining the content of chemical components in Panax notoginseng based on UPLC-MS / MS, wherein the chemical components are scopolamine, anisodamine, hyoscyamine, protocatechuic acid, hyoscyamine, scopolamine, p-hydroxybenzoic acid, caffeic acid, tropic acid, scopoletin, isoquercetin and scopolamine.

[0026] The method comprises the following steps:

[0027] (1) Scopolamine, anisodamine, hyoscyamine, protocatechuic acid, hyoscyamine, scopolamine, p-hydroxybenzoic acid, caffeic acid, tropic acid, scopolamine, isoquercetin, and scopolamine were mixed with methanol to obtain standard reference solutions of the chemical components;

[0028] (2) mixing each standard reference solution obtained in step (1) with methanol to obtain a mixed reference solution; measuring the mixed reference solution of different concentrations using UPLC-MS / MS, establishing a linear regression equation with the concentration of the chemical component as the abscissa and the peak area of ​​the chemical component as the ordinate;

[0029] (3) Extracting Panax notoginseng with methanol to obtain a Panax notoginseng medicinal material solution, filtering the extracted solution to obtain a test solution; measuring the test solution using UPLC-MS / MS to obtain the peak area of ​​the chemical components of the test solution, and combining the linear regression equation obtained in step (2) to obtain the concentration of the chemical components of the test solution.

[0030] In steps (1), (2) and (3) of the present invention, the volume fraction of the methanol is independently preferably 40 to 60%, more preferably 50 to 55%.

[0031] In step (1) of the present invention, the concentration of the standard reference solution of each chemical component is independently preferably 0.5 to 2 mg / mL, more preferably 1 to 1.5 mg / mL.

[0032] In the mixed reference solution of step (2) of the present invention, the concentrations of anisodamine, scopolamine, scopolamine lactone and hyoscyamine are preferably 40-60 μg / mL, more preferably 50-55 μg / mL; the concentration of scopolamine is preferably 180-220 μg / mL, more preferably 200-210 μg / mL; the concentration of scopolamine lactone is preferably 1-3 μg / mL, more preferably 2-2.5 μg / mL; the concentration of protocatechuic acid is preferably 7-9 μg / mL, more preferably 7.5-8 μg / mL; the concentrations of scopolamine, tropic acid, caffeic acid and p-hydroxybenzoic acid are preferably 15-25 μg / mL, more preferably 20-22 μg / mL; the concentration of isoquercetin is preferably 0.5-1.5 μg / mL, more preferably 0.8-1 μg / mL.

[0033] In step (2) of the present invention, the mixed reference solution of different concentrations is obtained by diluting the mixed reference solution in sequence by 2 times, 2.5 times, 2 times, 2 times, 2.5 times, 2 times, 2.5 times, 2 times, and 2 times; the volume fraction of methanol used for dilution is preferably 40-60%, and more preferably 50-55%.

[0034] In step (2) of the present invention, the establishment of the linear regression equation includes the following steps: using the weighted least squares method to perform regression calculation, the horizontal axis (X) is the concentration of the chemical component, the vertical axis (Y) is the peak area of ​​the chemical component, and the weight coefficient is 1 / X 2 The linear regression equations of scopolamine, anisodamine, hyoscyamine, protocatechuic acid, hyoscyamine, scopolamine, p-hydroxybenzoic acid, caffeic acid, tropic acid, scopolamine, isoquercetin and artemisinin were obtained respectively; the lowest limit of quantification (LLOQ) was the concentration of the chemical component calculated when the signal-to-noise ratio (S / N) was 10.

[0035] In step (3) of the present invention, the preparation of the Panax ginseng medicinal material solution includes the following steps: placing Panax ginseng medicinal material in a volumetric flask, adding methanol to the volume, and then extracting, cooling to room temperature, making up for weight loss and mixing to obtain the Panax ginseng medicinal material solution.

