A method for detecting the content of multiple chemical components in Elsholtzia ciliata.

Abstract

This invention provides a method for detecting the content of multiple chemical components in Elsholtzia ciliata, belonging to the field of analytical detection technology. Based on UPLC-MS / MS, this invention establishes a method for simultaneously determining the content of up to 14 chemical components in Elsholtzia ciliata. Using 70-100% lower alcohols as the extraction solvent, the chromatographic peak response is stronger after UPLC-MS / MS detection. Furthermore, the addition of 0.08-0.12 vol% formic acid to the mobile phase effectively improves peak tailing, enabling simultaneous determination of multiple chemical components in Elsholtzia ciliata within a short time (within 6 minutes). The detection method provided by this invention is simple, efficient, highly sensitive, and specific, exhibiting good precision, repeatability, and stability. It can reflect the differences in the content of chemical components measured in different batches of Elsholtzia ciliata, providing a reference for the quality evaluation of Elsholtzia ciliata.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, specifically to a method for detecting the content of multiple chemical components in Elsholtzia ciliata. Background Technology

[0002] Elsholtzia ciliata is a plant belonging to the Lamiaceae family. Mosla chinensis Maxim. Or Jiangxiangru Mosla chinensis The dried aerial parts of 'Jiangxiangru'. As a medicinal and edible herb, 'Xiangru' is classified in traditional Chinese medicine as a pungent and warm exterior-releasing herb, possessing the effects of inducing sweating to relieve exterior symptoms, resolving dampness, and harmonizing the middle jiao (spleen and stomach). It was traditionally considered a summer exterior-releasing herb, effective in inducing sweating to relieve summer heat, and also has diuretic properties, somewhat similar to ephedra, hence the name "summer ephedra." Clinically, it is often used for summer-heat colds, chills and fever, headache without sweating, abdominal pain, vomiting and diarrhea, edema, and difficulty urinating.

[0003] Studies have shown that the pharmacological activity of Elsholtzia ciliata is not determined by a single component, but rather by the synergistic effect of multiple chemical components (such as flavonoids, phenolic acids, and terpenes). Currently, quality evaluation studies of Elsholtzia ciliata mainly focus on volatile oil components, using gas chromatography for determination. However, for the non-volatile components of Elsholtzia ciliata, such as flavonoids and phenolic acids, which also exhibit significant anti-inflammatory and antioxidant activities, effective quality evaluation methods are lacking. Therefore, establishing an analytical method capable of simultaneously determining the content of multiple components in Elsholtzia ciliata is of great significance for the scientific and comprehensive evaluation of its quality. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for detecting the content of multiple chemical components in Elsholtzia ciliata. The detection method provided by this invention can simultaneously achieve accurate and highly sensitive detection of the content of multiple chemical components in Elsholtzia ciliata, thereby realizing the quality monitoring of Elsholtzia ciliata.

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

[0006] This invention provides a method for detecting the content of multiple chemical components in Elsholtzia ciliata, comprising the following steps:

[0007] The powder of Elsholtzia ciliata to be tested was extracted using an extractant to obtain a sample solution; the extractant included lower alcohols or aqueous solutions of lower alcohols; the volume fraction of the lower alcohols in the extractant was 70-100%;

[0008] The sample solution to be tested was subjected to high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) to obtain the detection results of the content of multiple chemical components in Elsholtzia ciliata.

[0009] The multi-index chemical components include at least five of the following: protocatechuic acid, protocatechuic aldehyde, caffeic acid, rutin, isoquercitrin, luteolin, p-coumaric acid, kaempferol-3-O-rutinoside, astragaloside, rosmarinic acid, luteolin, apigenin, baicalin, and baicalein-7-methyl ether.

[0010] The high-performance liquid chromatography-tandem mass spectrometry detection includes high-performance liquid chromatography separation and mass spectrometry detection;

[0011] The high-performance liquid chromatography (HPLC) separation conditions include: mobile phase A is 0.08~0.12 vol% formic acid aqueous solution, mobile phase B is acetonitrile, and the elution method is gradient elution. The gradient elution program is as follows: 0~3 min, the volume fraction of mobile phase B increases from 20% to 50%; 3~4 min, the volume fraction of mobile phase B increases from 50% to 70%; 4~6 min, the volume fraction of mobile phase B increases from 70% to 80%.

