A method for determining the content of effective components of dog spine medicinal materials and preparations thereof

Abstract

The application discloses a method for determining the content of effective components of dog spine medicinal materials and preparations thereof, and belongs to the technical field of Chinese medicinal material and Chinese medicinal preparation detection. The technical problems to be solved are that the quality control indexes of the dog spine medicinal materials and preparations thereof are single, the specificity is not strong, and the internal quality cannot be accurately and comprehensively reflected. Technical solution points are that the application adopts triple quadrupole liquid chromatography-mass spectrometry, uses methanol and water as mobile phases, and realizes the simultaneous detection of the effective components, i.e., chingpensitin and chingpensitin-2'-O-glucoside, in the dog spine medicinal materials and preparations thereof through gradient elution.

Description

Technical Field

[0001] This invention belongs to the field of detection technology of Chinese medicinal materials and preparations, and provides a method for determining the content of effective components in Cibotium barometz and its preparations. Background Technology

[0002] Cibotium barometz is a plant of the family Ceratophyllaceae, specifically Cibotium barometz (Golden- Cibotium barometz The dried rhizome of *Cibotium barometz* (L.) J. Sm. has the effects of dispelling wind and dampness, tonifying the liver and kidneys, and strengthening the waist and knees. It is often used to treat rheumatic pain, soreness and weakness of the waist and knees, and weakness of the lower limbs. There are more than 100 traditional Chinese medicine preparations with Cibotium barometz as the main ingredient, including 10 types of preparations such as pills, capsules, tablets, and medicated wines.

[0003] According to the 2025 edition of the Pharmacopoeia of the People's Republic of China, no content determination index has been established for Cibotium barometz (dog's spine) medicinal materials. The content determination index for its processed slices, "tang gouji" (hot-processed Cibotium barometz), is protocatechuic acid. Currently, for some traditional Chinese medicine preparations containing Cibotium barometz, some preparations do not involve specific determination of the components contained in Cibotium barometz, such as Bushen Qiangshen Capsules, Yishen Gujing Pills, and Jinwu Gutong Capsules. Other preparations, such as Zhuangyao Jianshen Tablets, Shujin Huoxue Preparations, and Bushen Qiangshen Tablets, although involving the determination of Cibotium barometz-related components, still use 5-hydroxymethylfurfural, protocatechuic acid, and protocatechuic aldehyde as content determination indicators.

[0004] For example, Chinese invention patent application CN109917045A (publication date: 2019.06.21) discloses an HPLC method for simultaneously determining the content of five components in processed Cibotium barometz slices. In addition to determining protocatechuic aldehyde and protocatechuic acid, it also selects to simultaneously determine three active ingredients, namely kojic acid, 5-HMF (5-hydroxymethylfurfural), and maltol, in order to control their quality, improve the specificity of the method, and ensure the efficacy.

[0005] For example, Chinese invention patent application CN118191158A (publication date: June 14, 2024) discloses a method for constructing a fingerprint spectrum of Cibotium barometz and a method for determining the content of its active ingredients. The fingerprint spectrum obtained by the construction method includes at least 12 characteristic peaks, including 5-hydroxymethylfurfural, protocatechuic acid, protocatechuic aldehyde, 1-O-caffeoyl-β-D-glucose, and protocatechuic acid-4-O-glucoside. The results show that the chromatographic baseline is stable, the characteristic peaks are well separated, and the relative retention times of each characteristic peak are calculated based on protocatechuic acid. This is beneficial for comprehensive quality testing and overall quality control of Cibotium barometz preparations, and helps to improve the safety and stability of drug use.

[0006] To date, chemical components isolated from Cibotium barometz include sugars and glycosides, aromatic compounds, volatile oils, pterosins, flavonoids, steroids, pyranones, amino acids, and inorganic elements. Among these, the chemical components reported to have biological activity are mainly sugars and glycosides, pterosins, and phenolic acids. Pterosins, as characteristic components of ferns, are specifically distributed in ferns. As early as the 21st century, researchers isolated several pterosins from Cibotium barometz, including onitin, onitin 2'-O-glucoside, and pterosin R, proving that Cibotium barometz contains these characteristic components. Pterosins have a natural advantage as a specific quality evaluation indicator for Cibotium barometz, but they have not yet been included in the content determination system in current quality standards or research.

[0007] Current research on the quality standards of Cibotium barometz and its traditional Chinese medicine preparations mainly focuses on phenolic acid components. The quality control indicators are relatively simple and lack specificity, making it difficult to accurately and comprehensively reflect their intrinsic quality. There is an urgent need to develop a detection method for the effective components of Cibotium barometz and its preparations, focusing on pteridin components, in order to achieve more comprehensive quality control. Summary of the Invention

[0008] The purpose of this invention is to provide: A method for determining the content of effective components in Cibotium barometz and its preparations, using fern extract and fern extract-2'-O-glucoside as indicators, aims to solve the problems of single quality control indicators, weak specificity, and difficulty in accurately and comprehensively reflecting the intrinsic quality of Cibotium barometz and its preparations in the existing technology.

[0009] Terminology Explanation: Unless otherwise defined, all technical terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter pertains.

[0010] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0011] The definition of the standard chemical term can be found in the reference "Pharmacopoeia of the People's Republic of China 2025 Edition, China Medical Publishing House, implemented on October 1, 2025".

[0012] Unless otherwise stated, conventional methods within the scope of the art shall be used.

[0013] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0014] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0015] The term "medicinal materials" as used in this article refers to raw medicinal materials derived from medicinal plants, medicinal animals, and medicinal minerals, as well as prepared slices of Chinese medicine obtained through conventional Chinese medicine processing such as cleaning, cutting, and roasting. It also includes medicinal raw materials obtained directly from the above-mentioned raw materials without undergoing subsequent deep preparation processing.

[0016] The term "preparation" as used in this article refers to a finished drug form with a defined dosage form, specification, and administration purpose, which is prepared by processing pharmaceutical active ingredients, medicinal materials or their processed products, and optional pharmaceutical excipients through a specific process. It covers all conventional dosage forms such as solids, semi-solids, and liquids suitable for pharmaceutical use, and also includes novel pharmaceutical preparations developed for specific routes of administration.