[0036] In step (3) of the present invention, the particle size of the Panax ginseng medicinal material is preferably ≥50 mesh, more preferably ≥65 mesh; the extraction is preferably ultrasonic extraction, and the extraction time is preferably 50 to 80 min, more preferably 60 to 70 min; the ultrasonic power is preferably 280 to 320 W, more preferably 300 to 310 W; the ultrasonic frequency is preferably 30 to 50 kHz, more preferably 40 to 45 kHz; the concentration of the Panax ginseng medicinal material solution is preferably 18 to 22 mg / mL, more preferably 19 to 20 mg / mL; the pore size of the filter membrane used for filtration is preferably 0.22 μm.

[0037] In steps (2) and (3) of the present invention, the chromatographic conditions of the UPLC-MS / MS are:

[0038] The chromatographic column is preferably ACQUITY UPLC C SHC 18Column; the specifications of the chromatographic column are preferably an inner diameter of 2.1 mm, a length of 100 mm, and a film thickness of 1.7 μm; in the mobile phase, phase A is preferably an aqueous formic acid solution with a volume fraction of 0.05 to 0.2%, and more preferably an aqueous formic acid solution with a volume fraction of 0.1%; phase B is preferably acetonitrile; the elution method is preferably gradient elution; the flow rate is preferably 0.2 to 0.4 mL / min, and more preferably 0.3 to 0.35 mL / min; the column temperature is preferably 28 to 32°C, and more preferably 30°C; the injection volume is preferably 1.9 to 2.2 μL, and more preferably 2 to 2.1 μL.

[0039] In the present invention, the gradient elution procedure is preferably: 0 min, 10% volume fraction of phase B, 90% volume fraction of phase A; 7 min, 55% volume fraction of phase B, 45% volume fraction of phase A; 8 min, 61% volume fraction of phase B, 39% volume fraction of phase A.

[0040] In steps (2) and (3) of the present invention, the mass spectrometry conditions of the UPLC-MS / MS are:

[0041] The ion source is preferably an electrospray ion source (ESI); the detection mode is preferably multiple reaction ion monitoring (MRM); the scanning mode is preferably positive and negative ion scanning mode; the gas temperature is preferably 280-320°C, more preferably 300-310°C; the gas flow rate is preferably 10-12 L / min, more preferably 11 L / min; the nebulizer pressure is preferably 32-38 Psig, more preferably 35-37 Psig.

[0042] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0043] The Chinese ginseng medicinal material used in the following examples is the dried root of Physochlainainfundibularis Kuang of the Solanaceae family, and its source information is shown in Table 1.

[0044] Table 1 Source information of Panax notoginseng used in Examples 1 to 10

[0045] Example batch Origin batch number Example 1 S1 Shaanxi 20221028 Example 2 S2 Shaanxi 20221202 Example 3 S3 Shaanxi 20221124 Example 4 S4 Shaanxi 20221208 Example 5 S5 Shaanxi 20221210 Example 6 S6 Shaanxi 20221212 Example 7 S7 Shaanxi 20230202 Example 8 S8 Shaanxi 20230205 Example 9 S9 Shaanxi 20230208 Example 10 S10 Shaanxi 20230210

[0046] The instruments and reagents used in the following examples are as follows:

[0047] Instruments: Agilent 1290 high-performance liquid chromatograph (Agilent, USA); Agilent 6470 triple quadrupole tandem mass spectrometer (Agilent, USA); Agilent Mass Hunter analysis software (Agilent, USA); Milli-QIQ 7005 ultrapure water preparation instrument (Millipore); AS 60 / 220.R2 1 / 100,000 balance (Radwag, Poland); G3KT18273 vortex mixer (Thermo Fisher Scientific).

[0048] Reagents: Methanol and acetonitrile (chromatographic grade) were purchased from Fisher, USA; formic acid was purchased from ROE, USA; water was prepared by Milli-QIQ7005 ultrapure water preparation instrument.