[0012] Preferably, the lower alcohol includes methanol and / or ethanol.

[0013] Preferably, the solid-liquid ratio of the Elsholtzia ciliata powder to the extractant is 1g:0.8~1.2L.

[0014] Preferably, the extraction includes ultrasonic extraction.

[0015] Preferably, the ultrasonic extraction power is 280~320W, the frequency is 30~50kHz, and the time is 50~70min.

[0016] Preferably, the chromatographic column used in the high-performance liquid chromatography separation is a C18 column. 18 The chromatographic column was set at a temperature of 28–32 °C, the injection volume was 1.8–2.2 μL, and the mobile phase flow rate was 0.28–0.32 mL / min.

[0017] Preferably, the mass spectrometry detection conditions include: 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 drying gas temperature is 340~360℃; the drying gas flow rate is 9~13L / min, the nebulizer pressure is 30~40psig, the fragmentation voltage is 93~237V, and the collision energy is 11~39eV.

[0018] Preferably, the quantitative ion pairs of protocatechuic acid, protocatechuic aldehyde, caffeic acid, rutin, isoquercitrin, luteolin, p-coumaric acid, kaempferol-3-O-rutinoside, astragaloside, rosmarinic acid, luteolin, apigenin, baicalin, and baicalein-7-methyl ether are 153.1 / 109.1, 137.1 / 108.1, 179.1 / 135.1, and 60, respectively. 9.1 / 300.1, 463.1 / 300.1, 447.1 / 285.1, 163.0 / 119.1, 593.2 / 285.1, 447.1 / 284.1, 358.9 / 161.0, 285.2 / 133.0, 269.2 / 117.1, 461.1 / 285.0, and 285.1 / 270.0.

[0019] Preferably, the collision energies of protocatechuic acid, protocatechuic aldehyde, caffeic acid, rutin, isoquercitrin, luteolin, p-coumaric acid, kaempferol-3-O-rutinoside, astragaloside, rosmarinic acid, luteolin, apigenin, baicalin, and baicalein-7-methyl ether are 12eV, 24eV, 14eV, 38eV, 27eV, 27eV, 11eV, 32eV, 26eV, 13eV, 36eV, 39eV, 24eV, and 32eV, respectively.

[0020] Preferably, the fragmentation voltages of protocatechuic acid, protocatechuic aldehyde, caffeic acid, rutin, isoquercitrin, luteolin, p-coumaric acid, kaempferol-3-O-rutinoside, astragaloside, rosmarinic acid, luteolin, apigenin, baicalin, and baicalein-7-methyl ether are 107V, 117V, 117V, 139V, 158V, 188V, 104V, 144V, 177V, 237V, 168V, 146V, 93V, and 141V, respectively.

[0021] This invention establishes a method for the simultaneous determination of multiple chemical components (up to 14) in *Elsholtzia ciliata* medicinal materials based on high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). Using 70-100% lower alcohols as the extraction solvent, the chromatographic peak response is stronger after UPLC-MS / MS detection. Furthermore, the addition of 0.08-0.12 vol% formic acid to the mobile phase effectively improves peak tailing. This method can simultaneously determine the content of multiple chemical components in *Elsholtzia ciliata* medicinal materials within a short time (within 6 minutes). The detection method provided by this invention is simple, efficient, highly sensitive, and specific, exhibiting good precision, repeatability, and stability. It can reflect the differences in the content of chemical components measured in different batches of *Elsholtzia ciliata* medicinal materials, providing a reference for the quality evaluation of *Elsholtzia ciliata* medicinal materials. As shown in the test results of the examples, the 14 chemical components in *Elsholtzia ciliata* medicinal materials showed good linearity within their respective determination ranges (r>0.9995), with an average recovery rate of 90.79-112.42%. This invention demonstrates that the detection method provided is accurate and reliable, and can be used for the quality evaluation of Elsholtzia ciliata medicinal materials. Attached Figure Description

[0022] Picture 1 The MRM diagrams for the standard reference (A) and the test sample (B) are shown below, where 1 is protocatechuic acid; 2 is protocatechuic aldehyde; 3 is caffeic acid; 4 is rutin; 5 isoquercitrin; 6 is luteolin; 7 is p-coumaric acid; 8 is kaempferol-3-O-rutinoside; 9 is astragaloside; 10 is rosmarinic acid; 11 is luteolin; 12 is baicalin; 13 is apigenin; and 14 is baicalin-7-methyl ether. Detailed Implementation

