Method for simultaneously determining contents of 21 components in radix tetrastigme based on LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) technology

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a method for simultaneously determining the contents of 21 components in radix tetrastigme based on an LC-MS / MS technology. The method comprises the following steps: preparing a mixed reference substance solution of 21 components, preparing a radix tetrastigme extracting solution as a test solution, and performing high performance liquid chromatography-mass spectrometry analysis on the test solution and the mixed reference substance solution under the same conditions; wherein the mixed reference substance solution is taken and diluted into multiple concentrations, a linear regression equation curve is drawn by taking the mass concentration of the mixed reference substance solution as an abscissa and the peak area as an ordinate, and the mass concentrations of the 21 components in the test solution are calculated through the peak area in a linear regression equation, so that the contents of the 21 components in the radix tetrastigme can be obtained. According to the method, efficient and accurate determination of multiple components can be realized, and reliable theoretical basis and data support are provided for quality evaluation and high-quality germplasm screening of radix tetrastigme.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a method for simultaneously determining the content of 21 components in Tripterygium wilfordii based on LC-MS / MS technology. Background Technology

[0002] San Ye Qing is derived from the fresh or dried tuberous roots of Tetrastigma hemsleyanum Diels et Gilg, a plant belonging to the genus Tetrastigma in the Vitaceae family. It is a rare and precious traditional Chinese medicine unique to China, with effects such as clearing heat and detoxifying, reducing swelling and relieving pain, resolving phlegm and dissipating nodules. It is often used to treat infantile high fever and convulsions, whooping cough, carbuncles and phlegm nodules, snake bites, and other ailments.

[0003] Currently, there are no national standards for the testing of *Tripterygium wilfordii*. Local drug standards include the 2015 edition of the *Zhejiang Province Standard for Processing Traditional Chinese Medicine*, the 2012 edition of the *Fujian Province Standard for Processing Traditional Chinese Medicine Pieces*, and the 2010 edition of the *Hunan Province Standard for Processing Traditional Chinese Medicine Pieces*. These standards only include testing items such as appearance, chemical reaction, microscopic identification, moisture and total ash content testing, and extractives determination. None of these standards include qualitative or quantitative indicators of characteristic components, making it difficult to effectively control the quality of *Tripterygium wilfordii* and ensure its clinical efficacy. Summary of the Invention

[0004] The present invention aims to provide a method for the efficient and accurate determination of multiple components in Tripterygium wilfordii, providing a reliable theoretical basis and data support for the quality evaluation and high-quality germplasm screening of Tripterygium wilfordii.

[0005] To achieve the above objectives, this invention provides a method for simultaneously determining the content of 21 components in *Tripterygium wilfordii* based on LC-MS / MS technology, comprising: (1) Prepare the mixed reference solution and the test solution; The preparation method of the mixed reference solution includes: accurately weighing the reference standards of 21 components in Tripterygium wilfordii, then adding the reference standard stock solutions made of methanol, and then accurately measuring each reference standard stock solution and adding methanol to obtain the mixed reference solution; The test solution was an extract of *Trifolium repens*. (2) Dilute the mixed reference solution to multiple mass concentrations, analyze the mixed reference solution at multiple mass concentrations by high performance liquid chromatography-mass spectrometry, and plot the linear regression equation curve with the mass concentration of the mixed reference solution as the abscissa and the peak area as the ordinate. (3) Perform high performance liquid chromatography-mass spectrometry analysis on the test solution under the same settings, and calculate the mass concentration of 21 components in the test solution through the linear regression equation obtained in (2) to obtain the content of 21 components in Trifolium repens. The 21 components are: trigonelline, succinic acid, uridine, guanosine, adenosine, protocatechuic acid, (-)-gallic acid, 5-hydroxymaltol, protocatechuic aldehyde, p-hydroxybenzoic acid, vanillyl glycol, proanthocyanidin B1, catechin, epicatechin, protocatechuic aldehyde methyl ester, salicylic acid, rutin, isoquercitrin, kaempferol-3-O-rutinoside, astragaloside, and kaempferol-7-O-α-L-rhamnoside.

[0006] In the above method, the high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis conditions are as follows: the chromatographic column is an Agilent RRHD Eclipse Plus C1000 LC4 ... 18 The column has an inner diameter of 2.1 mm, a length of 50 mm, and a packing particle size of 1.8 μm; the column temperature is 35 °C, the flow rate is 0.3 mL / min, and the injection volume is 1 μL. The mobile phases are mobile phase A and mobile phase B. Mobile phase A is methanol, and mobile phase B is a 5 mmol / L aqueous solution of ammonium formate containing 0.1% formic acid. The gradient elution conditions of the mobile phase were set in the software as follows: 0-5 min, the mass percentage of mobile phase A in the mobile phase increased from 3% to 5%; 5-11 min, the mass percentage of mobile phase A in the mobile phase increased from 5% to 11%; 11-12 min, the mass percentage of mobile phase A in the mobile phase increased from 11% to 40%; 12-17 min, the mass percentage of mobile phase A in the mobile phase increased from 40% to 70%; 17-18 min, the mass percentage of mobile phase A in the mobile phase increased from 70% to 90%; 18-20 min, the mass percentage of mobile phase A in the mobile phase was 90%.

[0007] In the above method, the mass spectrometry conditions for high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis are: electrospray ionization source; multiple reaction monitoring mode; sheath gas temperature 250℃; sheath gas flow rate 11 L / min. -1 The drying gas is N2, temperature 300℃, flow rate 5 L·min. -1 Positive and negative ion mode, capillary voltage 3500V.

[0008] The preparation method of the test solution in the above method is as follows: After pulverizing *Trifolium repens*, the powder is sieved to obtain powder. 1.0038~1.0481g of powder is accurately weighed and placed in a stoppered conical flask. 20mL of a 70% (v / v) methanol aqueous solution is accurately added, the flask is sealed, the weight is measured, and the mixture is sonicated for 45min. After cooling to room temperature, the weight is measured again. The weight loss is replenished with a 70% methanol aqueous solution, and the mixture is shaken well. The supernatant is taken and filtered through a 0.22μm microporous membrane. The filtrate is taken to obtain the test solution.