[0017] This invention is achieved through the following technical solutions: This invention provides a method for determining the content of effective components in Cibotium barometz and its preparations, specifically: using triple quadrupole liquid chromatography-mass spectrometry to simultaneously detect the effective components of Cibotium barometz and its preparations, namely, fern extract and fern extract-2'-O-glucoside. The liquid chromatography conditions include: Mobile phase composition: Mobile phase A is methanol, and mobile phase B is water; The gradient elution procedure is as follows: 0-2 min, 20% mobile phase A, 80% mobile phase B; 2-2.1 min, 20%→60% mobile phase A, 80%→40% mobile phase B; 2.1-6 min, 60% mobile phase A, 40% mobile phase B; 6-6.1 min, 60%→100% mobile phase A, 40%→0% mobile phase B; 6.1-8 min, 100% mobile phase A; 8-8.1 min, 100%→20% mobile phase A, 0%→80% mobile phase B; 8.1-12 min, 20% mobile phase A, 80% mobile phase B.

[0018] Furthermore, the pretreatment of the test sample, Cibotium barometz and its preparation, includes: adding the test sample to the extraction solvent for ultrasonic extraction, then centrifuging, and filtering the supernatant to obtain the final product.

[0019] Preferably, the frequency of the ultrasonic extraction is 16-32 kHz; the ultrasonic extraction time is 15-30 min.

[0020] More preferably, the frequency of the ultrasonic extraction is 32 kHz; and the ultrasonic extraction time is 30 min.

[0021] Furthermore, the liquid chromatography conditions also include: column temperature of 25-35℃; flow rate of 0.25-0.35 mL / min.

[0022] Preferably, the column temperature is 30°C and the flow rate is 0.3 mL / min.

[0023] Furthermore, the chromatographic column of the liquid chromatography is selected from any one of Poroshell 120 EC-C18, Shim-pack VeloxC18, and Kinetex C18.

[0024] Furthermore, the mass spectrometry conditions for the triple quadrupole liquid chromatography-mass spectrometry are as follows: ESI ion source, positive ion mode; capillary voltage: 4000 V (pos); nozzle voltage: 400 V (pos).

[0025] Furthermore, the mass spectrometry conditions for the triple quadrupole liquid chromatography-mass spectrometry also include: a drying gas temperature of 315-385℃ and a drying gas flow rate of 4-6 L / min.

[0026] Preferably, the temperature of the drying gas is 350°C; and the flow rate of the drying gas is 5 L / min.

[0027] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention is the first to establish a method for determining the effective components of Cibotium barometz and its preparations by using fern extract and fern extract-2'-O-glucoside as monitoring indicators. The method is rapid, efficient, highly repeatable, accurate, specific, has a wide linear detection range, and low detection and quantitation limits. It solves the problem that the quality control indicators of Cibotium barometz and its preparations are single in the prior art and cannot achieve comprehensive quality control. (2) The pretreatment method of the test sample in the detection process of the present invention is simple and efficient, requires no special separation or purification treatment, and has high stability. Attached Figure Description

[0028] Figure 1 This is the liquid chromatogram of the blank solution obtained by high performance liquid chromatography in Example 1 of the present invention; Figure 2 The image shows the liquid chromatogram of the working solution of the reference standard of *Gynostemma pentaphyllum*-2'-O-glucoside, obtained by high performance liquid chromatography in Example 1 of this invention (elution time 6.483 min). Figure 3 The image shows the liquid chromatogram of the working solution of the reference standard of *Gynostemma pentaphyllum* obtained by high performance liquid chromatography in Example 1 of this invention (elution time 10.007 min). Figure 4 This is the liquid chromatogram of the working solution of fern extract-2'-O-glucoside and fern extract mixed reference standard obtained by high performance liquid chromatography in Example 1 of the present invention; Figure 5 This is a chromatogram of the mixed reference standard working solution obtained by triple quadrupole liquid chromatography-mass spectrometry in Example 1 of the present invention (peak 1 is ginsenoside-2'-O-glucoside, peak 2 is ginsenoside, the same below); Figure 6 This is a chromatogram of the Cibotium barometz extract obtained in Example 3 of the present invention using 50% methanol solution as the extraction solvent; Figure 7 This is a chromatogram of the Cibotium barometz extract obtained in Example 3 of the present invention using 80% methanol solution as the extraction solvent; Figure 8 This is a chromatogram of the Cibotium barometz extract obtained in Example 3 of the present invention using 100% methanol solution as the extraction solvent; Figure 9 This is a chromatogram of the Cibotium barometz extract obtained in Example 3 of the present invention using 80% acetonitrile solution as the extraction solvent; Figure 10 This is the chromatogram of the Cibotium barometz extract obtained by isocratic elution in Example 4 of the present invention; Figure 11 This is the chromatogram of the Cibotium barometz extract in Example 4 of the present invention after gradient elution 1; Figure 12 This is the chromatogram of the Cibotium barometz extract obtained by gradient elution 2 in Example 4 of the present invention; Figure 13 The chromatogram of the Cibotium barometz extract obtained by analysis using a Kinetex C18 column (100×2.1mm, 2.6μm) in Example 5 of this invention is shown below. Figure 14 The chromatogram of the Cibotium barometz extract obtained in Example 5 of this invention was obtained by analysis using a Poroshell 120 EC-C18 column (150×3.0mm, 2.7μm). Figure 15Mass spectra of different parent and daughter ion parameters under the mass spectrometry conditions in Example 6 of this invention; a is the chromatogram corresponding to 249.1 / 189.0; b is the chromatogram corresponding to 249.1 / 231.0; c is the chromatogram corresponding to 249.1 / 161.0; d is the chromatogram corresponding to 411.0 / 249.0; Figure 16 This is a blank solution spectrum used in the specificity assessment of the method of this invention. Figure 17 This is a chromatogram of the mixed reference standard working solution used in the specificity assessment of the method of this invention; Figure 18 This is a chromatogram of the test solution used in the specificity assessment of the method of the present invention. Figure 19 This is a chromatogram of the spiked solution of the test sample used in the specificity assessment of the method of this invention; Figure 20 The standard curve of fern extract-2'-O-glucoside in the linearity and range test of the method of the present invention; Figure 21 The standard curve of *Gynostemma pentaphyllum* is used to examine the linearity and range of the method of this invention. Detailed Implementation

[0029] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0030] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0031] Unless otherwise specified, all percentages in this invention are volume percentages.