[0049] Example 1

[0050] Take appropriate amounts of scopolamine, anisodamine hydrobromide, hyoscyamine, protocatechuic acid, hyoscyamine, scopolamine, p-hydroxybenzoic acid, caffeic acid, tropic acid, scopolamine, isoquercetin, and scopolamine, dissolve them in 50% methanol to prepare 1 mg / mL standard reference solution, and store in a refrigerator at 4°C until use;

[0051] The source information of the above raw materials is as follows:

[0052] Scopolamine (Batch No.: DST220614-163), Anisodamine Hydrobromide (Batch No.: DST220721-478), Hyoscyamine (Batch No.: DSTDL011501), Protocatechuic Acid (Batch No.: DSTDY008101), Fascicloside (Batch No.: DST230209-020), Scopolamine (Batch No.: DST220215-056), Parahydroxybenzoic Acid (Batch No.: DSTDL011501), STDD011401), caffeic acid (batch number: DST191030-013), tropic acid (batch number: DSTDT023201), scopoletin (batch number: DST191018-064), isoquercetin (batch number: DSTDY000601) and scopolamine (batch number: DST200627-031) were purchased from Chengdu Desit Biotechnology Co., Ltd. with a purity of >98%.

[0053] Take each standard reference solution and mix it with 50% methanol by volume to prepare a mixed reference solution. Keep the concentrations of anisodamine, scopolamine, scopolamine lactone and hyoscyamine at 50 μg / mL, the concentration of hyoscyamine at 200 μg / mL, the concentration of artemisinin at 2 μg / mL, the concentration of protocatechuic acid at 8 μg / mL, the concentrations of scopolamine, tropic acid, caffeic acid and p-hydroxybenzoic acid at 20 μg / mL, and the concentration of isoquercetin at 20 μg / mL. The mixed reference solution was diluted with 50% methanol by volume in sequence of 2 times, 2.5 times, 2 times, 2 times, 2.5 times, 2 times, 2.5 times, 2 times, and 2 times to obtain mixed reference solutions of different concentrations. The mixed reference solutions of each concentration were taken for UPLC-MS / MS analysis. The weighted least squares method was used for regression calculation, with the abscissa (X) representing the concentration of the chemical component and the ordinate (Y) representing the peak area of ​​the chemical component, and the weight coefficient being 1 / X. 2 , the linear regression equations for scopolamine, anisodamine, hyoscyamine, protocatechuic acid, hyoscyamine, scopolamine, p-hydroxybenzoic acid, caffeic acid, tropic acid, scopolamine, isoquercetin, and scopolamine were obtained; the concentrations of the chemical components calculated at a signal-to-noise ratio (S / N) of 10 were used as the lower limit of quantification (LLOQ). The results are shown in Table 2;

[0054] Table 2 Standard regression equations and minimum quantification limits of chemical components

[0055]

[0056]

[0057] The Chinese ginseng medicinal materials of batch S1 were passed through a No. 3 sieve (50 mesh), 200 mg of the sieve material was taken and placed in a 10 mL volumetric flask, and 50% volume fraction of methanol was added to the scale line. The mixture was ultrasonically treated at a power of 300 W and a frequency of 40 kHz for 1 hour, cooled to room temperature, made up to the weight loss, shaken, and passed through a 0.22 μm microporous filter membrane to obtain the test solution; the test solution was determined by UPLC-MS / MS to obtain the peak area of ​​the chemical components of the test solution, and the concentration of the chemical components of the test solution was obtained by combining the linear regression equations of the chemical components obtained above.

[0058] The chromatographic conditions of UPLC-MS / MS described in this example are as follows:

[0059] Column: ACQUITY UPLC C SHC 18Column (2.1mm×100mm, 1.7μm); mobile phase: phase A is a 0.1% volume fraction of formic acid in water, phase B is acetonitrile; gradient elution, gradient elution program: 0min, 10% volume fraction of phase B, 90% volume fraction of phase A; 7min, 55% volume fraction of phase B, 45% volume fraction of phase A; 8min, 61% volume fraction of phase B, 39% volume fraction of phase A; flow rate: 0.3mL / min; column temperature: 30℃; injection volume: 2μL.