[0023] This invention provides a method for detecting the content of multiple chemical components in Elsholtzia ciliata, comprising the following steps:

[0024] The powder of Elsholtzia ciliata to be tested was extracted using an extractant to obtain a sample solution; the extractant included lower alcohols or aqueous solutions of lower alcohols; the volume fraction of the lower alcohols in the extractant was 70-100%;

[0025] The sample solution to be tested was subjected to high performance liquid chromatography-tandem mass spectrometry to obtain the detection results of the content of multiple chemical components in Elsholtzia ciliata.

[0026] The multi-index chemical components include at least five of the following: protocatechuic acid, protocatechuic aldehyde, caffeic acid, rutin, isoquercitrin, luteolin, p-coumaric acid, kaempferol-3-O-rutinoside, astragaloside, rosmarinic acid, luteolin, apigenin, baicalin, and baicalein-7-methyl ether.

[0027] The high-performance liquid chromatography-tandem mass spectrometry detection includes high-performance liquid chromatography separation and mass spectrometry detection;

[0028] The high-performance liquid chromatography (HPLC) separation conditions include: mobile phase A is 0.08~0.12 vol% formic acid aqueous solution, mobile phase B is acetonitrile, and the elution method is gradient elution. The gradient elution program is as follows: 0~3 min, the volume fraction of mobile phase B increases from 20% to 50%; 3~4 min, the volume fraction of mobile phase B increases from 50% to 70%; 4~6 min, the volume fraction of mobile phase B increases from 70% to 80%.

[0029] This invention utilizes an extractant to extract the powder of Elsholtzia ciliata to obtain a sample solution; the extractant includes lower alcohols or aqueous solutions of lower alcohols; the volume fraction of lower alcohols in the extractant is 70-100%.

[0030] In this invention, the Elsholtzia powder to be tested is preferably obtained by crushing Elsholtzia and then sieving it, and the sieving is preferably through a No. 4 sieve.

[0031] In this invention, the volume fraction of lower alcohols in the extractant is 70-100%, preferably 70-90%, more preferably 70-80%, and may specifically be 70%, 75%, 80%, 85%, 90%, 95% or 100%; the lower alcohols preferably include methanol and / or ethanol, more preferably methanol.

[0032] In this invention, the solid-liquid ratio of the tested Elsholtzia powder (dry weight) to the extractant is preferably 1g:0.8~1.2L, more preferably 1g:0.9~1.1L, and even more preferably 1g:1L.

[0033] In this invention, the extraction preferably includes ultrasonic extraction; the power of the ultrasonic extraction is preferably 280~320W, more preferably 290~310W, and even more preferably 300W; the frequency of the ultrasonic extraction is preferably 30~50kHz, more preferably 35~45kHz, and even more preferably 40kHz; the time of the ultrasonic extraction is preferably 50~70min, more preferably 55~65min, and even more preferably 60min.

[0034] After the extraction is completed, the present invention preferably further includes: cooling the obtained extraction system to room temperature, making up for weight loss, shaking thoroughly, centrifuging, and filtering the obtained supernatant through a 0.22 μm microporous membrane to obtain the test solution. In the present invention, the centrifugation speed is preferably 10000~16000 rpm, more preferably 12000~15000 rpm, and even more preferably 13000~14000 rpm; the centrifugation time is preferably 5~15 min, more preferably 8~12 min, and even more preferably 10 min.

[0035] After obtaining the sample solution to be tested, the present invention performs high performance liquid chromatography-tandem mass spectrometry on the sample solution to obtain the detection results of the content of multiple chemical components in Elsholtzia ciliata.

[0036] In this invention, the multi-index chemical components include at least five of the following: protocatechuic acid, protocatechuic aldehyde, caffeic acid, rutin, isoquercitrin, luteolin, p-coumaric acid, kaempferol-3-O-rutinoside, astragaloside, rosmarinic acid, luteolin, apigenin, baicalin, and baicalein-7-methyl ether. Specifically, it is preferred to include five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen of the above substances.

[0037] In this invention, the high performance liquid chromatography-tandem mass spectrometry detection includes high performance liquid chromatography separation and mass spectrometry detection.