[0032] Triple quadrupole LC-MS / MS (Agilent Technologies, USA, Model: 1290-G6470A); chromatographic columns: AgelaTechnologies Venusil XBP C18 (4.6 × 250 mm, 5 μm), Kinetex C18 (2.1 × 100 mm, 2.6 μm), Poroshell 120 EC-C18 (3.0 × 150 mm, 2.7 μm), Poroshell 120 EC-C18 (3.0 × 150 mm, 2.7 μm); electronic analytical balance (Mettler-Toledo, Switzerland, Model: XSR205DU); ultrapure water system (Sartorius, Germany, Model: Comfort Ⅰ); ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., Model: KQ-500DE).

[0033] Chromatographic grade methanol (batch number: 2899901, chromatographic purity) was purchased from Tianjin Kelude Company; Jinfenjuting reference standard (batch number: PS014399A, purity: 99.58%) and Jinfenjuting-2'-O-glucoside reference standard (PS014398A, purity: 99.08%) were both purchased from Chengdu Pusi Biotechnology Co., Ltd.

[0034] The seven batches of Cibotium barometz (YC01-YC07) were all collected in Fenghuang Town, Dinghu District, Zhaoqing City, Guangdong Province. The administrative permit number for their collection is Yue (Zhao) Lin Cai Xu Zhun

[2024] No. 1. The rhizomes were cleaned of mud and sand and dried to obtain Cibotium barometz medicinal material.

[0035] Scalded Cibotium barometz (YC08-YC010): Take Cibotium barometz (YC05-YC07), and scald it with sand until it puffs up according to the stir-frying method (Pharmacopoeia of the People's Republic of China, 2020 edition, General 0213). After cooling, remove any remaining hairs to obtain the product.

[0036] See Table 1 below for specific sample information.

[0037] Table 1. Information on samples of Cibotium barometz and Cibotium barometz.

[0038] Example 1: Investigation using instrumental methods 1. High Performance Liquid Chromatography 1.1 Preparation of reference solution 1.1.1 Preparation of the working solution of the reference standard for *Gynostemma pentaphyllum*: Accurately weigh 1 mg of *Pteris vittatum* reference standard into a 10 mL volumetric flask, dissolve it in methanol by sonication, and dilute to the mark. Shake well to obtain a 100 mg / L *Pteris vittatum* reference standard solution. Accurately measure 100 μL of the above reference standard solution into a 10 mL volumetric flask, dissolve it in methanol by sonication, and dilute to the mark. Shake well to obtain a 1 mg / L *Pteris vittatum* reference standard working solution.

[0039] 1.1.2 Preparation of the working solution of the reference standard of *Gynostemma pentaphyllum*-2'-O-glucoside: Accurately weigh 1 mg of *Gynostemma pentaphyllum*-2'-O-glucoside reference standard into a 10 mL volumetric flask, dissolve in methanol by sonication, and dilute to the mark. Shake well to obtain a 100 mg / L solution of *Gynostemma pentaphyllum*-2'-O-glucoside reference standard. Accurately measure 100 μL of the above reference standard solution into a 10 mL volumetric flask, dissolve in methanol by sonication, and dilute to the mark. Shake well to obtain a 1 mg / L working solution of *Gynostemma pentaphyllum*-2'-O-glucoside reference standard.

[0040] 1.1.3 Preparation of the mixed reference standard working solution: Accurately measure 1 mL each of the 1 mg / L fern fungus reference working solution prepared under “1.1.1 Preparation of fern fungus reference working solution” and the 1 mg / L fern fungus-2'-O-glucoside reference working solution prepared under “1.1.2 Preparation of fern fungus-2'-O-glucoside reference working solution” into 5 mL volumetric flasks. Dilute to the mark with 80% methanol and shake well to obtain a mixed reference working solution with a concentration of 200 μg / L.

[0041] 1.2 Instrument Conditions Chromatographic conditions: Agela Technologies Venusil XBP C18 (4.6 × 250 mm, 5 μm) column; column temperature 30 ℃; constant flow rate 1.00 mL / min; injection volume 10 μL; mobile phase: methanol (A) and water (B), elution 1:1 isocratic for 60 min. UV detector, wavelength 254 nm.

[0042] 1.3 Results Analysis The blank solution, the working solution of fern extract-2'-O-glucoside reference standard, the working solution of fern extract reference standard, and the mixed reference standard working solution were analyzed according to "1.2 Instrument Conditions". The results are as follows: Figures 1-4 As shown.

[0043] Figure 1The blank solution chromatogram shows a sharp peak signal at approximately 3-5 min, in addition to the solvent peak. This is a background interference peak introduced by the chromatographic system or solvent. Figure 2 The results showed that the background interference peak was not sufficiently separated from the glucoside 2'-O-glucoside (6.483 min), and baseline separation could not be achieved. This directly interfered with the qualitative identification and quantitative integration of the target analyte, indicating that the high performance liquid chromatography method could not effectively distinguish between background interference and the target analyte signal. Figure 3 The results showed that under these chromatographic conditions, the peak of *Gynostemma pentaphyllum* (10.007 min) was symmetrical, with no tailing or leading-out phenomenon; the resolution between it and adjacent impurity peaks was greater than 1.5, achieving baseline separation and meeting the requirements for quantitative analysis. Figure 4 Showing Figure 2 Similarly, the results showed that the ginseng-2'-O-glucoside was affected by background interference peaks and could not meet the requirements for quantitative analysis.

[0044] In summary, under these high-performance liquid chromatography (HPLC) conditions, both the blank solution and the working solution of the reference standard exhibit background interference peaks, and the separation from the target peak of ginseng-2'-O-glucoside is poor, failing to meet the requirements for determining the content of the target peak. Therefore, this HPLC method is not suitable for simultaneously determining the content of ginseng-2'-O-glucoside and ginseng.

[0045] 2. Triple quadrupole liquid chromatography-mass spectrometry (LC-MS) method 2.1 Preparation of working solution for reference standard Prepare the mixed reference standard working solution according to the "1.1.3 Preparation of Mixed Reference Standard Working Solution" section.

[0046] 2.2 Instrument Conditions Chromatographic conditions: Kinetex C18 (2.6 μm, 100 × 2.1 mm) column was used; column temperature was 30 ℃; constant flow rate was 0.30 mL / min; mobile phase was methanol (A): water (B) = 6:4, isocratic elution for 12 min.