[0060] The mass spectrometry conditions of UPLC-MS / MS described in this example are as follows:

[0061] Ion source: electrospray ion source (ESI); detection mode: multiple reaction ion monitoring (MRM); scan mode: positive and negative ion scan mode; gas temperature (Gas Temp): 300°C; gas flow rate (Gas Flow): 11 L / min; nebulizer pressure (Nebulizer): 35 Psig; the quantitative analysis ion pairs, mass spectrometry parameters and ion mode are shown in Table 3.

[0062] Table 3 Mass spectrometry parameters of 12 chemical components

[0063]

[0064]

[0065] The MRM diagram of the mixed control sample solution and the test solution in this example is as follows Figure 1 shown.

[0066] Depend on Figure 1 It can be seen that the peak shapes of the 12 chemical components described in the present invention are good and do not interfere with each other.

[0067] Example 2

[0068] The Panax ginseng medicinal materials of batch S1 in Example 1 were replaced with Panax ginseng medicinal materials of batch S2, and the rest were the same as in Example 1.

[0069] Example 3

[0070] The Panax ginseng medicinal materials of batch S1 in Example 1 were replaced with Panax ginseng medicinal materials of batch S3, and the rest were the same as in Example 1.

[0071] Example 4

[0072] The Panax ginseng medicinal materials of batch S1 in Example 1 were replaced with Panax ginseng medicinal materials of batch S4, and the rest were the same as in Example 1.

[0073] Example 5

[0074] The Panax ginseng medicinal materials of batch S1 in Example 1 were replaced with Panax ginseng medicinal materials of batch S5, and the rest were the same as in Example 1.

[0075] Example 6

[0076] The Panax ginseng medicinal materials of batch S1 in Example 1 were replaced with Panax ginseng medicinal materials of batch S6, and the rest were the same as in Example 1.

[0077] Example 7

[0078] The Panax ginseng medicinal materials of batch S1 in Example 1 were replaced with Panax ginseng medicinal materials of batch S7, and the rest were the same as in Example 1.

[0079] Example 8

[0080] The Panax ginseng medicinal materials of batch S1 in Example 1 were replaced with Panax ginseng medicinal materials of batch S8, and the rest were the same as in Example 1.

[0081] Example 9

[0082] The Panax ginseng medicinal materials of batch S1 in Example 1 were replaced with Panax ginseng medicinal materials of batch S9, and the rest were the same as in Example 1.

[0083] Example 10

[0084] The Panax ginseng medicinal materials of batch S1 in Example 1 were replaced with Panax ginseng medicinal materials of batch S10, and the rest were the same as in Example 1.

[0085] The contents of the 12 chemical components in the different batches of Panax notoginseng medicinal materials described in Examples 1 to 10 are shown in Tables 4, 5 and 6.

[0086] Table 4 Contents of four chemical components in different batches of Panax notoginseng (μg / g, n=3)

[0087] batch Scopolamine Anisodamine scopolamine Protocatechuic acid S1 156.76±7.65 408.22±7.91 637.41±59.46 9.69±0.91 S2 141.41±1.94 396.09±4.47 570.58±26.06 17.51±0.96 S3 148.04±3.60 386.45±3.26 398.11±17.80 8.88±0.30 S4 155.71±2.67 387.87±5.03 407.00±20.60 10.16±0.34 S5 130.51±3.40 384.27±5.73 520.18±23.21 10.87±0.24 S6 153.70±1.99 396.79±4.90 409.91±19.01 9.39±0.31 S7 165.29±4.04 372.63±5.28 527.16±23.38 13.23±0.30 S8 146.40±3.42 396.91±6.98 418.48±20.70 8.64±0.34 S9 147.43±2.89 380.02±5.51 374.16±18.25 9.20±0.20 S10 133.13±3.50 377.90±5.84 367.51±16.93 9.16±0.17

[0088] Table 5 Contents of four chemical components in different batches of Panax notoginseng (μg / g, n=3)

[0089]

[0090]