[0038] In this invention, the high-performance liquid chromatography separation conditions include: the chromatographic column is preferably C12. 18 The chromatographic column is preferably an ACQUITY UPLC® BEH column. A C18 column; the column temperature is preferably 28~32℃, more preferably 29~31℃, and even more preferably 30℃; mobile phase A is 0.08~0.12 vol% formic acid aqueous solution, more preferably 0.09~0.11 vol% formic acid aqueous solution, and even more preferably 0.1 vol% formic acid aqueous solution; mobile phase B is acetonitrile; the mobile phase flow rate is preferably 0.28~0.32 mL / min, more preferably 0.29~0.31 mL / min, and even more preferably 0.3 mL / min; the elution method is gradient elution, and the gradient elution program is as follows: 0~3 min, the volume fraction of mobile phase B increases from 20% to 50%; 3~4 min, the volume fraction of mobile phase B increases from 50% to 70%; 4~6 min, the volume fraction of mobile phase B increases from 70% to 80%; the injection volume is preferably 1.8~2.2 μL, more preferably 1.9~2.1 μL, and even more preferably 2 μL.

[0039] In this invention, the preferred mass spectrometry detection conditions include: an electrospray ionization (ESI) source, a multiple reaction monitoring (MRM) detection mode, a positive and negative ion scanning mode, and a drying gas temperature of 340-360°C; a drying gas flow rate of 9-13 L / min, more preferably 10-12 L / min, and even more preferably 10-11 L / min; a nebulizer pressure of 30-40 psig, more preferably 32-38 psig, and even more preferably 35-36 psig; a fragmentation voltage of 93-237 V; a collision energy of 11-39 eV; and a collision gas preferably high-purity nitrogen. The nebulizer gas is preferably high-purity nitrogen. The quantitative analysis ion pairs, mass spectrometry parameters, and ion modes are shown in Table 1.

[0040] Table 1. Mass Spectrometry Parameters of 14 Chemical Components

[0041]

[0042] In this invention, the preferred method for obtaining the content of multiple chemical components is the standard curve method. Specifically, the mixed reference linear solution is detected under the conditions of ultra-high performance liquid chromatography-tandem mass spectrometry to obtain the chromatographic peak area. A standard curve is plotted with the concentration of the mixed reference linear solution as the independent variable and the chromatographic peak area as the dependent variable to obtain the standard curve regression equation. The chromatographic peak area of ​​the sample solution to be tested is then substituted into the standard curve regression equation to obtain the detection results of the content of multiple chemical components.

[0043] In this invention, the method for preparing the mixed reference linear solution preferably includes the following steps: preparing a standard reference stock solution, diluting the reference stock solution to obtain a mixed reference solution; and gradually diluting the mixed reference solution to obtain a mixed reference linear solution.

[0044] In this invention, the preferred method for preparing the reference stock solution includes the following steps: dissolving the reference standards of protocatechuic acid, protocatechuic aldehyde, caffeic acid, rutin, isoquercitrin, luteolin, p-coumaric acid, kaempferol-3-O-rutinoside, astragaloside, rosmarinic acid, luteolin, apigenin, baicalin, and baicalein-7-methyl ether in methanol and making up to volume with methanol to prepare a reference stock solution of 14 single reference standards, each with a concentration of 1 mg / mL.

[0045] In this invention, the preferred method for preparing the mixed reference linear solution includes the following steps: accurately measuring each reference stock solution and placing it in the same volumetric flask, diluting it with 70 vol% methanol aqueous solution to obtain the mixed reference solution; in the mixed reference solution, the concentrations are as follows: protocatechuic acid 2 ng / mL, protocatechuic aldehyde and rutin 0.4 μg / mL, p-coumaric acid 0.2 μg / mL, baicalin 0.04 μg / mL, baicalein-7-methyl ether, luteolin and isoquercitrin 4 μg / mL, caffeic acid 1.6 μg / mL, rosmarinic acid 20 μg / mL, luteolin and apigenin 0.32 μg / mL, kaempferol-3- O - The concentration of rutin was 0.16 μg / mL, and the concentration of astragalin was 0.8 μg / mL; the mixed reference solution was diluted sequentially with 70 vol% methanol aqueous solution by 2 times, 2 times, 2 times, 2.5 times, 2 times, 2.5 times, 2 times, and 2 times to obtain a linear solution of the mixed reference solution with 9 concentrations.

