A method for simultaneous determination of 13 chemical components in Equisetum by HPLC-MS / MS

Abstract

The application belongs to the technical field of analytical chemistry, and particularly relates to a method for simultaneously determining 13 chemical components in Equisetum by HPLC-MS / MS method. The application provides a method for simultaneously determining 13 chemical components in Equisetum by HPLC-MS / MS method, the method has good linear relationship (the 13 chemical components in Equisetum have good linear relationship (r>0.999) in respective determination ranges, good precision and repeatability, and high recovery rate (the average recovery rate of sample addition is 86.67%~110.91%), can simultaneously and accurately determine the content of the 13 chemical components in Equisetum, and meets the demand of comprehensively evaluating the quality of Equisetum.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a method for the simultaneous determination of 13 chemical components in horsetail based on HPLC-MS / MS. Background Technology

[0002] Equisetum is a plant of the Equisetaceae family (Equisetaceae). Equisetum hyemale The dried aerial parts of *Equisetum hyemale* (L.) are abundant in my country, mainly distributed in Heilongjiang, Liaoning, Jilin, and Anhui provinces. The 2020 edition of the *Chinese Pharmacopoeia* records that *Equisetum hyemale* has a sweet and bitter taste, enters the lung and liver meridians, and has the effects of dispersing wind-heat and improving eyesight. The chemical composition of *Equisetum hyemale* is complex, mainly containing flavonoids, organic acids, and volatile oils, among which flavonoids are the main active medicinal components. Studies have shown that *Equisetum hyemale* has antioxidant, anti-inflammatory, anti-tumor, and hepatoprotective effects.

[0003] In recent years, research on horsetail has mainly focused on flavonoids. The Chinese Pharmacopoeia (2020 edition) only uses kaempferol as the sole indicator for determining the content of chemical components in horsetail. However, the components of traditional Chinese medicine are complex, and determining the content of only a single component cannot comprehensively reflect its quality. Therefore, there is an urgent need to establish a scientific and reasonable method for the quality evaluation of horsetail. Summary of the Invention

[0004] The purpose of this invention is to provide a method for the simultaneous determination of 13 chemical components in horsetail based on HPLC-MS / MS. The detection method provided by this invention can simultaneously determine the content of 13 chemical components in horsetail, and the method is accurate and reliable, meeting the needs of comprehensive quality evaluation of horsetail.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for the simultaneous determination of 13 chemical components in horsetail based on HPLC-MS / MS, comprising the following steps:

[0007] The horsetail to be tested was extracted with a methanol-water solution to obtain the test solution;

[0008] The test solution was analyzed by HPLC-MS / MS to obtain the peak areas of each chemical component in Equisetum hyemale.

[0009] The content of each analyte in horsetail was obtained based on the peak area of ​​each analyte and the corresponding predetermined standard curve. The predetermined standard curve for each analyte represents a linear relationship between the concentration and peak area of ​​that analyte. The analytes include kaempferol-3- O -Sophorabolic acid-7- O -Glucoside, Kaempferol-3-O -Sophoroside, Kaempferol-7- O - Glucoside 、 gallic acid 、 caffeic acid 、 vanillic acid 、 Protocatechuic acid 、 Protocatechuic aldehyde 、 Kaempferol 、 p-coumaric acid 、 chlorogenic acid 、 Ferulic acid and vanillin;

[0010] For HPLC-MS / MS detection, the chromatographic separation conditions include: mobile phase A is formic acid aqueous solution; mobile phase B is acetonitrile; and the chromatographic column is C. 18 The column; the flow rates of mobile phase A and mobile phase B are 0.4 mL / min;

[0011] The elution method is gradient elution.

[0012] Preferably, the gradient elution procedure is as follows:

[0013] 0-5 min: The volume percentage of mobile phase B increases from 5% to 80%;

[0014] Within 5-12 minutes, the volume percentage of the dynamic phase B increases from 80% to 95%.