[0091] Table 6 Contents of four chemical components in different batches of Panax notoginseng (μg / g, n=3)

[0092] batch Tropicic acid Scopoletin Isoquercetin Scoparone S1 86.31±3.05 314.42±7.12 0.25±0.03 0.99±0.06 S2 112.07±4.15 281.53±5.31 0.00±0.00 5.09±0.28 S3 155.46±6.03 279.54±7.30 0.05±0.01 2.29±0.14 S4 126.37±4.49 310.90±10.33 0.00±0.00 1.64±0.11 S5 87.01±4.23 337.66±8.90 0.13±0.02 2.17±0.16 S6 109.11±5.96 191.91±6.44 0.06±0.01 3.32±0.20 S7 79.40±3.90 237.29±7.23 0.00±0.00 3.15±0.15 S8 147.51±7.13 278.53±7.37 0.00±0.00 4.15±0.23 S9 134.44±6.69 272.02±6.19 0.11±0.01 1.67±0.13 S10 132.70±7.21 256.79±5.46 0.00±0.00 1.90±0.12

[0093] The instrument used in the present invention was subjected to a precision test, and the test method and results are as follows:

[0094] Precision test (intraday): The test solution was prepared by the method described in Example 1, and the sample was injected 6 times continuously. The RSD value of the peak area of ​​each chemical component was calculated. The results are shown in Tables 7 and 8.

[0095] Table 7 Intra-day precision results of 12 chemical components (n=6)

[0096]

[0097]

[0098] Table 8 Intra-day precision results of 12 chemical components (n=6)

[0099] chemical composition 5 6 average value RSD (%) Scopolamine 2856795.26 2860314.32 2902472.83 1.98 Anisodamine 8926708.21 8942464.99 9070719.74 1.74 scopolamine 2618002.98 2621462.92 2605768.88 0.54 Protocatechuic acid 104212.24 103671.54 104770.88 1.64 Fabirin 2475944.20 2481428.43 2463040.89 0.59 Scopoletin 7633005.97 7558404.41 7668896.86 1.07 Parabens 75714.98 77207.06 78735.53 4.76 Caffeic acid 3457469.40 3449383.21 3520613.55 1.52 Tropicic acid 492789.97 488274.38 499790.88 1.49 Scopoletin 4140559.85 4167864.79 4005988.81 3.39 Isoquercetin 1195.19 1222.19 1234.55 3.25 Scoparone 55906.33 53913.06 55972.74 2.81

[0100] It can be seen from Tables 7 and 8 that the intra-day precision of the instrument used in the present invention is good.

[0101] Precision test (day): The test solution was prepared using the method described in Example 1. The injection was repeated twice for three consecutive days, and the RSD value of the peak area of ​​each chemical component was calculated. The results are shown in Tables 9 and 10.

[0102] Table 9 Inter-day precision results of 12 chemical components (n=6)

[0103] chemical composition 1 2 3 4 Scopolamine 2896619.96 2869129.58 2850700.16 2844394.34 Anisodamine 9085692.71 8961428.48 8904890.31 8822382.06 scopolamine 2592327.24 2608353.24 2636989.20 2639178.48 Protocatechuic acid 105707.05 107710.28 104733.54 105937.57 Fabirin 2450095.37 2447857.87 2454906.88 2443288.34 Scopoletin 7669111.14 7634975.18 7312043.09 7279007.48 Parabens 83493.59 80721.43 76053.90 73787.08 Caffeic acid 3558785.17 3540341.48 3431866.91 3345382.70 Tropicic acid 506064.49 501670.14 474620.57 472765.12 Scopoletin 3967263.52 4042465.45 4201045.37 4213174.56 Isoquercetin 1257.60 1304.79 1222.19 1335.82 Scoparone 54376.44 56409.81 55457.85 54681.55

[0104] Table 10 Inter-day precision results of 12 chemical components (n=6)

[0105]

[0106]

[0107] It can be seen from Tables 9 and 10 that the intraday precision of the instrument used in the present invention is good.