[0046] To further illustrate the present invention, the method for detecting the content of multiple chemical components in Elsholtzia ciliata provided by the present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0047] In embodiments of the present invention, the specific instruments are as follows: Agilent 6470 triple quadrupole mass spectrometer; Agilent 1200 high-performance liquid chromatograph; Agilent MassHunter analysis software (Agilent Technologies, USA); Milli-Q IQ 7005 ultrapure water preparation system (Millipore, France); G3KT18273 vortex mixer (Thermo Fisher Scientific, USA); 5424R high-speed centrifuge (Eppendorf, Germany); ZZ-L6DT ultrasonic cleaner (Tianjin Zhizhu Technology Co., Ltd., China); A 60 / 220.R2 0.0001 g balance (Radiwag, Poland).

[0048] The materials are as follows: Reference standards: protocatechuic acid (batch number: DSTDY008101), protocatechuic aldehyde (batch number: DSTDY008002), caffeic acid (batch number: DSTDK001301), rutin (batch number: DSTDL001702), isoquercitrin (batch number: DSTDY000604), luteolin (batch number: DSTDM003201), luteolinoglycoside (batch number: DSTDM001602), p-coumaric acid (batch number: DSTDD005) 701), kaempferol-3-O-rutin (batch number: DSTDS007501), astragalin (batch number: DSTDZ000102), rosmarinic acid (batch number: DST220301), baicalin (batch number: DSTDH002602), apigenin (batch number: DSTDQ002602), and baicalin-7-methyl ether (batch number: DST240509-139) were purchased from Chengdu Desite Biotechnology Co., Ltd., China, with a purity greater than 98%. Methanol (chromatographic grade) and acetonitrile (chromatographic grade) were purchased from Fisher Scientific, USA; formic acid (chromatographic grade) was purchased from ROE, USA; ultrapure water was prepared using a Milli-Q 7005 ultrapure water preparation system. Elsholtzia ciliata is a plant of the Lamiaceae family, Elsholtzia ciliata. Mosla chinensis Maxim. or Jiangxiangru Mosla chinensis The specific source information for the dried aerial parts of 'Jiangxiangru' is shown in Table 2.

[0049] Table 2. Specific Source Information for Elsholtzia ciliata Medicinal Material

[0050]

[0051] The UPLC chromatographic conditions are as follows:

[0052] Chromatographic column: ACQUITY UPLC® BEH C18 column (2.1×100mm, 1.7μm); mobile phase: 0.1 vol% formic acid water (A) - acetonitrile (B); elution gradient: 0~3 min, the volume fraction of mobile phase B increases from 20% to 50%; 3~4 min, the volume fraction of mobile phase B increases from 50% to 70%; 4~6 min, the volume fraction of mobile phase B increases from 70% to 80%; mobile phase flow rate: 0.3 mL / min; injection volume: 2 μL; column temperature: 30℃.

[0053] The mass spectrometry conditions were as follows: the ion source was an electrospray ionization source (ESI); the detection mode was multiple reaction monitoring (MRM); the scanning mode was positive and negative ion scanning mode; the drying gas temperature (Gas Temp) was 350℃; the gas flow rate (Gas Flow) was 11 L / min; the nebulizer pressure (Nebulizer) was 35 psig; the quantitative analysis ion pairs, mass spectrometry parameters and ion modes are shown in Table 1.

[0054] Example 1

[0055] Methodological Examination—Standard Curve Regression Equation

[0056] Preparation of reference stock solutions: The reference standards of protocatechuic acid, protocatechuic aldehyde, caffeic acid, rutin, isoquercitrin, luteolin, p-coumaric acid, kaempferol-3-O-rutinoside, astragaloside, rosmarinic acid, luteolin, apigenin, baicalin, and baicalein-7-methyl ether were dissolved in methanol and diluted to volume with methanol to prepare reference stock solutions of 14 single reference standards with a concentration of 1 mg / mL.