[0015] Preferably, the mass spectrometry detection conditions during HPLC-MS / MS detection include: ion source: electrospray ion source; detection mode: multiple reaction ion detection; scanning mode: positive and negative ion scanning mode; drying temperature: 320℃; gas flow rate: 11 L / min; nebulizer pressure: 20 psi.

[0016] Preferably, during HPLC-MS / MS detection, the chromatographic column is a CORTECS UPLC C18 column; the column temperature is 30 ℃; and the injection volume is 2 μL.

[0017] Preferably, the volume concentration of the formic acid aqueous solution is 0.1%.

[0018] Preferably, the volume concentration of the methanol aqueous solution is 70%.

[0019] Preferably, the mass-to-volume ratio of the horsetail to be tested and the methanol aqueous solution is 1.00 g: 20 mL.

[0020] Preferably, the mass spectrometry data of each chemical component to be tested are kaempferol-3- O -Sophorabolic acid-7- O - Glucoside m / z : 771.1, 609.0; Kaempferol-3-O - Sophoroside 609.1, 283.9; Kaempferol-7- O - Glucoside m / z : 447.1, 285.0; gallic acid m / z : 169.1, 125.3; caffeic acid m / z : 179.0, 135.0; vanillic acid m / z : 166.9, 108.0; protocatechuic acid m / z : 153.0, 108.9; protocatechuic aldehyde m / z : 137.0, 108.0; Kaempferol m / z : 285.1, 117.1; p-coumaric acid m / z : 163.0, 119.0; chlorogenic acid m / z : 353.0, 190.8; ferulic acid m / z : 193.0, 134.0; vanillin m / z : 153.0, 93.0.

[0021] This invention provides a method for the simultaneous determination of 13 chemical components in Equisetum hyemale based on HPLC-MS / MS. This method exhibits good linearity (the 13 chemical components in Equisetum hyemale show good linearity within their respective determination ranges (r>0.999)), good precision and repeatability, and high recovery rate (average recovery rate of 86.67%~110.91%). It can simultaneously and accurately determine the content of 13 chemical components in Equisetum hyemale, meeting the needs for comprehensive quality evaluation of Equisetum hyemale. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The MRM diagrams are for the reference standard (A) and the sample (B). Detailed Implementation

[0024] This invention provides a method for the simultaneous determination of 13 chemical components in horsetail based on HPLC-MS / MS, comprising the following steps:

[0025] The horsetail to be tested was extracted with a methanol-water solution to obtain the test solution;

[0026] The test solution was analyzed by HPLC-MS / MS to obtain the peak areas of each chemical component in Equisetum hyemale.

[0027] The content of each analyte in horsetail was obtained based on the peak area of ​​each analyte and the corresponding predetermined standard curve. The predetermined standard curve for each analyte represents a linear relationship between the concentration and peak area of ​​that analyte. The analytes include kaempferol-3- O -Sophorabolic acid-7- O -Glucoside, Kaempferol-3- O -Sophoroside, Kaempferol-7- O - Glucoside 、 gallic acid 、 caffeic acid 、 vanillic acid 、 Protocatechuic acid 、 Protocatechuic aldehyde 、 Kaempferol 、 p-coumaric acid 、 chlorogenic acid 、 Ferulic acid and vanillin.

[0028] The structures of the 13 chemical components are as follows:

[0029]

[0030] In one embodiment of the present invention, the volume concentration of the methanol aqueous solution can be 70%; the mass-to-volume ratio of the horsetail to be tested and the methanol aqueous solution can be 1.00 g: 20 mL; the extraction can be ultrasonic extraction; and the ultrasonic extraction time can be 45 min.

[0031] In one embodiment of the present invention, the chromatographic separation conditions for HPLC-MS / MS detection include: mobile phase A is formic acid aqueous solution; mobile phase B is acetonitrile; the flow rates of mobile phase A and mobile phase B can be 0.4 mL / min; the chromatographic column is a C18 column, specifically a CORTECS UPLC C18 column; the column temperature can be 30 ℃; the injection volume can be 2 μL; and the elution method can be gradient elution.