[0009] The mass spectrometry parameters for the 21 components described above are as follows: The retention time of trigonelline was 0.516 min, the precursor ion was 138.1 m / z, the fragment voltage was 116 V, and the product ion was 92.1. * 94.1 m / z, collision energy of 24.24 V, ionization mode is positive ion; The retention time of succinic acid was 1.091 min, the precursor ion was 117.0 m / z, the fragmentation voltage was 61 V, and the daughter ion was 73.1 V. * 99.1 m / z, collision energy of 8.8 V, ionization mode is negative ion; The retention time of uridine was 1.186 min, the precursor ion was 289.1 m / z, the fragment voltage was 49 V, and the daughter ion was 243.0 V. * 200.0 m / z, collision energy of 4.16 V, ionization mode of negative ions; The retention time of guanosine was 2.22 min, the precursor ion was 282.1 m / z, the fragmentation voltage was 100 V, and the product ion was 150.0 V. * 133.0 m / z, collision energy of 16.32 V, ionization mode is negative ion; The retention time of adenosine was 2.62 min, the precursor ion was 268.1 m / z, the fragment voltage was 85 V, and the daughter ion was 136.1 m / z. * 119.1 m / z, collision energy of 16,50 V, ionization mode is positive ion; The retention time of protocatechuic acid was 3.397 min, the precursor ion was 153.0 m / z, the fragmentation voltage was 90 V, and the daughter ion was 109.1. * 108.1 m / z, collision energy of 12.28 V, ionization mode is negative ion; The retention time of (-)-gallic catechin was 3.914 min, the precursor ion was 307.1 m / z, the fragmentation voltage was 68 V, and the daughter ion was 139.0. * 163.0 m / z, collision energy of 20,12 V, ionization mode is positive ion; The retention time of 5-hydroxymaltol was 4.242 min, the precursor ion was 143.0 m / z, the fragmentation voltage was 91 V, and the product ion was 41.3 V. * 39.3 m / z, collision energy of 26.38 V, ionization mode is positive ion; The retention time of protocatechuic aldehyde was 5.544 min, the precursor ion was 137.0 m / z, the fragmentation voltage was 100 V, and the daughter ion was 108.0 V. * 92.1 m / z, collision energy of 24.24 V, ionization mode is negative ion; The retention time of p-hydroxybenzoic acid was 6.031 min, the precursor ion was 137.0 m / z, the fragment voltage was 68 V, and the product ion was 93.1. * 65.2 m / z, collision energy of 16.32 V, ionization mode is negative ion; The retention time of vanillyl glycol was 8.677 min, the precursor ion was 216.1 m / z, the fragmentation voltage was 53 V, and the daughter ion was 137.0. * 94.1 m / z, collision energy of 10,46 V, ionization mode is positive ion; The retention time of proanthocyanidin B1 was 9.58 min, the precursor ion was 579.2 m / z, the fragmentation voltage was 98 V, and the daughter ion was 127.0 V. * 289.1 m / z, collision energy of 12.28 V, ionization mode is positive ion; The retention time of catechins was 10.259 min, the precursor ion was 289.1 m / z, the fragmentation voltage was 125 V, and the daughter ion was 245.1 m / z. * 203.0 m / z, collision energy of 12,20 V, ionization mode of negative ions; The retention time of epicatechin was 12.827 min, the precursor ion was 291.1 m / z, the fragmentation voltage was 92 V, and the daughter ion was 139.0. * ,122.9 m / z, collision energy of 12.28 V, ionization mode is positive ion; The retention time of protocatechuic aldehyde methyl ester was 12.834 min, the precursor ion was 167.0 m / z, the fragment voltage was 96 V, and the daughter ion was 108.1 V. * 152.0 m / z, collision energy of 22.14 V, ionization mode is negative ion; The retention time of salicylic acid was 12.877 min, the precursor ion was 137.0 m / z, the fragmentation voltage was 63 V, and the daughter ion was 93.1. * 65.2 m / z, collision energy of 16.36 V, ionization mode is negative ion; The retention time of rutin was 13.811 min, the precursor ion was 611.2 m / z, the fragment voltage was 121 V, and the daughter ion was 303.1. * 85.1 m / z, collision energy of 20,50 V, ionization mode is positive ion; The retention time of isoquercitrin was 13.814 min, the precursor ion was 465.1 m / z, the fragmentation voltage was 83 V, and the daughter ion was 303.1. * 85.1 m / z, collision energy of 8.32 V, ionization mode is positive ion; The retention time of kaempferol-3-O-rutin was 14.278 min, the precursor ion was 595.2 m / z, the fragment voltage was 53 V, and the daughter ion was 287.1. * 449.1 m / z, collision energy of 8,20 V, ionization mode is positive ion; The retention time of astragaloside was 14.282 min, the precursor ion was 449.1 m / z, the fragment voltage was 53 V, and the daughter ion was 287.0. * 85.1 m / z, collision energy of 8.32 V, ionization mode is positive ion; The retention time of kaempferol-7-O-α-L-rhamnoside was 15.519 min, the precursor ion was 433.1 m / z, the fragment voltage was 88 V, and the product ion was 287.0. * 71.1 m / z, collision energy of 8.28 V, ionization mode is positive ion; in," * "This refers to quantitative ions."