[0108] The test method of the present invention was subjected to a repeatability test, and the test method and results are as follows:

[0109] Test method: 6 test solutions were prepared using the method described in Example 1, and the samples were injected separately. The RSD values ​​of the concentrations of the chemical components were calculated. The results are shown in Tables 11 and 12.

[0110] Table 11 Repeatability results of 12 chemical components (ng / mL, n=6)

[0111]

[0112]

[0113] Table 12 Repeatability results of 12 chemical components (ng / mL, n=6)

[0114] chemical composition 5 6 average value RSD (%) Scopolamine 3191.85 3187.60 3193.02 0.52 Anisodamine 8093.52 7942.30 8009.10 0.71 scopolamine 13070.18 13112.82 13133.28 0.81 Protocatechuic acid 209.77 205.39 201.77 3.24 Fabirin 64011.65 63052.64 60700.72 3.66 Scopoletin 16746.50 16822.42 16967.21 2.45 Parabens 155.73 159.19 155.97 2.39 Caffeic acid 4817.11 4858.44 4780.10 1.19 Tropicic acid 1732.46 1686.42 1702.67 2.27 Scopoletin 6108.31 6210.89 6387.64 2.93 Isoquercetin 4.76 4.79 4.83 2.33 Scoparone 18.16 16.80 17.83 4.75

[0115] It can be seen from Tables 11 and 12 that the test method of the present invention has good repeatability.

[0116] The chemical composition of the present invention was subjected to a stability test, and the test method and results are as follows:

[0117] Test method: The test solution was prepared by the method described in Example 1, and the sample was injected at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, and the RSD value of the peak area of ​​each chemical component was calculated. The results are shown in Tables 13 and 14.

[0118] Table 13 Stability results of 12 chemical components (n=6)

[0119] chemical composition 1 2 3 4 Scopolamine 2767207.59 2762828.70 2778454.25 2781496.88 Anisodamine 8694871.59 8771126.51 8693895.31 8691941.63 scopolamine 2676568.12 2684076.79 2675417.22 2676569.58 Protocatechuic acid 97045.50 102453.85 99640.79 95532.24 Fabirin 2268104.32 2353225.20 2258658.06 2212726.92 Scopoletin 6332301.67 6905217.75 6639397.53 6240547.18 Parabens 63643.50 67558.30 66534.77 64147.68 Caffeic acid 3000214.29 3135249.54 3081306.14 2958181.83 Tropicic acid 406127.20 435361.36 410395.94 401039.61 Scopoletin 4307253.53 4288953.93 4226687.70 4202317.17 Isoquercetin 1538.00 1700.23 1482.48 1584.81 Scoparone 53696.54 59701.04 60330.82 58101.89

[0120] Table 14 Stability results of 12 chemical components (n=6)

[0121]

[0122]

[0123] It can be seen from Tables 13 and 14 that the chemical components described in the present invention have good stability.

[0124] The chemical components of the present invention were subjected to a sample recovery test, and the test method and results are as follows:

[0125] Experimental method: The Chinese ginseng medicinal material of batch S1 was passed through a No. 3 sieve (50 mesh), 100 mg of the sieve was taken, placed in a 10 mL volumetric flask, and the mixed reference solution obtained in Example 1 was added to the scale line. The mixture was ultrasonically treated at a power of 300 W and a frequency of 40 kHz for 1 h, cooled to room temperature, and the weight loss was supplemented with the mixed reference solution obtained in Example 1. The mixture was shaken and filtered through a 0.22 μm microporous membrane to obtain a sample. The sample was injected, and the sample recovery rate of each chemical component was calculated. The results are shown in Table 15.

[0126] Table 15 Recovery results of 12 chemical components (n=6)

[0127]

[0128]

[0129] As can be seen from Table 15, the recovery rates of the chemical components described in the present invention are high.