[0032] The gradient elution procedure can be as follows:

[0033] 0-5 min: The volume percentage of mobile phase B increases from 5% to 80%;

[0034] Within 5-12 minutes, the volume percentage of the dynamic phase B increases from 80% to 95%.

[0035] Mass spectrometry detection conditions may include: ion source: electrospray ion source; detection mode: multiple reaction ion detection; scanning mode: positive and negative ion scanning mode; drying temperature: 320℃; gas flow rate: 11 L / min; nebulizer pressure: 20 psi.

[0036] To further illustrate the present invention, the following detailed description of the invention's solutions, in conjunction with the accompanying drawings and embodiments, is provided, but should not be construed as limiting the scope of protection of the present invention.

[0037] Agilent 1200 high-performance liquid chromatograph (Agilent Technologies, USA); Agilent 6430 triple quadrupole tandem mass spectrometer (Agilent Technologies, USA); Agilent Mass Hunter analysis software (Agilent Technologies, USA); Milli-Q IQ 7005 ultrapure water preparation system (Millipore); 5424 R high-speed centrifuge (Eppendorf, Germany); AS60 / 220. R2 0.0001 ppm balance (Radiwag, Poland); G3KT 18273 vortex mixer (Thermo Fisher Scientific).

[0038] Kaempferol-3- O -Sophorabolic acid-7- O - Glucoside (batch number: DSTDS011501), Kaempferol-3- O - Sophoroside (batch number: DSTDS001701), Kaempferol-7- O - Glucoside (batch number: DST180829-077), caffeic acid (batch number: DSTDK001301), vanillic acid (batch number: DSTDX008801), protocatechuic acid (batch number: DSTDY008101), p-coumaric acid (batch number: DST210508-057), ferulic acid (batch number: DSTDL002101), gallic acid (batch number: DST240122-008), protocatechuic aldehyde (batch number: DST190627-80), vanillin (batch number: DSTDX009001), kaempferol (batch number: DST181226-056), and chlorogenic acid (batch number: DSTDL002101) were all purchased from Dester Biotechnology Co., Ltd. (all with a purity greater than 98.0%). Formic acid, methanol, and acetonitrile (chromatographic grade) were purchased from Fisher Scientific, and the water was ultrapure water (prepared using a Milli-Q ultrapure water system). The 20 batches of horsetail medicinal materials were sourced from Heilongjiang, Jilin, Liaoning, Anhui, and Inner Mongolia, respectively. Specific source information is shown in Table 1.

[0039] Table 1. Information Sources for 20 Batches of Horsetail Medicinal Materials

[0040]

[0041] Example 1

[0042] 2.1 Chromatographic conditions

[0043] Chromatographic column: CORTECS C18 column (2.1 mm × 100 mm, 2.7 μm); mobile phase: 0.1% formic acid water (A) - acetonitrile (B); gradient elution: 0-5 min, 5%-80% B; 5-12 min, 80%-95% B; flow rate: 0.4 mL / min; column temperature: 30℃; injection volume: 2 μL.

[0044] 2.2 Mass Spectrometry Conditions

[0045] Ion source: electrospray ionization source; detection mode: multiple reaction ion detection (MRM); scanning mode: positive and negative ion scanning mode; drying temperature: 320℃; gas flow rate: 11 L / min; nebulizer pressure: 20 psi; mass spectrometry parameters and ion modes of 13 components are shown in Table 2.

[0046] Table 2 Mass spectrometry parameters of 13 chemical components in horsetail

[0047]

[0048] 2.3 Solution Preparation

[0049] 2.3.1 Preparation of reference solution

[0050] Accurately weigh kaempferol-3- O -Sophorabolic acid-7- O -Glucoside, Kaempferol-3- O -Sophoroside, Kaempferol-7- O Dissolve appropriate amounts of glucoside, caffeic acid, vanillic acid, protocatechuic acid, p-coumaric acid, ferulic acid, gallic acid, protocatechuic aldehyde, vanillin, kaempferol, and chlorogenic acid reference standards in 70 vol.% methanol to prepare a 1 mg / mL reference solution.