[0010] In the above method, the detection limit was determined by the concentration with a signal-to-noise ratio (S / N) of 3, and the quantitation limit was determined by the concentration with a S / N of 10. The linear equations, correlation coefficients, linear ranges, detection limits, and quantitation limits of the 21 compounds are shown below: The linear equation for trigonelline is Y1 = 520.80X1 + 255145.30, with a linear range of 33.50–3350.38 ng·mL. -1 The detection limit is 0.09 ng / mL. -1 The limit of quantitation is 0.31 ng / mL. -1 ; The linear equation for succinic acid is Y² = 5.01X² + 5007.95, with a linear range of 251.10–15065.81 ng·mL. -1 The detection limit is 12.15 ng / mL. -1 The limit of quantitation is 40.50 ng / mL. -1 ; The linear equation for uridine was Y3 = 9.62X3 + 1045.93, with a linear range of 56.44–5643.64 ng / mL. -1 The detection limit is 3.94 ng / mL. -1 The limit of quantitation is 13.12 ng / mL. -1 ; The linear equation for guanosine was Y4 = 4.73X4 + 331.51, with a linear range of 54.74–5473.73 ng / mL. -1 The detection limit is 0.86 ng / mL. -1 The limit of quantitation is 2.87 ng / mL.-1 ;; The linear equation for adenosine was Y5 = 392.81X5 + 105717.10, with a linear range of 45.66–4566.26 ng / mL. -1 The detection limit is 0.08 ng / mL. -1 The limit of quantitation is 0.26 ng / mL. -1 ; The linear equation for protocatechuic acid is Y6 = 19.61X6 + 2575.88, with a linear range of 68.92–6892.19 ng·mL. -1 The detection limit is 9.40 ng / mL. -1 The limit of quantitation is 31.33 ng / mL. -1 ; The linear equation for (-)-gallic acid catechin is Y7 = 29.11X7 + 2068.58, with a linear range of 51.65~5164.60 ng·mL. -1 The detection limit is 0.48 ng / mL. -1 The limit of quantitation is 1.60 ng / mL. -1 ; The linear equation for 5-hydroxymaltol was Y8 = 5.29X8 - 1010.84, with a linear range of 383.95–7679.28 ng·mL. -1 The detection limit is 76.79 ng·mL. -1 The limit of quantitation is 255.96 ng / mL. -1 ; The linear equation for protocatechuic aldehyde is Y9 = 25.40X9 + 1367.63, with a linear range of 18.76–1876.35 ng·mL. -1 The detection limit is 0.83 ng / mL. -1 The limit of quantitation is 2.76 ng / mL. -1 ; The linear equation for p-hydroxybenzoic acid is Y 10 =39.08X 10 +682.35, linear range 16.29~1628.74 ng·mL -1 The detection limit is 1.14 ng / mL. -1 The limit of quantitation is 3.79 ng / mL. -1 ; The linear equation for vanillyl glycol is Y 11 =3.34X 11 -189.31, linear range 166.52~16652.16 ng·mL -1 The detection limit is 26.29 ng / mL. -1The limit of quantitation is 87.64 ng / mL. -1 ; The linear equation for proanthocyanidin B1 is Y 12 =2.91X 12 +2430.45, linear range 419.60~16783.94 ng·mL -1 The detection limit is 2.62 ng / mL. -1 The limit of quantitation is 8.74 ng / mL. -1 ; The linear equation for catechins is Y 13 =8.24X 13 +10039.83, linear range 223.20~17856.00 ng·mL -1 The detection limit is 12.88 ng / mL. -1 The limit of quantitation is 42.92 ng / mL. -1 ; The linear equation for epicatechin is Y 14 =6.58X 14 -54.75, linear range 40.98~4097.67 ng·mL -1 The detection limit is 0.31 ng / mL. -1 The limit of quantitation is 1.04 ng / mL. -1 ; The linear equation for protocatechuic aldehyde methyl ester is Y 15 =147.90X 15 +2340.90, linear range 3.66~366.03 ng·mL -1 The detection limit is 0.22 ng / mL. -1 The limit of quantitation is 0.73 ng / mL. -1 ; The linear equation for salicylic acid is Y. 16 =64.23X 16 +17071.74, linear range 41.08~4107.77 ng·mL -1 The detection limit is 2.93 ng / mL. -1 The limit of quantitation is 9.78 ng / mL. -1 ; The linear equation for rutin is Y 17 =5.24X 17 +564.89, linear range 419.06~16762.56 ng·mL -1 The detection limit is 0.61 ng / mL. -1 The limit of quantitation is 2.04 ng / mL. -1 ; The linear equation for isoquercitrin is Y18 =14.37X 18 -1.19, linear range 40.10~4009.50 ng·mL -1 The detection limit is 0.99 ng / mL. -1 The limit of quantitation is 3.29 ng / mL. -1 ; The linear equation for kaempferol-3-O-rutin is Y. 19 =5.16X 19 +2176.77, linear range 430.40~17216.06 ng·mL -1 The detection limit is 0.43 ng / mL. -1 The limit of quantitation is 1.42 ng / mL. -1 ; The linear equation for astragaloside is Y 20 =7.76X 20 +231.29, linear range 42.49~4249.15 ng·mL -1 The detection limit is 0.82 ng / mL. -1 The limit of quantitation is 2.72 ng / mL. -1 ; The linear equation for kaempferol-7-O-α-L-rhamnoside is Y. 21 =10.21X 21 +347.91, linear range 29.52~2952.25 ng·mL -1 The detection limit is 1.38 ng / mL. -1 The limit of quantitation is 4.61 ng / mL. -1 .

[0011] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: 1. This invention establishes an effective method for simultaneous quantitative detection of multiple components in *Tripterygium wilfordii* based on LC-MS, covering a total of 21 active ingredients, including proanthocyanidins, flavonoids, organic acids, and nucleosides. This method overcomes the shortcomings of existing provincial and municipal processing standards that rely solely on morphological identification and basic examinations while lacking quantitative indicators for components, thus filling the technological gap in precise quality control of medicinal materials.