[0047] Mass spectrometry conditions: ESI ion source, positive ion mode; capillary voltage: 4000V (pos); nozzle voltage: 400V (pos); dryer temperature: 350℃; dryer flow rate: 5L / min; nebulizer pressure: 45psi. See Table 2 for specific ion pair parameters.

[0048] Table 2 Ion pair parameters

[0049] 2.3 Results Analysis The working solutions of ginsenoside-2'-O-glucoside and ginsenoside-mixed reference standard were simultaneously detected using a triple quadrupole LC-MS / MS instrument according to "2. Triple quadrupole LC-MS / MS method 2.2 Instrument conditions". The target analytes ginsenoside-2'-O-glucoside and ginsenoside were well separated, with no interference from other impurity peaks. The results are shown in the figure. Figure 5 .

[0050] Therefore, triple quadrupole liquid chromatography-mass spectrometry was chosen as the method for simultaneously determining the content of ferns-2'-O-glucoside and ferns.

[0051] Example 2: Examination of Preprocessing-Extraction Methods (1) Ultrasonic extraction method Accurately weigh 2 g of the test sample powder (number: YC01) that has passed through a No. 3 sieve into a 50 mL polystyrene stoppered centrifuge tube, add 10 mL of water, vortex to mix, then add 40 mL of methanol, sonicate at 32 kHz for 30 min, centrifuge at 4000 r / min for 5 min, take the supernatant, and filter it through a 0.22 μm microporous membrane.

[0052] (2) Homogenization extraction method Accurately weigh 2 g of the test sample powder (number: YC01) that has passed through a No. 3 sieve into a 150 mL polystyrene stoppered centrifuge tube, add 10 mL of water, vortex to mix, then add 40 mL of methanol, homogenize at 12000 rpm for 3 min, centrifuge at 4000 r / min for 5 min, take the supernatant, and filter through a 0.22 μm microporous membrane.

[0053] Two parallel samples were weighed and extracted using the ultrasonic extraction method and homogenization extraction method described above, respectively. The responses of the target analytes were compared after analysis according to "2. Triple quadrupole liquid chromatography-mass spectrometry method 2.2 Instrument conditions". The results are shown in Table 3.

[0054] Table 3 Results of the investigation on extraction methods

[0055] Table 3 shows that the response values ​​of the target compounds extracted by homogenization extraction were all lower than those extracted by ultrasonic extraction. Specifically, the response value of ginseng-2'-O-glucoside extracted by ultrasonic extraction was 336,429, which was 96.11% higher than that of homogenization extraction; the response value of ginseng extracted by ultrasonic extraction was 1,128,232, which was 44.24% higher than that of homogenization extraction. This may be because ginseng-2'-O-glucoside and ginseng undergo hydrolysis during homogenization extraction due to excessively high temperatures, resulting in lower extraction efficiency compared to ultrasonic extraction. Therefore, ultrasonic extraction is the preferred method.

[0056] Example 3: Optimization of Preprocessing Method 2.4.1 Selection of extraction vessel Accurately weigh 2 g of Cibotium barometz powder (sample number: YC01) that has passed through a No. 3 sieve into a 150 mL polystyrene stoppered centrifuge tube and a 150 mL stoppered glass Erlenmeyer flask. Add 10 mL of water, vortex to mix, then add 40 mL of methanol. Sonicate at 32 kHz for 30 min, centrifuge at 4000 r / min for 5 min, take the supernatant, filter through a 0.22 μm microporous membrane, and analyze according to “2. Triple quadrupole liquid chromatography-mass spectrometry 2.2 Instrument conditions”. Compare the response of the target analytes. The results are shown in Table 4.

[0057] Table 4. Results of the investigation of extraction vessels

[0058] As shown in Table 4, the response values ​​of fern extract-2'-O-glucoside and fern extract obtained using a stoppered glass Erlenmeyer flask are higher than those obtained using polystyrene stoppered centrifuge tubes. Therefore, a stoppered glass Erlenmeyer flask is preferred as the extraction vessel.

[0059] 2.4.2 Selection of Ultrasound Time 2 g of Cibotium barometz powder (sample number: YC01) that has passed through a No. 3 sieve was accurately weighed into a 150 mL stoppered glass Erlenmeyer flask. 10 mL of water was added, and the mixture was vortexed. Then 40 mL of methanol was added. The mixture was sonicated at 32 kHz for 0 min, 15 min, and 30 min, respectively. After centrifugation at 4000 r / min for 5 min, the supernatant was collected and filtered through a 0.22 μm microporous membrane. The results were compared with the response of the target analyte after analysis according to "2. Triple quadrupole liquid chromatography-mass spectrometry 2.2 Instrument conditions". The results are shown in Table 5.

[0060] Table 5 Results of the ultrasound time study

[0061] As shown in Table 5, when using an ultrasonic duration of 30 min, the target compounds, ginsenoside-2'-O-glucoside and ginsenoside, had the highest response values ​​in the extracted sample solution.

[0062] 2.4.3 Selection of Ultrasonic Frequency Accurately weigh 2 g of Cibotium barometz sample powder (No.: YC01) that has passed through a No. 3 sieve into a 150 mL stoppered glass Erlenmeyer flask, add 10 mL of water, vortex to mix, then add 40 mL of 80% methanol, sonicate at 16 kHz, 24 kHz, and 32 kHz for 30 min respectively, centrifuge at 4000 r / min for 5 min, take the supernatant, filter through a 0.22 μm microporous membrane, and analyze according to "2. Triple quadrupole liquid chromatography-mass spectrometry 2.2 Instrument conditions" and compare the response of the target analyte. The results are shown in Table 6.

[0063] Table 6 Results of the investigation of ultrasonic frequencies

[0064] Table 6 shows that the responses of *Gynostemma pentaphyllum* at ultrasonic frequencies of 16 kHz, 24 kHz, and 32 kHz are not significantly different, while *Gynostemma pentaphyllum*-2'-O-glucoside exhibits the largest response value at 32 kHz. Considering all factors, 32 kHz is the preferred ultrasonic frequency.

[0065] 2.4.4 Investigation of Extraction Solvents Considering the easy hydrolysis of the compound ginsenoside-2'-O-glucoside, further investigation was conducted on the extraction solvent.