[0051] 2.3.2 Preparation of the test solution

[0052] Accurately weigh 1.00 g of each of 20 batches of horsetail powder (passed through a No. 3 sieve), add 20 mL of 70 vol.% methanol, sonicate for 45 min, cool, shake well, take 1 mL of the solution, centrifuge at 14000 rpm for 10 min, take the supernatant, filter through a microporous membrane to obtain the test solution.

[0053] 2.4 Methodological Examination

[0054] 2.4.1 Standard Curve Regression Equation

[0055] Accurately pipette an appropriate amount of the prepared reference solution and dilute kaempferol-3- with 70 vol.% methanol. O -Sophorabolic acid-7- O-Glucoside, Kaempferol-3- O -Sophoroside, caffeic acid, kaempferol-7- O A mixed reference solution containing glucosinolate, vanillic acid, protocatechuic acid, p-coumaric acid, ferulic acid, gallic acid, protocatechuic aldehyde, vanillin, kaempferol, and chlorogenic acid at concentrations of 50,000 ng / mL, 40,000 ng / mL, 15,000 ng / mL, 4,000 ng / mL, 2,000 ng / mL, 2,000 ng / mL, 10,000 ng / mL, 10,000 ng / mL, 900 ng / mL, 240 ng / mL, 3,000 ng / mL, 180 ng / mL, and 180 ng / mL, respectively, was prepared. This solution was then sequentially diluted with 70 vol.% methanol by 2, 2.5, 2, 2, 2, 2.5, 2, and 2 times to obtain a series of mixed reference solutions, which were then injected for analysis.

[0056] Table 3. Standard curve regression equations, limits of detection, and limits of quantitation for 13 compounds.

[0057]

[0058] 2.4.2 Precision Test

[0059] Intra-day precision: 1.00 g of batch A1 horsetail powder was accurately weighed, and the test solution was prepared according to the above method. The sample was injected 6 times consecutively, and the RSD values ​​of the peak areas of 13 compounds were calculated. The results are shown in Table 4, indicating that the instrument has good intra-day precision.

[0060] Table 4. Intra-day precision results for 13 chemical components (n = 6)

[0061]

[0062] Daytime precision: 1.00 g of batch A1 horsetail powder was accurately weighed and the test solution was prepared according to the above method. The sample was injected continuously for 3 days and twice a day. The RSD values ​​of the peak areas of 13 compounds were calculated. The results are shown in Table 5, indicating that the instrument has good daytime precision.

[0063] Table 5. Daytime precision of 13 components (n = 6)

[0064]

[0065] 2.4.3 Repeatability Test

[0066] Six portions of batch A1 horsetail powder, each weighing 1.00 g, were accurately weighed and prepared into test solutions according to the above method. The solutions were injected separately, and the RSD values ​​of the contents of 13 compounds were calculated. The results are shown in Table 6, indicating that the method has good repeatability.

[0067] Table 6. Repeatability of 13 components (μg / g, n = 6)

[0068]

[0069] 2.4.4 Stability Test

[0070] Accurately weigh 1.00 g of batch A1 horsetail powder and prepare the test solution according to the above method. Inject the sample at 0, 2, 4, 8, 12 and 24 h respectively, and calculate the RSD value of the peak area of ​​13 compounds. The results are shown in Table 7, indicating that each compound has good stability.

[0071] Table 7. Stability of 13 components (n = 6)

[0072]

[0073] 2.4.5 Recovery Test

[0074] Accurately weigh 6 portions of A1 horsetail powder, each 0.50 g, add a certain amount of mixed reference solution, and prepare 6 test solutions in parallel according to the above method. Inject the samples separately and calculate the recovery rates of 13 compounds. The results are shown in Table 8.