[0012] 2. The determination method provided by this invention can be systematically verified, demonstrating its high stability and reliability. It can clearly quantify the differences in the content of key components in *Tripterygium wilfordii* samples from different origins. The obtained data provides core scientific basis for the quality grading of authentic medicinal materials and the screening of high-quality germplasm.

[0013] 3. The present invention provides a method for simultaneously determining the content of 21 components in Tripterygium wilfordii based on LC-MS / MS technology, which significantly improves efficiency compared with traditional component screening technology. A single analysis can complete the accurate quantification of 21 target components. The calculation mechanism based on external standard method and standard curve ensures the accuracy of the results, providing the industry with a solution that combines high efficiency and high reliability. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0015] Figure 1 A1 is a multiple reaction monitoring chromatogram of a mixed reference solution; A2 is a multiple reaction monitoring chromatogram of a mixed reference solution containing C1, C2, C4~C7, C9~C12, C15~C17, C19, and C21; A3 is a multiple reaction monitoring chromatogram of a mixed reference solution containing C3, C8, C13, C14, C18, and C20. Figure 2 B1 is the multiple reaction monitoring chromatogram of the test solution; B2 is the multiple reaction monitoring chromatogram of the test solution C1, C2, C4~C7, C9~C12, C15~C17, C19, C21; B3 is the multiple reaction monitoring chromatogram of the test solution C3, C8, C13, C14, C18, C20. Among them, C1 is trigonelline; C2 is succinic acid; C3 is uridine; C4 is guanosine; C5 is adenosine; C6 is protocatechuic acid; C7 is (-)-gallicatechin; C8 is 5-hydroxymaltol; C9 is protocatechuic aldehyde; C10 is p-hydroxybenzoic acid; C11 is vanillyl glycol; C12 is proanthocyanidin B1; C13 is catechin; C14 is epicatechin; C15 is protocatechuic aldehyde methyl ester; C16 is salicylic acid; C17 is rutin; C18 isoquercitrin; C19 is kaempferol-3-O-rutinoside; C20 is astragaloside; and C21 is kaempferol-7-O-α-L-rhamnoside. Detailed Implementation

[0016] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods in each embodiment are conventional methods; reagents and materials, unless otherwise specified, are commercially available. Unless otherwise specified, all percentages in the following embodiments refer to mass percentages. Unless otherwise specified, all proportions in the following embodiments refer to mass ratios.

[0017] Instruments and reagents: Agilent UHPLC-QQQ-MS / MS (1290-6470) system (Agilent Technologies, Inc., USA); XS105DU electronic balance (Mettler-Toledo International AG, Switzerland); DL-360D intelligent ultrasonic cleaner (Shanghai Zhixin Instrument Co., Ltd., China); Milli-Q Advantage A10 Ultrapure Water System (Merck Millipore GmbH, Germany).

[0018] Methanol (chromatographic grade, Merck KGaA, Germany); Formic acid (chromatographic grade, Aladdin Biochemical Technology Co., Ltd., Shanghai, China); Ammonium formate (mass spectrometry grade, Sigma-Aldrich, USA).

[0019] Reference standards for 21 components in *Trifolium repens*: trigonelline (batch number A28IB224336, mass fraction 98.0%) and succinic acid (batch number O25IB230020, mass fraction 98.0%), were all purchased from Shanghai Yuanye Biotechnology Co., Ltd. (-)- Gallic catechin (batch number WP24051302, mass fraction 98.0%), purchased from Sichuan Weikeqi Biotechnology Co., Ltd.; Proanthocyanidin B1 (batch number 250039-202112, mass fraction 97.27%) and protocatechuic aldehyde methyl ester (batch number 250462-202302, mass fraction 98.0%) were both purchased from Shanghai Hongyong Biotechnology Co., Ltd. Kaempferol-3-O-rutin (batch number PS012599, mass fraction 99.63%) was purchased from Chengdu Pusi Biotechnology Co., Ltd. Astragaloside (batch number AT-CC0703, mass fraction 99.57%) was purchased from Chengdu Efa Biotechnology Co., Ltd. 5-Hydroxymaltol (batch number 202508, mass fraction 98.0%) and vanillyl glycol (batch number 202508, mass fraction 98.0%) were both prepared in-house. Uric acid (batch number 110887-201803, mass fraction 99.5%), guanosine (batch number 111977-201501, mass fraction 93.6%), adenosine (batch number 110879-201703, mass fraction 99.7%), protocatechuic acid (batch number 110809-202207, mass fraction 97.5%), protocatechuic aldehyde (batch number 110810-201608, mass fraction 99.7%), catechins (batch number 11... The following ingredients were purchased from the National Institutes for Food and Drug Control: epicatechin (batch number 110878-201703, mass fraction 99.7%), salicylic acid (batch number 100106-202106, mass fraction 99.8%), isoquercitrin (batch number 111809-201804, mass fraction 97.2%), and rutin (batch number 100080-201610, mass fraction 91.9%). p-Hydroxybenzoic acid (batch number 2418996, mass fraction 99.8%) was purchased from Shanghai Anpu Cuishi Standard Technical Service Co., Ltd. Kaempferol-7-O-α-L-rhamnoside (batch number DST250926-437, mass fraction 98.0%) was purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.

[0020] The five batches of *Tetrastigma hemsleyanum* medicinal materials were all collected from Lishui City, Zhejiang Province (numbered ZSYQ1, ZSYQ2, ZSYQ3, ZSYQ4, and ZSYQ5), with harvesting periods of November 2, 2023, May 20, 2024, June 23, 2024, December 1, 2024, and July 7, 2025, respectively. They were identified by Yu Le, Deputy Chief Pharmacist of Traditional Chinese Medicine at the Lishui Municipal Institute of Quality Inspection and Testing, as dried tuberous roots of *Tetrastigma hemsleyanum* Diels et Gilg, a plant belonging to the genus *Tetrastigma* of the Vitaceae family.