[0057] Preparation of mixed reference linear solutions (linear solutions 1-9): Accurately measure each reference stock solution and place it in the same volumetric flask. Dissolve and dilute to volume with diluent to obtain mixed reference solutions (linear solution 1); wherein, the concentration of protocatechuic acid is 2 ng / mL, the concentration of protocatechuic aldehyde and rutin is 0.4 μg / mL, the concentration of p-coumaric acid is 0.2 μg / mL, the concentration of baicalin is 0.04 μg / mL, the concentration of baicalein-7-methyl ether, luteolin and isoquercitrin is 4 μg / mL, the concentration of caffeic acid is 1.6 μg / mL, the concentration of rosmarinic acid is 20 μg / mL, the concentration of luteolin and apigenin is 0.32 μg / mL, and the concentration of kaempferol-3- O - The concentration of rutin was 0.16 μg / mL, and the concentration of astragalin was 0.8 μg / mL. Linear solution 1 was diluted 2-fold to obtain linear solution 2; linear solution 2 was diluted 2-fold to obtain linear solution 3; linear solution 3 was diluted 2-fold to obtain linear solution 4; linear solution 4 was diluted 2.5-fold to obtain linear solution 5; linear solution 5 was diluted 2-fold to obtain linear solution 6; linear solution 6 was diluted 2.5-fold to obtain linear solution 7; linear solution 7 was diluted 2-fold to obtain linear solution 8; and linear solution 8 was diluted 2-fold to obtain linear solution 9. The diluent used for dilution was a 70 vol% methanol aqueous solution.

[0058] Preparation of the test solution: Accurately weigh 10 mg of coarse powder of Elsholtzia ciliata (passed through a No. 4 sieve), place it in a 10 mL volumetric flask, add 70 vol% methanol aqueous solution to make up to the mark, sonicate at 300 W and 40 kHz for 1 h, cool, make up the weight loss, shake well, centrifuge at 14000 rpm for 10 min, take the supernatant and filter it through a 0.22 μm microporous membrane to obtain the test solution.

[0059] The above solutions were analyzed by UPLC-MS / MS. Weighted least squares regression was performed, with the x-axis (X) representing the analyte concentration and the y-axis (Y) representing the analyte peak area. Linear regression equations were obtained for protocatechuic acid, protocatechuic aldehyde, caffeic acid, rutin, isoquercitrin, luteolin, p-coumaric acid, kaempferol-3-O-rutinoside, astragaloside, rosmarinic acid, luteolin, apigenin, baicalin, and baicalein-7-methyl ether. The limits of detection (LOD) and quantitation (LOQ) of each reference standard were set at signal-to-noise ratios (S / N) of 3 and 10, respectively. The results are shown in Table 3.

[0060] Table 3. Standard curve regression equations, linear ranges, correlation coefficients, limits of detection, and limits of quantitation for 14 chemical components.

[0061]

[0062] As shown in Table 3, the 14 chemical components in Elsholtzia ciliata showed good linearity (r>0.9995) within their respective determination ranges, and the linear range was wide.

[0063] Picture 1 The MRM chromatograms for the standard reference (A) and the test sample (B) are shown, where 1 represents protocatechuic acid; 2 represents protocatechuic aldehyde; 3 represents caffeic acid; 4 represents rutin; 5 represents isoquercitrin; 6 represents luteolin; 7 represents p-coumaric acid; 8 represents kaempferol-3-O-rutinoside; 9 represents astragaloside; 10 represents rosmarinic acid; 11 represents luteolin; 12 represents baicalin; 13 represents apigenin; and 14 represents baicalin-7-methyl ether. Picture 1 It can be seen that the peaks of each compound have good shapes and do not interfere with each other.

[0064] Example 2

[0065] Methodological investigation—precision testing

[0066] Intra-day precision: The test solution was prepared according to Example 1. Six consecutive injections were performed, and the RSD values ​​of the peak areas of each compound were calculated. The results are shown in Table 4. All RSDs were ≤4.49%, indicating good intra-day precision of the instrument.

[0067] Daytime precision: The test solution was prepared according to Example 1. Injections were repeated twice over three consecutive days, and the RSD values ​​of the peak areas of each compound were calculated. The results are shown in Table 5. All RSDs were ≤4.96%, indicating good daytime precision of the instrument.

[0068] Table 4. Intra-day precision results for 14 chemical components (n=6)

[0069]

[0070] Table 5. Daytime precision results for 14 chemical components (n=6)

[0071]

[0072] Example 3

[0073] Methodological investigation—repeatability testing

[0074] Six test solutions were prepared in parallel according to Example 1, and analyzed by UPLC-MS / MS. The RSD values ​​of each compound concentration were calculated. The results are shown in Table 6. The RSD values ​​were all ≤4.77%, indicating that the method has good repeatability.