[0066] 2 g of the test sample powder (sample number: YC02) passing through a No. 3 sieve was accurately weighed. Extraction solvents were prepared as follows: 50% methanol solution (25 mL water + 25 mL methanol), 80% methanol solution (10 mL water + 40 mL methanol), 100% methanol (50 mL methanol), and 80% acetonitrile. Ultrasonic extraction was performed according to "Example 2". The responses of the target analytes were compared after analysis according to "2. Triple quadrupole liquid chromatography-mass spectrometry 2.2 Instrument conditions". The results are shown in Table 7. Figures 6-9 .

[0067] Table 7 Results of the investigation of extraction solvents

[0068] According to Table 7, Figures 6-9 It can be seen that ginseng-2'-O-glucoside and ginseng showed the best response in the sample solution extracted with ultrasonication in 80% methanol solution. Therefore, 80% methanol solution is preferred for extraction.

[0069] Example 4: Test Method – Examination of the Elution Procedure For the pretreatment of the Cibotium barometz sample (No.: YC03), an ultrasonic extraction with 80% methanol solution was selected. Based on "2. Triple quadrupole liquid chromatography-mass spectrometry method 2.2 Instrument conditions", the elution program of the mobile phase was further investigated.

[0070] 4.1 Isocratic elution: Methanol A: Water B = 6:4.

[0071] 4.2 Gradient elution procedure 1: 0-2 min, 30%A; 2-4 min, 30%→70%A; 4-6 min, 70% A; 6-6.1 min, 70%→100%A; 6.1-8 min, 100% A.

[0072] 4.3 Gradient elution procedure 2: 0-2 min, 20% A; 2-2.1 min, 20%→60%A; 2.1-6 min, 60% A; 6-6.1 min, 60%→100%A; 6.1-8 min, 100% A.

[0073] The mixed reference standard working solution was separately pipetted onto the instrument, and the effects of gradient elution, isocratic elution 1, and isocratic elution 2 on peak elution time and peak shape were analyzed. The results are shown in the figure. Figures 10-12 .

[0074] Depend on Figure 10 and Figure 12 It can be seen that: using isocratic elution and gradient elution program 1 results in faster elution times for the target peaks, with resolutions below 1.5, and the chromatographic column is prone to residual impurities, affecting sequence operation; while gradient elution program 2 has a more reasonable elution time for the target peaks and a resolution greater than 1.5. Therefore, gradient elution program 2 is the preferred mobile phase.

[0075] Example 5: Test Methods – Investigation of Chromatographic Columns The mixed reference standard working solution was analyzed using Kinetex C18 (100 × 2.1 mm, 2.6 μm) and Poroshell 120 EC-C18 (150 × 3.0 mm, 2.7 μm) columns respectively, and the responses of the target analytes were compared after analysis under the conditions of "4.3 Gradient Elution Program 2". The results are shown in [Figure number missing]. Figure 13 , Figure 14 .

[0076] Depend on Figure 13It can be seen that the theoretical plate number of *Gynostemma pentaphyllum*-2'-O-glucoside obtained by analysis using a Kinetex C18 column (2.6 μm, 100 × 2.1 mm) is 1869, with a peak resolution of 1.0; the theoretical plate number of *Gynostemma pentaphyllum* is 1986, with a peak resolution of 1.2. Therefore, the system suitability does not meet the relevant requirements, namely, a resolution of not less than 3000 and a resolution of not less than 1.5.

[0077] Depend on Figure 14 It can be seen that the theoretical plate number of *Gynostemma pentaphyllum*-2'-O-glucoside obtained by analysis using a Poroshell 120 EC-C18 column (150 × 3.0 mm, 2.7 μm) is 6804, and the peak resolution is 3.3; the theoretical plate number of *Gynostemma pentaphyllum* is 3883, and the peak resolution is 3.1. Therefore, the system suitability meets the relevant requirements, namely, a resolution of not less than 3000 and a resolution of not less than 1.5.

[0078] Example 6: Test Method – Investigation of Mass Spectrometry Ion Pair Parameters This embodiment compares the peak elution of the target compounds, ferruginous ginsenoside-2'-O-glucose and ferruginous ginsenoside, under different parent and daughter ion parameters (as shown in Table 8) in mass spectrometry conditions. The results are shown in Table 8. Figure 15 .

[0079] Table 8 Ion pair parameters

[0080] Depend on Figure 15 It can be seen that the elution effect is best when the parent ion is 249.1 m / z and the daughter ion is 189.0 m / z or 231.0 m / z, which are the preferred ion pairs for mass spectrometry.

[0081] Through the examination of the above-mentioned instrumental methods, the examination of pretreatment methods, and the optimization of testing methods, the "optimal process" was finally determined as follows: The *Cibotium barometz* sample was extracted with 80% methanol by ultrasound, and the contents of *Pteris vittata*-2'-O-glucoside and *Pteris vittata* were analyzed by gradient elution using triple quadrupole liquid chromatography-mass spectrometry.

[0082] Chromatographic conditions: A Poroshell 120 EC-C18 (150×3.0 mm, 2.7 μm) column was used; column temperature was 30℃; constant flow rate was 0.30 mL / min; mobile phase was methanol (A) and water (B) = 6:4, gradient elution for 12 min. The gradient was set as follows: 0–2 min, 20% A; 2–2.1 min, 20%→60% A; 2.1–6 min, 60% A; 6–6.1 min, 60%→100% A; 6.1–8 min, 100% A.

[0083] Mass spectrometry conditions: ESI ion source, positive ion mode; capillary voltage: 4000 V (pos); nozzle voltage: 400 V (pos); dryer temperature: 350 ℃; dryer flow rate: 5 L / min; nebulizer pressure: 45 psi.

[0084] The specific ion pair parameters are shown in Table 9.

[0085] Table 9 Ion pair parameters

[0086] Test Example 1: Methodology Validation 1. Solution preparation 1.1 Preparation of the stock solution of *Gynostemma pentaphyllum* reference standard: Accurately weigh 5 mg of the reference standard, Pteris vittatum, into a 5 mL volumetric flask, dissolve it in methanol by sonication, and dilute to the mark. Shake well to prepare a stock solution of the reference standard containing 1000 mg / L of Pteris vittatum.

[0087] 1.2 Preparation of the stock solution of 2'-O-glucoside reference standard: Accurately weigh 5 mg of the reference standard, ginseng-2'-O-glucoside, into a 5 mL volumetric flask, dissolve it in methanol by sonication, and dilute to the mark. Shake well to prepare a stock solution of ginseng-2'-O-glucoside containing 1000 mg / L.