[0075] Table 8 Recovery rates of 13 chemical components (n = 6)

[0076]

[0077] 2.5 Content Determination

[0078] 1.00 g of each of 20 batches of horsetail powder was accurately weighed, and test solutions were prepared according to the above method. The solutions were injected separately, and the contents of 13 compounds in different batches of horsetail were calculated. The results are shown in Table 9.

[0079] Table 9. Content of horsetail components from different origins (µg / g) (n = 3)

[0080]

[0081] Table 10. Content of horsetail components from different origins (µg / g) (n = 3)

[0082]

[0083] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for the simultaneous determination of 13 chemical components in horsetail based on HPLC-MS / MS, characterized in that, Includes the following steps: The horsetail to be tested was extracted with a methanol-water solution to obtain the test solution; The test solution was analyzed by HPLC-MS / MS to obtain the peak areas of each chemical component in Equisetum hyemale. The content of each analyte in horsetail was obtained based on the peak area of ​​each analyte and the corresponding predetermined standard curve; the predetermined standard curve for each analyte represents the linear relationship between the concentration and peak area of ​​each analyte; the analyte is kaempferol-3- O -Sophorabolic acid-7- O -Glucoside, Kaempferol-3- O -Sophoroside, Kaempferol-7- O - Glucoside 、 gallic acid 、 caffeic acid 、 vanillic acid 、 Protocatechuic acid 、 Protocatechuic aldehyde 、 Kaempferol 、 p-coumaric acid 、 chlorogenic acid 、 Ferulic acid and vanillin; During HPLC-MS / MS detection, the chromatographic separation conditions include: mobile phase A being formic acid aqueous solution; and mobile phase B being acetonitrile. The chromatographic column was a C18 column; the flow rates of mobile phase A and mobile phase B were 0.4 mL / min; the chromatographic column was a CORTECS UPLC C18 column. The elution method is gradient elution; the gradient elution procedure is as follows: 0-5 min: The volume percentage of mobile phase B increases from 5% to 80%; Within 5-12 minutes, the volume percentage of the dynamic phase B increased from 80% to 95%. The mass spectrometry data of each chemical component to be tested are kaempferol-3- O -Sophorabolic acid-7- O - Glucoside m / z : 771.1, 609.0; Kaempferol-3- O - Sophoroside 609.1, 283.9; Kaempferol-7- O - Glucoside m / z : 447.1, 285.0; gallic acid m / z : 169.1, 125.3; caffeic acid m / z : 179.0, 135.0; vanillic acid m / z : 166.9, 108.0; protocatechuic acid m / z : 153.0, 108.9; protocatechuic aldehyde m / z : 137.0, 108.0; Kaempferol m / z : 285.1, 117.1; p-coumaric acid m / z : 163.0, 119.0; chlorogenic acid m / z : 353.0, 190.8; ferulic acid m / z : 193.0, 134.0; vanillin m / z : 153.0, 93.

0.

2. The method as claimed in claim 1, characterized in that, For HPLC-MS / MS detection, the mass spectrometry detection conditions include: ion source: electrospray ion source; detection mode: multiple reaction ion detection; scanning mode: positive and negative ion scanning mode; drying temperature: 320℃; gas flow rate: 11 L / min; nebulizer pressure: 20 psi.

3. The method as described in claim 1, characterized in that, For HPLC-MS / MS detection, the column temperature was 30 ℃ and the injection volume was 2 μL.

4. The method as claimed in claim 1, characterized in that, The volume concentration of the formic acid aqueous solution is 0.1%.

5. The method as claimed in claim 1, characterized in that, The volume concentration of the methanol aqueous solution is 70%.

6. The method as claimed in claim 1 or 5, characterized in that, The mass-to-volume ratio of the tested horsetail and the methanol-water solution was 1.00 g: 20 mL.

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