[0075] Table 6. Repeatability results for 14 chemical components (n=6, ng / mL)

[0076]

[0077] Example 4

[0078] Methodological investigation—stability testing

[0079] The test solution prepared according to Example 1 was analyzed by UPLC-MS / MS at 0h, 2h, 4h, 8h, 12h and 24h respectively. The RSD value of the peak area of ​​each compound was calculated and the results are shown in Table 7. The RSD is ≤4.83%, which indicates that each compound has good stability.

[0080] Table 7. Stability results of 14 chemical components (n=6)

[0081]

[0082] Example 5

[0083] Methodological investigation—recovery test

[0084] Preparation of spiked test solution: Accurately weigh 10 mg of crude powder of Elsholtzia ciliata (passed through a No. 4 sieve), place it in a 10 mL volumetric flask, add different amounts (see Table 8) of the mixed reference solution (linear solution 1) prepared in Example 1, add 70 vol% methanol aqueous solution to make up to the mark, sonicate at 300 W and 40 kHz for 1 h, cool, make up the weight loss, shake well, centrifuge at 14000 rpm for 10 min, take the supernatant and filter it through a 0.22 μm microporous membrane to obtain the spiked test solution.

[0085] Each spiked test solution was analyzed by UPLC-MS / MS six times, and the recovery rate of each compound was calculated. The results are shown in Table 8. The average recovery rate of each component was 90.79-112.42%, and the RSD was ≤8.61%, indicating that the method provided by this invention has a high recovery rate.

[0086] Table 8. Recovery results of 14 chemical components (n=6)

[0087]

[0088] Example 6

[0089] Content determination

[0090] Accurately weigh each batch (S1~S20) of Elsholtzia ciliata crude powder, prepare test solution according to Example 1, inject into UPLC-MS / MS for analysis, record the concentration of 14 compounds and calculate the content using a standard curve, and express the results as average value and standard deviation. The results are shown in Tables 9-10.

[0091] Table 9. Content of 7 chemical components in different batches of Elsholtzia ciliata (μg / g, n=3)

[0092]

[0093] Table 10 Content of 7 chemical components in different batches of Elsholtzia ciliata (μg / g, n=3)

[0094]

[0095] Example 7

[0096] Investigation of extractant and chromatographic conditions

[0097] Accurately weigh 10 mg of Elsholtzia ciliata crude powder (passed through a No. 4 sieve) and place it in a 10 mL volumetric flask. Add extraction solvent (30 vol% methanol aqueous solution, 50 vol% methanol aqueous solution, 70 vol% methanol aqueous solution, and methanol, respectively) to the mark. Sonicate at 300 W and 40 kHz for 1 h, cool, make up the weight loss, shake well, and filter through a 0.22 μm microporous membrane to obtain the test solution. Inject the test solution into UPLC-MS / MS for analysis. Mobile phases A through B were water-methanol, 0.1 vol% formic acid aqueous solution-methanol, water-acetonitrile, and 0.1 vol% formic acid aqueous solution-acetonitrile, respectively. The results showed that when 70 vol% methanol aqueous solution was used as the extraction solvent and water-acetonitrile was used as the mobile phase, the chromatographic peak response was stronger, and the addition of 0.1 vol% formic acid to mobile phase A improved the peak tailing phenomenon. Therefore, a UPLC-MS / MS method was established using 70 vol% methanol aqueous solution as the extraction solvent and 0.1 vol% formic acid aqueous solution-acetonitrile as the mobile phase to achieve simultaneous determination of the content of multiple chemical components in Elsholtzia ciliata.

[0098] In summary, this invention establishes an analytical method for the simultaneous determination of 14 chemical components in Elsholtzia ciliata using UPLC-MS / MS technology, and applies it to the content determination of different batches of Elsholtzia ciliata. This method can achieve simultaneous quantification of multiple components in a short time (within 6 minutes). The detection method established in this invention exhibits good linearity, high precision, and accurate and reliable results, reflecting the differences in the content of chemical components in different batches of Elsholtzia ciliata, and providing a reference for comprehensively improving the quality control standards of Elsholtzia ciliata.