[0021] A method for preparing 5-hydroxymaltol is provided herein for use in the following examples: S1. First, 3.0 kg of dried tuberous roots of *Trifolium repens* were crushed and ultrasonically extracted with 70% ethanol aqueous solution for 1 hour, followed by 3 extractions, each lasting 5 days, to obtain the extract. The extract was then concentrated under reduced pressure and a suitable amount of water was added to form a suspension. The suspension was then separated using an Agilent 1260 preparative liquid chromatograph with AB-8 macroporous adsorption resin. Finally, the extract was eluted sequentially with pure water, 20% ethanol aqueous solution, 70% ethanol aqueous solution, and 95% ethanol aqueous solution. The eluted fraction with 70% ethanol aqueous solution was collected and concentrated to obtain 85 g of *Trifolium repens* extract. S2. The *Trifolium repens* extract and silica gel were mixed at a volume ratio of 1:1, air-dried, and packed into a column. Elution was first performed with 0.5 L of dichloromethane, followed by gradient elution with dichloromethane-methanol at ratios of 19:1, 9:1, 4:1, 7:3, 3:2, and 1:1 (v / v), 0.5 L per gradient. 100 mL fractions were collected in Erlenmeyer flasks. After TLC analysis, identical fractions were combined and concentrated under reduced pressure, then placed in sample vials to obtain five fractions, labeled as fraction I, fraction II, fraction III, fraction IV, and fraction V. TLC analysis showed that fraction V contained characteristic spots of the target compound. S3. Fraction V was subjected to Sephadex LH-20 gel column chromatography, eluted with 1:1 (v / v) methanol-dichloromethane. 28 fractions were collected in 10 mL test tubes, analyzed by TLC, and then concentrated under reduced pressure. The fractions were then separated using a semi-preparative HPLC column. The mobile phase was 22:78 (v / v) acetonitrile-water. Detection wavelengths were 220 nm and 254 nm; flow rate was 1.5 mL / min; column temperature was 30 °C. 5-hydroxymaltol (6.5 mg; t) was obtained. R =14min).

[0022] Example 1 This embodiment presents a method for simultaneously determining the content of 21 components in *Tripterygium wilfordii* based on LC-MS / MS technology, including: (1) Prepare the mixed reference solution and the test solution; The preparation method of the mixed reference solution includes: accurately weighing each reference standard, and then adding methanol to dilute to a concentration of 457.5375~814.3680 μg·mL. -1 The reference stock solutions were prepared, and then each reference stock solution was accurately measured and placed in the same 50 mL volumetric flask. Methanol was added to the mark and the mixture was shaken well to obtain a mixed reference solution. The specific preparation data are shown in Table 1. Table 1: Process data for preparing mixed standard solutions The test solution was prepared by ultrasonic extraction of *Tripterygium wilfordii*. The specific preparation method is as follows: *Tripterygium wilfordii* powder was passed through a No. 3 sieve, and approximately 1.0 g was accurately weighed. It was placed in a stoppered conical flask, and 20 mL of 70% methanol-water (v / v) solution was accurately added. After sealing the flask, the weight was measured. The solution was ultrasonically treated for 45 minutes, cooled to room temperature, weighed again, and the weight loss was replenished with 70% methanol-water solution. After shaking well, the supernatant was filtered through a 0.22 μm microporous membrane, and the filtrate was collected.

[0023] All five batches of *Trifolium repens* medicinal materials numbered ZSYQ1 to ZSYQ5 were prepared using the same method, and two samples were prepared in parallel. The average value was then used to record the results.

[0024] (2) Dilute the mixed reference solution to multiple mass concentrations, and analyze the mixed reference solution at multiple mass concentrations using high performance liquid chromatography-mass spectrometry (HPLC-MS); wherein the HPLC conditions are: Agilent RRHDEclipse Plus C column. 18 The column has an inner diameter of 2.1 mm, a length of 50 mm, and a packing particle size of 1.8 μm. Mobile phase A is methanol, and mobile phase B is a 5 mmol / L ammonium formate aqueous solution containing 0.1% formic acid. The column temperature is 35℃, the flow rate is 0.3 mL / min, and the injection volume is 1 μL. The concentrations of the mixed standards in the mixed reference solution are shown in Table 2: Table 2: Concentration of mixed standard in the mixed standard solution The gradient elution conditions were as follows: 0-5 min, A was 3%→5%; 5-11 min, A was 5%→11%; 11-12 min, A was 11%→40%; 12-17 min, A was 40%→70%; 17-18 min, A was 70%→90%; 18-20 min, A was 90%.

[0025] Mass spectrometry conditions were: electrospray ionization source; multiple reaction monitoring mode; sheath gas temperature 250℃, sheath gas flow rate 11 L·min. -1 The drying gas is N2, temperature 300℃, flow rate 5 L·min. -1 The mass spectrometry parameters for the 21 components were measured in positive and negative ion mode, with a capillary voltage of 3500 V. The multiple reaction monitoring chromatograms are shown in Table 3. Figures 1-2 : Table 3: Mass spectrometry parameters for quantitative analysis of 21 compounds Note:" * "Quantitative ions" Peak area (Y) versus mass concentration (X, ng·mL) -1 Linear regression was performed, and the limit of detection was determined at the concentration with a signal-to-noise ratio (S / N) of 3, and the limit of quantitation was determined at the concentration with a S / N of 10, as shown in Table 4. Table 4: Linear equations, correlation coefficients, linear ranges, limits of detection, and limits of quantitation for 21 compounds. (3) The test solution was analyzed by high performance liquid chromatography-mass spectrometry under the same conditions. The concentrations of the 21 components in the test solution were calculated by substituting the peak areas into the linear regression equation. The contents of the 21 components in *Trifolium repens* can be obtained, as shown in Table 5. Table 5: Results of determination of the content of 21 components in the test solution Precision experiments were performed on the method in this embodiment: 1 μL of the same mixed standard solution was accurately pipetted and injected six times consecutively under the high-performance liquid chromatography-mass spectrometry (HPLC-MS) conditions described in this embodiment, and the peak areas were recorded. The results are shown in Table 6. Table 6: Precision Experiment Results Data Table The results showed that the peak area RSD of the 21 components was 1.51% to 4.79%, indicating that the instrument had good precision.