[0088] 1.3 Preparation of mixed reference standard stock solution: Accurately measure 100 μL each of the reference stock solution of *Gynostemma pentaphyllum* and the reference stock solution of *Gynostemma pentaphyllum*-2'-O-glucoside into 10 mL volumetric flasks, dilute to the mark with methanol, and shake well to prepare mixed reference stock solutions containing 10 μg / mL of *Gynostemma pentaphyllum*-2'-O-glucoside and *Gynostemma pentaphyllum*.

[0089] 1.4 Preparation of the working solution of the reference standard for *Gynostemma pentaphyllum*: Accurately weigh 1 mg of *Pteris vittatum* reference standard into a 10 mL volumetric flask, dissolve it in methanol by sonication, and dilute to the mark. Shake well to obtain a 100 mg / L *Pteris vittatum* reference standard solution. Accurately measure 100 μL of the above reference standard solution into a 10 mL volumetric flask, dissolve it in methanol by sonication, and dilute to the mark. Shake well to obtain a 1 mg / L *Pteris vittatum* reference standard working solution.

[0090] 1.5 Preparation of the working solution of fern extract-2'-O-glucoside reference standard: Accurately weigh 1 mg of *Gynostemma pentaphyllum*-2'-O-glucoside reference standard into a 10 mL volumetric flask, dissolve in methanol by sonication, and dilute to the mark. Shake well to obtain a 100 mg / L solution of *Gynostemma pentaphyllum*-2'-O-glucoside reference standard. Accurately measure 100 μL of the above reference standard solution into a 10 mL volumetric flask, dissolve in methanol by sonication, and dilute to the mark. Shake well to obtain a 1 mg / L working solution of *Gynostemma pentaphyllum*-2'-O-glucoside reference standard.

[0091] 1.6 Preparation of the mixed reference standard working solution: Accurately measure 1 mL each of the 1 mg / L fern fungus reference working solution prepared under “1.4 Preparation of fern fungus reference working solution” and the 1 mg / L fern fungus-2'-O-glucoside reference working solution prepared under “1.5 Preparation of fern fungus-2'-O-glucoside reference working solution” into 5 mL volumetric flasks. Dilute to the mark with 80% methanol and shake well to obtain a mixed reference working solution with a concentration of 200 μg / L.

[0092] 1.7 Preparation of System Suitability Solution: Accurately measure 70 μL of the mixed reference standard stock solution into a 10 mL volumetric flask, dilute to the mark with 80% methanol, shake well, and prepare system suitability solutions containing 70 ng / mL of each of the following: ginseng and ginseng-2'-O-glucoside.

[0093] 1.8 Preparation of the test solution: Accurately weigh 2 g of the test sample powder (number: YC01) that has passed through a No. 3 sieve into a 150 mL stoppered glass Erlenmeyer flask, add 10 mL of water, vortex to mix, then add 40 mL of 80% methanol, sonicate at 32 kHz for 30 min, centrifuge at 4000 r / min for 5 min, take the supernatant, and filter it through a 0.22 μm microporous membrane.

[0094] 1.9 Preparation of blank matrix solution: Add 10 mL of water directly to a 150 mL stoppered glass Erlenmeyer flask, vortex to mix, then add 40 mL of 80% methanol, sonicate at 32 kHz for 30 min, centrifuge at 4000 r / min for 5 min, take the supernatant, filter through a 0.22 μm microporous membrane to prepare a blank matrix solution.

[0095] 1.10 Preparation of blank solution: Except for the absence of a sample, the preparation of the test solution is the same.

[0096] 1.11 Preparation of negative control solution: Accurately weigh 2 g each of the muscle-relaxing negative control preparation (No.: YX01) and the waist-strengthening negative control preparation (No.: YX02), and prepare negative control solutions according to the preparation method of the test solution.

[0097] 1.12 Preparation of linear solutions of reference standards: Accurately measure 20 μL, 50 μL, 70 μL, 100 μL, 120 μL, and 150 μL of the mixed reference standard stock solution into 10 mL volumetric flasks, dilute to the mark with blank matrix solution, and mix well to obtain the blank matrix reference standard linear solution. The concentrations of the reference standard linear solutions STD1-STD7 are 20 ng / mL, 50 ng / mL, 70 ng / mL, 100 ng / mL, 120 ng / mL, and 150 ng / mL, respectively.

[0098] 1.13 Preparation of spiking solution for test sample: Accurately weigh 2 g of the test sample powder (number: YC01) that has passed through a No. 3 sieve into 3 50 mL stoppered glass Erlenmeyer flasks. Accurately add 250 μL, 350 μL, and 500 μL of the mixed reference standard stock solution (10 μg / mL), respectively. Add 10 mL of water, then add 40 mL of methanol. Sonicate at 32 kHz for 30 min, centrifuge at 4000 r / min for 5 min, collect the supernatant, and filter through a 0.22 μm microporous membrane to obtain low, medium, and high spiking solutions of the test sample at 50 ng / mL, 70 ng / mL, and 100 ng / mL.

[0099] 2. Determination Method 2.1 High Performance Liquid Chromatography-Tandem Mass Spectrometry Accurately pipette 1 µL each of the mixed reference working solution and the test solution into a high-performance liquid chromatography-tandem mass spectrometer, and calculate using the external standard curve method to obtain the result.

[0100] 2.2 Instrument Conditions The contents of fern extract-2'-O-glucoside and fern extract were analyzed by gradient elution using triple quadrupole liquid chromatography-mass spectrometry.

[0101] Chromatographic conditions: A Poroshell 120 EC-C18 column (150 × 3.0 mm, 2.7 μm) was used; column temperature was 30℃; constant flow rate was 0.30 mL / min; mobile phase was methanol (A) and water (B) = 6:4, with gradient elution for 12 min. The gradient was set as follows: 0–2 min, 20% A; 2–2.1 min, 20% → 60% A; 2.1–6 min, 60% A; 6–6.1 min, 60% → 100% A; 6.1–8 min, 100% A.

[0102] Mass spectrometry conditions: ESI ion source, positive ion mode; capillary voltage: 4000V (pos); nozzle voltage: 400V (pos); dryer temperature: 350℃; dryer flow rate: 5L / min; nebulizer pressure: 45psi.