[0099] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting the content of multiple chemical components in Elsholtzia ciliata, comprising the following steps: The powder of Elsholtzia ciliata to be tested was extracted using an extractant to obtain a sample solution; the extractant included lower alcohols or aqueous solutions of lower alcohols; the volume fraction of the lower alcohols in the extractant was 70-100%; The sample solution to be tested was subjected to high performance liquid chromatography-tandem mass spectrometry to obtain the detection results of the content of multiple chemical components in Elsholtzia ciliata. The multi-index chemical components include protocatechuic acid, protocatechuic aldehyde, caffeic acid, rutin, isoquercitrin, luteolin, p-coumaric acid, kaempferol-3-O-rutinoside, astragaloside, rosmarinic acid, luteolin, apigenin, baicalin and baicalin-7-methyl ether. The high-performance liquid chromatography-tandem mass spectrometry detection includes high-performance liquid chromatography separation and mass spectrometry detection; The high-performance liquid chromatography (HPLC) separation conditions include: an ACQUITY UPLC® BEH C18 column; mobile phase A is 0.08–0.12 vol% formic acid aqueous solution, mobile phase B is acetonitrile, and the elution method is gradient elution. The gradient elution program is as follows: 0–3 min, the volume fraction of mobile phase B increases from 20% to 50%; 3–4 min, the volume fraction of mobile phase B increases from 50% to 70%; 4–6 min, the volume fraction of mobile phase B increases from 70% to 80%.

2. The detection method according to claim 1, characterized in that, The lower alcohols include methanol and / or ethanol.

3. The detection method according to claim 1 or 2, characterized in that, The solid-liquid ratio of the tested Elsholtzia powder to the extractant is 1g:0.8~1.2L.

4. The detection method according to claim 1 or 2, characterized in that, The extraction includes ultrasonic extraction.

5. The detection method according to claim 4, characterized in that, The ultrasonic extraction power is 280~320W, the frequency is 30~50kHz, and the time is 50~70min.

6. The detection method according to claim 1, characterized in that, The column temperature for the high-performance liquid chromatography separation is 28~32℃, the injection volume is 1.8~2.2μL, and the mobile phase flow rate is 0.28~0.32mL / min.

7. The detection method according to claim 1, characterized in that, The mass spectrometry detection conditions include: 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 drying gas temperature is 340~360℃, the drying gas flow rate is 9~13L / min, the nebulizer pressure is 30~40psig, the fragmentation voltage is 93~237V, and the collision energy is 11~39eV.

8. The detection method according to claim 1 or 7, characterized in that, The quantitative ion pairs of protocatechuic acid, protocatechuic aldehyde, caffeic acid, rutin, isoquercitrin, luteolin, p-coumaric acid, kaempferol-3-O-rutinoside, astragaloside, rosmarinic acid, luteolin, apigenin, baicalin, and baicalein-7-methyl ether are 153.1 / 109.1, 137.1 / 108.1, 179.1 / 135.1, and 609, respectively. 1 / 300.1, 463.1 / 300.1, 447.1 / 285.1, 163.0 / 119.1, 593.2 / 285.1, 447.1 / 284.1, 358.9 / 161.0, 285.2 / 133.0, 269.2 / 117.1, 461.1 / 285.0, and 285.1 / 270.

0.

9. The detection method according to claim 1 or 7, characterized in that, The collision energies of protocatechuic acid, protocatechuic aldehyde, caffeic acid, rutin, isoquercitrin, luteolin, p-coumaric acid, kaempferol-3-O-rutinoside, astragaloside, rosmarinic acid, luteolin, apigenin, baicalin, and baicalein-7-methyl ether are 12 eV, 24 eV, 14 eV, 38 eV, 27 eV, 27 eV, 11 eV, 32 eV, 26 eV, 13 eV, 36 eV, 39 eV, 24 eV, and 32 eV, respectively.

10. The detection method according to claim 1 or 7, characterized in that, The fragmentation voltages of the protocatechuic acid, protocatechuic aldehyde, caffeic acid, rutin, isoquercitrin, luteolin, p-coumaric acid, kaempferol-3-O-rutinoside, astragaloside, rosmarinic acid, luteolin, apigenin, baicalin, and baicalein-7-methyl ether are 107V, 117V, 117V, 139V, 158V, 188V, 104V, 144V, 177V, 237V, 168V, 146V, 93V, and 141V, respectively.

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