[0026] To test the repeatability of the assay method in this embodiment, ZSYQ4 was used as the sample to be tested, and six parallel samples were taken. The test solution was prepared according to the preparation method in this embodiment, injected, and the peak area was recorded and the content calculated. The results are shown in Table 7. Table 7: Data Table of Repeatability Experiment Results The results showed that the RSD of the contents of the 21 components ranged from 1.57% to 4.52%, indicating that the method had good repeatability.

[0027] Stability tests were performed on the determination method in this embodiment: 1 μL of the test solution from the repeatability experiment was accurately pipetted and measured at 0, 5, 10, 15, 20, and 24 h, respectively. The peak areas were recorded, and the results are shown in Table 8. Table 8: Stability Test Results Data Table The results showed that the peak area RSD of the 21 components ranged from 2.25% to 8.93%, indicating that the test solution was basically stable within 24 hours.

[0028] A spiking recovery test was conducted on the determination method in this embodiment: Six parallel samples of ZSYQ4 with known content were accurately weighed and placed in stoppered conical flasks. An appropriate amount of reference solution equal to the amount of each analyte was added to each flask. The spiking recovery solution was prepared according to the method for preparing the test solution in this embodiment. The samples were injected for determination, the peak area was recorded, and the content was calculated. The results are shown in Table 9. Table 9: Recovery Rate Test Results Data Table The results showed that the average recoveries of the 21 components ranged from 82.30% to 117.02%, with RSDs ranging from 2.54% to 9.77%.

[0029] In summary, the LC-MS / MS simultaneous analysis method constructed in this embodiment achieved accurate quantitative analysis of 21 chemical components in *Tripterygium wilfordii*, covering key components such as trigonelline and succinic acid. Method validation showed excellent instrument precision, good repeatability in parallel sample tests, and RSD consistently maintained below 4.43%. The test solution exhibited reliable stability within 24 hours, with a peak area RSD below 8.83%. The recovery rate ranged from 81.74% to 118.05%, validating the method's accuracy. The results of testing five batches of medicinal materials showed that the trigonelline content ranged from 18.8 to 40.0 μg·g⁻¹. -1 The concentration of succinic acid is 87.3~177.8 μg·g. -1 The content of vanillylglycol ranged from 108.0 to 264.1 μg·g. -1 Exhibiting excellent sensitivity, combined with a linear correlation coefficient of 0.993~1.000 and stable reproducibility, this method, based on LC-MS / MS technology, verifies that it establishes a reliable technical platform for the multi-component quality control of *Tripterygium wilfordii*.

[0030] The above description effectively illustrates the basic principles, main features, and advantages of the present invention. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention. The present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A method for simultaneously determining the content of 21 components in *Tripterygium wilfordii* based on LC-MS / MS technology, characterized in that, include: (1) Prepare the mixed reference solution and the test solution; The method for preparing the mixed reference solution includes: accurately weighing the reference standards of 21 components in *Trifolium repens*, then adding methanol to each to prepare reference standard stock solutions, and then accurately measuring each of the reference standard stock solutions and adding methanol to obtain the mixed reference solution. The test solution is an extract of *Trifolium repens*. (2) Dilute the mixed reference solution to multiple mass concentrations, perform high performance liquid chromatography-mass spectrometry analysis on the mixed reference solution at multiple mass concentrations, and plot the linear regression equation curve with the mass concentration of the mixed reference solution as the abscissa and the peak area as the ordinate. (3) The test solution is analyzed by high performance liquid chromatography-mass spectrometry under the same setting conditions. The mass concentration of the 21 components in the test solution is calculated by the linear regression equation obtained in (2), and the content of the 21 components in Trifolium repens can be obtained. The 21 components are: trigonelline, succinic acid, uridine, guanosine, adenosine, protocatechuic acid, (-)-gallic acid, 5-hydroxymaltol, protocatechuic aldehyde, p-hydroxybenzoic acid, vanillyl glycol, proanthocyanidin B1, catechin, epicatechin, protocatechuic aldehyde methyl ester, salicylic acid, rutin, isoquercitrin, kaempferol-3-O-rutinoside, astragaloside, and kaempferol-7-O-α-L-rhamnoside.

2. The method according to claim 1, characterized in that, In the high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis, the HPLC conditions are as follows: the chromatographic column is an Agilent RRHD Eclipse Plus C1000 LC4 ... 18 The column has an inner diameter of 2.1 mm, a length of 50 mm, and a packing particle size of 1.8 μm; the column temperature is 35 °C, the flow rate is 0.3 mL / min, and the injection volume is 1 μL. The mobile phase consists of mobile phase A and mobile phase B, wherein mobile phase A is methanol and mobile phase B is a 5 mmol / L aqueous solution of ammonium formate containing 0.1% formic acid; The gradient elution conditions of the mobile phase are set in the software as follows: 0-5 min, the mass percentage of mobile phase A in the mobile phase increases from 3% to 5%; 5-11 min, the mass percentage of mobile phase A in the mobile phase increases from 5% to 11%; 11-12 min, the mass percentage of mobile phase A in the mobile phase increases from 11% to 40%; 12-17 min, the mass percentage of mobile phase A in the mobile phase increases from 40% to 70%; 17-18 min, the mass percentage of mobile phase A in the mobile phase increases from 70% to 90%; 18-20 min, the mass percentage of mobile phase A in the mobile phase is 90%.

3. The method according to claim 1, characterized in that, In the high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis, the mass spectrometry conditions were: electrospray ionization source; multiple reaction monitoring mode; sheath gas temperature 250℃; sheath gas flow rate 11 L·min. -1 The drying gas is N2, temperature 300℃, flow rate 5 L·min. -1 Positive and negative ion mode, capillary voltage 3500V.