[0130] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for determining the content of chemical components in Panax notoginseng based on UPLC-MS / MS, characterized in that: The chemical components are scopolamine, anisodamine, hyoscyamine, protocatechuic acid, hyoscyamine, scopolamine, p-hydroxybenzoic acid, caffeic acid, tropic acid, scopolamine, isoquercetin and scopolamine; The method comprises the following steps: (1) Scopolamine, anisodamine, hyoscyamine, protocatechuic acid, hyoscyamine, scopolamine, p-hydroxybenzoic acid, caffeic acid, tropic acid, scopolamine, isoquercetin and scopolamine were mixed with methanol to obtain standard reference solutions of the chemical components; (2) mixing the standard reference solution obtained in step (1) with methanol to obtain a mixed reference solution; UPLC-MS / MS was used to measure mixed reference solutions of different concentrations, and a linear regression equation was established with the concentration of the chemical components as the horizontal axis and the peak area of ​​the chemical components as the vertical axis. (3) Extracting the Panax notoginseng medicinal material with methanol to obtain a Panax notoginseng medicinal material solution, and filtering the extracted solution to obtain a test solution; The test solution is measured using UPLC-MS / MS to obtain the peak area of ​​the chemical components of the test solution, and the concentration of the chemical components of the test solution is obtained by combining the linear regression equation obtained in step (2); In steps (1), (2) and (3), the volume fraction of methanol is independently 40-60%; In step (3), the particle size of the Panax notoginseng medicinal material is ≥50 mesh; the extraction is ultrasonic extraction, the extraction time is 50-80 min, the ultrasonic power is 280-320 W, and the ultrasonic frequency is 30-50 kHz; the concentration of the Panax notoginseng medicinal material solution is 18-22 mg / mL; the pore size of the filter membrane used for filtration is 0.22 μm; In steps (2) and (3), the chromatographic conditions of the UPLC-MS / MS are: The chromatographic column is ACQUITY UPLC CSH C 18 Column; Phase A was a 0.05-0.2% volume fraction of formic acid in water, and Phase B was acetonitrile; the elution method was gradient elution; the flow rate was 0.2-0.4 mL / min; the column temperature was 28-32°C; the injection volume was 1.9-2.2 μL; The gradient elution procedure is as follows: 0 min, 10% volume fraction of phase B, 90% volume fraction of phase A; 7 min, 55% volume fraction of phase B, 45% volume fraction of phase A; 8 min, 61% volume fraction of phase B, 39% volume fraction of phase A; In steps (2) and (3), the mass spectrometry conditions of the UPLC-MS / MS are: The ion source is an electrospray ion source; the detection mode is multiple reaction ion monitoring; the scanning mode is positive and negative ion scanning mode; the gas temperature is 280~320℃; the gas flow rate is 10~12L / min; and the nebulizer pressure is 32~38Psig.

2. The method for determining the content of chemical components in Panax notoginseng based on UPLC-MS / MS according to claim 1, characterized in that: In step (1), the concentration of the standard reference solution of each chemical component is independently 0.5~2 mg / mL.

3. The method for determining the content of chemical components in Panax notoginseng based on UPLC-MS / MS according to claim 1 or 2, characterized in that: In the mixed reference solution of step (2), the concentrations of anisodamine, scopolamine, scopolamine lactone, and hyoscyamine are 40-60 μg / mL, the concentration of hyoscyamine is 180-220 μg / mL, the concentration of artemisinin is 1-3 μg / mL, the concentration of protocatechuic acid is 7-9 μg / mL, the concentrations of scopolamine, tropic acid, caffeic acid, and p-hydroxybenzoic acid are 15-25 μg / mL, and the concentration of isoquercetin is 0.5-1.5 μg / mL.

4. The method for determining the content of chemical components in Panax notoginseng based on UPLC-MS / MS according to claim 3, characterized in that: The mixed reference solution of different concentrations in step (2) is obtained by diluting the mixed reference solution in sequence by 2 times, 2.5 times, 2 times, 2 times, 2.5 times, 2 times, 2.5 times, 2 times, and 2 times; the volume fraction of methanol used for dilution is 40-60%.

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