[0103] The specific ion pair parameters are shown in Table 10.

[0104] Table 10 Ion pair parameters

[0105] 2.3 Result Calculation Formula for calculating the content of the analyte in a sample:

[0106] In the formula: X The content of the substance to be tested is expressed in mg / kg. b The intercept of the linear regression equation; a The slope of the linear regression equation; c The concentration of the target component in the sample is ng / mL; V The volume is constant, in mL; N is the dilution factor; m is the mass, in grams.

[0107] 3. Specificity Verification Take the prepared blank solution, mixed reference working solution, test solution, and test spiked solution, and inject them once each according to "2.2 Instrument Conditions". See the attached image for the peak elution status of the instrument chromatograms. Figures 16-19 The specific results of the specificity study are shown in Table 11.

[0108] Table 11 Results of Specificity Examination

[0109] Depend on Figures 16-19 As shown in Table 11, no target peak appeared in the blank solution spectrum; the mixed reference working solution spectrum had a clear target peak and no other interfering peaks; the test solution and the spiked test solution spectrum had clear target peaks, and the elution positions of the target peaks were consistent with those of the reference solution spectrum, indicating good specificity.

[0110] 4 System Applicability Take a system suitability solution and inject it six times consecutively according to "2.2 Instrument Conditions". Calculate the relative standard deviation of the peak area for each of the six injections. RSD The results are shown in Table 12.

[0111] Table 12 System Applicability Assessment

[0112] As shown in Table 12, after six consecutive injections of the system suitability solution, the relative standard deviation of the retention time of the peak of *Gynostemma pentaphyllum* was 0.1%, and the relative standard deviation of the peak area measurement was 7.9%; the theoretical plate number was ≥3906, and the resolution was ≥2.9. The relative standard deviation of the retention time of the peak of *Gynostemma pentaphyllum*-2'-O-glucoside was 0.1%, and the relative standard deviation of the peak area measurement was 7.1%; the theoretical plate number was ≥3432, and the resolution was ≥2.8, which meets the requirements that the resolution should be greater than 1.5 and the relative standard deviation of the peak area measurement should not be greater than 15%.

[0113] 4. Limit of detection and limit of quantitation Take a series of mixed reference standard solutions and determine them according to "2.2 Instrument Conditions". The limit of detection (LOD) is the concentration with a signal-to-noise ratio (S / N) of approximately 3, and the limit of quantitation (LOQ) is the concentration with a signal-to-noise ratio (S / N) of approximately 10. Take working solutions of mixed reference standard solutions with corresponding concentrations of LOD and LOQ, and inject them six times according to "2.2 Instrument Conditions". Calculate the relative standard deviation of the peak area of ​​the six injections for both ginseng-2'-O-glucoside and ginseng-2'-O-glucoside. RSD The results are shown in Tables 13 and 14.

[0114] Table 13 Limit of Detection and Limit of Quantification of *Gynostemma pentaphyllum*-2'-O-glucoside

[0115] Table 14. Investigation of the detection limit and quantitation limit of *Gynostemma pentaphyllum*

[0116] As shown in Table 13, the detection limit concentration of *Gynostemma pentaphyllum*-2'-O-glucoside is 1 ng / mL (equivalent to a content of 0.025 mg / kg), and the relative standard deviation of the six peak area measurements is 10.6%; the quantitation limit concentration of *Gynostemma pentaphyllum*-2'-O-glucoside is 5 ng / mL (equivalent to a content of 0.125 mg / kg), and the relative standard deviation of the six peak area measurements is 3.6%, both meeting the requirements. RSD ≤15%).

[0117] As shown in Table 14, the detection limit of *Gynostemma pentaphyllum* is 1 ng / mL (equivalent to a content of 0.025 mg / kg), and the relative standard deviation of the six peak area measurements is 8.2%; the quantitation limit of *Gynostemma pentaphyllum* is 5 ng / mL (equivalent to a content of 0.125 mg / kg), and the relative standard deviation of the six peak area measurements is 5.8%, both meeting the requirements. RSD ≤15%).

[0118] 5. Linearity and Range Accurately pipette 1 μL of each linear solution of the reference standard and inject it into the high-performance liquid chromatography-tandem mass spectrometer (HPLC-MS / MS). Perform the determination according to "2.2 Instrument Conditions". Plot a standard curve with the concentration of the reference standard as the abscissa (x) and the absorption peak area as the ordinate (y). The results are shown in [Figure 1]. Figure 20 , Figure 21 See Table 15.

[0119] Table 15 Linearity Examination of Standard Curve

[0120] Depend on Figure 20 , Figure 21 As shown in Table 15, the linear regression equation for *Pteris vittata*-2'-O-glucose is y=901x+10036, with a correlation coefficient r of 0.9964, which meets the requirement (r≥0.99). The linear regression equation for *Pteris vittata* is y=2411x+1347, with a correlation coefficient r of 0.9970, which also meets the requirement (r≥0.99). The results indicate that the correlation coefficients of *Pteris vittata* and *Pteris vittata*-2'-O-glucose both meet the requirement of r≥0.99, and within the concentration range of 20 ng / mL-150 ng / mL, their peak area shows a good linear relationship with concentration.

[0121] 6. Sample recovery Take three portions each of the low-concentration, medium-concentration, and high-concentration spiked solutions of the test sample, and inject them sequentially according to "2.2 Instrument Conditions". Calculate the spiked recovery rate and the relative standard deviation of the recovery rate. RSD The results are shown in Table 16.

[0122] Table 16 Results of Spiking Recovery Determination

[0123] Table 16 shows that under low, medium, and high spiking conditions, the recoveries of *Gynostemma pentaphyllum*-2'-O-glucoside ranged from 99.8% to 113.1%, with a relative standard deviation (RSD) of 4.7% for the nine spiking solutions. The recoveries of *Gynostemma pentaphyllum* ranged from 96.3% to 109.8%, with a RSD of 5.1% for the nine spiking solutions. The recoveries of both compounds met the requirements of 75%-120% and an RSD ≤ 15%.

[0124] 7. Repeatability Take six spiked solutions of the test sample at different concentrations and inject them sequentially according to "2.2 Instrument Conditions". Calculate the relative standard deviation of the spiked sample content. RSD The results are shown in Table 17.