4. The method according to claim 1, characterized in that, The preparation method of the test solution is as follows: the *Trifolium repens* is pulverized and sieved to obtain powder. 1.0038~1.0481g of the powder is accurately weighed and placed in a stoppered conical flask. 20mL of a 70% (v / v) methanol aqueous solution is accurately added, the flask is sealed, the weight is measured, and the flask is sonicated for 45min. After cooling to room temperature, the weight is measured again. The weight loss is replenished with a 70% methanol aqueous solution, and the flask is shaken well. The supernatant is taken and filtered through a 0.22μm microporous membrane. The filtrate is collected to obtain the test solution.

5. The method according to claim 1 or 3, characterized in that, The mass spectrometry parameters of the 21 components are as follows: The retention time of trigonelline was 0.516 min, the precursor ion was 138.1 m / z, the fragment voltage was 116 V, and the product ion was 92.

1. * 94.1 m / z, collision energy of 24.24 V, ionization mode is positive ion; The retention time of succinic acid was 1.091 min, the precursor ion was 117.0 m / z, the fragmentation voltage was 61 V, and the daughter ion was 73.1 V. * 99.1 m / z, collision energy of 8.8 V, ionization mode is negative ion; The retention time of uridine was 1.186 min, the precursor ion was 289.1 m / z, the fragment voltage was 49 V, and the daughter ion was 243.0 V. * 200.0 m / z, collision energy of 4.16 V, ionization mode of negative ions; The retention time of guanosine was 2.22 min, the precursor ion was 282.1 m / z, the fragmentation voltage was 100 V, and the product ion was 150.0 V. * 133.0 m / z, collision energy of 16.32 V, ionization mode is negative ion; The retention time of adenosine was 2.62 min, the precursor ion was 268.1 m / z, the fragment voltage was 85 V, and the daughter ion was 136.1 m / z. * 119.1 m / z, collision energy of 16,50 V, ionization mode is positive ion; The retention time of protocatechuic acid was 3.397 min, the precursor ion was 153.0 m / z, the fragmentation voltage was 90 V, and the daughter ion was 109.

1. * 108.1 m / z, collision energy of 12.28 V, ionization mode is negative ion; The retention time of (-)-gallic catechin was 3.914 min, the precursor ion was 307.1 m / z, the fragmentation voltage was 68 V, and the daughter ion was 139.

0. * 163.0 m / z, collision energy of 20,12 V, ionization mode is positive ion; The retention time of 5-hydroxymaltol was 4.242 min, the precursor ion was 143.0 m / z, the fragmentation voltage was 91 V, and the product ion was 41.3 V. * 39.3 m / z, collision energy of 26.38 V, ionization mode is positive ion; The retention time of protocatechuic aldehyde was 5.544 min, the precursor ion was 137.0 m / z, the fragmentation voltage was 100 V, and the daughter ion was 108.0 V. * 92.1 m / z, collision energy of 24.24 V, ionization mode is negative ion; The retention time of p-hydroxybenzoic acid was 6.031 min, the precursor ion was 137.0 m / z, the fragment voltage was 68 V, and the product ion was 93.

1. * 65.2 m / z, collision energy of 16.32 V, ionization mode is negative ion; The retention time of vanillyl glycol was 8.677 min, the precursor ion was 216.1 m / z, the fragmentation voltage was 53 V, and the daughter ion was 137.

0. * 94.1 m / z, collision energy of 10,46 V, ionization mode is positive ion; The retention time of proanthocyanidin B1 was 9.58 min, the precursor ion was 579.2 m / z, the fragmentation voltage was 98 V, and the daughter ion was 127.0 V. * 289.1 m / z, collision energy of 12.28 V, ionization mode is positive ion; The retention time of catechins was 10.259 min, the precursor ion was 289.1 m / z, the fragmentation voltage was 125 V, and the daughter ion was 245.1 m / z. * 203.0 m / z, collision energy of 12,20 V, ionization mode of negative ions; The retention time of epicatechin was 12.827 min, the precursor ion was 291.1 m / z, the fragmentation voltage was 92 V, and the daughter ion was 139.

0. * ,122.9 m / z, collision energy of 12.28 V, ionization mode is positive ion; The retention time of protocatechuic aldehyde methyl ester was 12.834 min, the precursor ion was 167.0 m / z, the fragment voltage was 96 V, and the daughter ion was 108.1 V. * 152.0 m / z, collision energy of 22.14 V, ionization mode is negative ion; The retention time of salicylic acid was 12.877 min, the precursor ion was 137.0 m / z, the fragmentation voltage was 63 V, and the daughter ion was 93.

1. * 65.2 m / z, collision energy of 16.36 V, ionization mode is negative ion; The retention time of rutin was 13.811 min, the precursor ion was 611.2 m / z, the fragment voltage was 121 V, and the daughter ion was 303.

1. * 85.1 m / z, collision energy of 20,50 V, ionization mode is positive ion; The retention time of isoquercitrin was 13.814 min, the precursor ion was 465.1 m / z, the fragmentation voltage was 83 V, and the daughter ion was 303.

1. * 85.1 m / z, collision energy of 8.32 V, ionization mode is positive ion; The retention time of kaempferol-3-O-rutin was 14.278 min, the precursor ion was 595.2 m / z, the fragment voltage was 53 V, and the daughter ion was 287.

1. * 449.1 m / z, collision energy of 8,20 V, ionization mode is positive ion; The retention time of astragaloside was 14.282 min, the precursor ion was 449.1 m / z, the fragment voltage was 53 V, and the daughter ion was 287.

0. * 85.1 m / z, collision energy of 8.32 V, ionization mode is positive ion; The retention time of kaempferol-7-O-α-L-rhamnoside was 15.519 min, the precursor ion was 433.1 m / z, the fragment voltage was 88 V, and the product ion was 287.

0. * 71.1 m / z, collision energy of 8.28 V, ionization mode is positive ion; in," * "This refers to quantitative ions." 6. The method according to claim 1, characterized in that, In the linear regression equation curves plotted, the detection limit was determined by the concentration with a signal-to-noise ratio (S / N) of 3, and the quantitation limit was determined by the concentration with a S / N of 10. The linear equations, correlation coefficients, linear ranges, detection limits, and quantitation limits for the 21 compounds are shown below: The linear equation for trigonelline is Y1 = 520.80X1 + 255145.30, with a linear range of 33.50–3350.38 ng·mL. -1 The detection limit is 0.09 ng / mL. -1 The limit of quantitation is 0.31 ng / mL. -1 ; The linear equation for succinic acid is Y² = 5.01X² + 5007.95, with a linear range of 251.10–15065.81 ng·mL. -1 The detection limit is 12.15 ng / mL. -1 The limit of quantitation is 40.50 ng / mL. -1 ; The linear equation for uridine was Y3 = 9.62X3 + 1045.93, with a linear range of 56.44–5643.64 ng / mL. -1 The detection limit is 3.94 ng / mL. -1 The limit of quantitation is 13.12 ng / mL. -1 ; The linear equation for guanosine was Y4 = 4.73X4 + 331.51, with a linear range of 54.74–5473.73 ng / mL. -1 The detection limit is 0.86 ng / mL. -1 The limit of quantitation is 2.87 ng / mL. -1 ;; The linear equation for adenosine was Y5 = 392.81X5 + 105717.10, with a linear range of 45.66–4566.26 ng / mL. -1 The detection limit is 0.08 ng / mL. -1 The limit of quantitation is 0.26 ng / mL. -1 ; The linear equation for protocatechuic acid is Y6 = 19.61X6 + 2575.88, with a linear range of 68.92–6892.19 ng·mL. -1 The detection limit is 9.40 ng / mL. -1 The limit of quantitation is 31.33 ng / mL. -1 ; The linear equation for (-)-gallic acid catechin is Y7 = 29.11X7 + 2068.58, with a linear range of 51.65~5164.60 ng·mL. -1 The detection limit is 0.48 ng / mL. -1 The limit of quantitation is 1.60 ng / mL. -1 ; The linear equation for 5-hydroxymaltol was Y8 = 5.29X8 - 1010.84, with a linear range of 383.95–7679.28 ng·mL. -1 The detection limit is 76.79 ng·mL. -1 The limit of quantitation is 255.96 ng / mL. -1 ; The linear equation for protocatechuic aldehyde is Y9 = 25.40X9 + 1367.63, with a linear range of 18.76–1876.35 ng·mL. -1 The detection limit is 0.83 ng / mL. -1 The limit of quantitation is 2.76 ng / mL. -1 ; The linear equation for p-hydroxybenzoic acid is Y 10 =39.08X 10 +682.35, linear range 16.29~1628.74 ng·mL -1 The detection limit is 1.14 ng / mL. -1 The limit of quantitation is 3.79 ng / mL. -1 ; The linear equation for vanillyl glycol is Y 11 =3.34X 11 -189.31, linear range 166.52~16652.16 ng·mL -1 The detection limit is 26.29 ng / mL. -1 The limit of quantitation is 87.64 ng / mL. -1 ; The linear equation for proanthocyanidin B1 is Y 12 =2.91X 12 +2430.45, linear range 419.60~16783.94 ng·mL -1 The detection limit is 2.62 ng / mL. -1 The limit of quantitation is 8.74 ng / mL. -1 ; The linear equation for catechins is Y 13 =8.24X 13 +10039.83, linear range 223.20~17856.00 ng·mL -1 The detection limit is 12.88 ng / mL. -1 The limit of quantitation is 42.92 ng / mL. -1 ; The linear equation for epicatechin is Y 14 =6.58X 14 -54.75, linear range 40.98~4097.67 ng·mL -1 The detection limit is 0.31 ng / mL. -1 The limit of quantitation is 1.04 ng / mL. -1 ; The linear equation for protocatechuic aldehyde methyl ester is Y 15 =147.90X 15 +2340.90, linear range 3.66~366.03 ng·mL -1 The detection limit is 0.22 ng / mL. -1 The limit of quantitation is 0.73 ng / mL. -1 ; The linear equation for salicylic acid is Y. 16 =64.23X 16 +17071.74, linear range 41.08~4107.77 ng·mL -1 The detection limit is 2.93 ng / mL. -1 The limit of quantitation is 9.78 ng / mL. -1 ; The linear equation for rutin is Y 17 =5.24X 17 +564.89, linear range 419.06~16762.56 ng·mL -1 The detection limit is 0.61 ng / mL. -1 The limit of quantitation is 2.04 ng / mL. -1 ; The linear equation for isoquercitrin is Y 18 =14.37X 18 -1.19, linear range 40.10~4009.50 ng·mL -1 The detection limit is 0.99 ng / mL. -1 The limit of quantitation is 3.29 ng / mL. -1 ; The linear equation for kaempferol-3-O-rutin is Y. 19 =5.16X 19 +2176.77, linear range 430.40~17216.06 ng·mL -1 The detection limit is 0.43 ng / mL. -1 The limit of quantitation is 1.42 ng / mL. -1 ; The linear equation for astragaloside is Y 20 =7.76X 20 +231.29, linear range 42.49~4249.15 ng·mL -1 The detection limit is 0.82 ng / mL. -1 The limit of quantitation is 2.72 ng / mL. -1 ; The linear equation for kaempferol-7-O-α-L-rhamnoside is Y. 21 =10.21X 21 +347.91, linear range 29.52~2952.25 ng·mL -1 The detection limit is 1.38 ng / mL. -1 The limit of quantitation is 4.61 ng / mL. -1 .

Images