[0125] Table 17 Repeatability Test

[0126] As shown in Table 17, the relative standard deviation of the concentration spiked solution of fern extract-2'-O-glucoside in the 6 parallel preparations was 3.4%; the relative standard deviation of the content of fern extract was 3.4%, indicating good repeatability (RSD≤15%).

[0127] 8. Intermediate Precision Personnel A and Personnel B each prepared 6 spiking solutions of the test sample at different concentrations. These solutions were injected sequentially according to "2.2 Instrument Conditions". The relative standard deviations of the concentrations in the 6 spiking solutions prepared by Personnel A and Personnel B were calculated respectively. RSD ), and calculate the relative standard deviation of the content of these 12 spiked solutions ( RSD The results are shown in Tables 18 and 19.

[0128] Table 18 Intermediate Precision Study of *Gynostemma pentaphyllum*-2'-O-glucoside

[0129] Table 19. Precision Study of the Intermediate Level of the Golden Powder Fern Pavilion

[0130] As shown in Table 18, the relative standard deviations of the concentrations of ginsenoside-2'-O-glucoside in the six test samples prepared by the two testing personnel were 3.4% and 2.8%, respectively, with a total relative standard deviation of 5.3%, which meets the requirements (RSD≤15%).

[0131] As shown in Table 19, the relative standard deviations of the concentrations of *Gynostemma pentaphyllum* in the six test samples prepared by the two testing personnel were 3.4% and 1.4%, respectively, with a total relative standard deviation of 4.6%, which meets the requirements. RSD ≤15%).

[0132] 9. Solution stability Six portions of the mixed reference working solution and six portions of the test spiked solution were taken and sampled at 0, 6, 12, 18, 24, and 30 hours respectively. Analysis was performed according to "2.2 Instrument Conditions," and the relative standard deviation of the six peak area measurements of the reference solution and the test spiked solution was calculated. RSD The results are shown in Table 20.

[0133] Table 20 Solution stability study

[0134] As shown in Table 20, during the six observation periods, the relative standard deviation of the peak area of ​​the reference solution for ginseng-2'-O-glucoside was 10.4%, and the relative standard deviation of the peak area of ​​ginseng was 8.6%, both meeting the requirements. RSD ≤15%). Meanwhile, the relative standard deviation of the peak area of ​​*Pteris vittatum*-2'-O-glucoside in the spiked solution of the test sample was 12.3%, and the relative standard deviation of the peak area of ​​*Pteris vittatum* was 9.4%, both meeting the requirements. RSD (≤15%). This indicates that the target compound, ginsenoside-2'-O-glucoside, is stable in both the reference solution and the spiked solution of the test sample for at least 30 hours.

[0135] 10. Durability Take the mixed reference standard working solution and the spiked solution of the test sample at different concentrations, select five key factors affecting the detection results, and calculate the spiked recovery rate of the spiked solution of the test sample to examine the robustness of the instrument method.

[0136] The assessment factors and levels are set as follows: ① Column type: Shim-pack Velox C18, Poroshell 120 EC-C18, Kinetex C18; ② Column temperature: 25℃, 30℃, 35℃; ③ LC flow rate: 0.25 mL / min, 0.30 mL / min, 0.35 mL / min; ④ MS drying gas temperatures: 315℃, 350℃, 385℃; ⑤ MS drying gas flow rate: 4 L / min, 5 L / min, 6 L / min.

[0137] The recovery rate experimental results for each factor under different levels are detailed in Tables 21 and 22.

[0138] Table 21 Arrangement of factors for evaluating the durability of *Gynostemma pentaphyllum*-2'-O-glucoside

[0139] Table 22 Arrangement of Durability Assessment Factors for the Golden Powder Fern Pavilion

[0140] As shown in Tables 21 and 22, under the conditions of changing the chromatographic column, column temperature, LC flow rate, MS drying gas temperature, and MS drying gas flow rate, the spiked recovery rate of ginsenoside-2'-O-glucoside in the spiked solution of the test sample was within 81.2%-103.1%, and the spiked recovery rate of ginsenoside was within 85.4%-105.9%, both of which met the requirements (75%-120%).

[0141] Example 7: Determination of the content of effective components in Cibotium barometz. The samples in Table 1 were prepared into test solutions according to the "optimal process" and tested separately. The determination results of fern-2'-O-glucoside and fern extract in the samples can be obtained, as shown in Table 23.

[0142] Table 23. Determination of the contents of ferns-2'-O-glucoside and ferns-2'-glucoside in Cibotium barometz samples.

[0143] Table 23 shows that the content of *Gnaphalium affine*-2'-O-glucoside in the seven batches of *Cibotium barometz* (YC01-YC07) ranged from 10.4 to 151.2 mg / kg, and the content of *Gnaphalium affine* ranged from 12.2 to 93.1 mg / kg. In contrast, the content of *Gnaphalium affine*-2'-O-glucoside in the three batches of *Cibotium barometz* treated with heat treatment (YC08-YC10) ranged from 66.2 to 157.2 mg / kg, and the content of *Gnaphalium affine* ranged from 5.68 to 53.9 mg / kg. This demonstrates that there are significant differences in the content of active ingredients between different batches of *Cibotium barometz* and heat-treated *Cibotium barometz*. The method of this invention can accurately obtain the batch-to-batch differences in *Cibotium barometz* medicinal materials or preparations.

[0144] Example 8: Determination of the content of active ingredients in Cibotium barometz preparations Specific information on the Cibotium barometz preparation sample in this embodiment is shown in Table 24. Among them, the muscle-relaxing and anti-inflammatory preparation (YX01) is prepared by omitting Cibotium barometz, and using the other ingredients according to the prescription for Shujin Jianyao Wan (Muscle-Relaxing and Waist-Strengthening Pill) in Volume 12 of the Ministry-issued Standard for Traditional Chinese Medicine Preparations (WS3-B-2441-97). The preparation method is the same as the self-made reference preparation. Specifically, Rosa laevigata, Spatholobus suberectus, Smilax glabra, and the woody part of Smilax china are decocted twice with water (excluding Cibotium barometz), filtered, and the filtrates are combined and concentrated into a thick paste. The remaining eight ingredients, including Smilax glabra and the bark of Smilax china, are pulverized into coarse powder, added to the above thick paste, mixed, dried, pulverized into fine powder, sieved, and mixed. For every 100g of powder, 35-40g of refined honey and an appropriate amount of water are used to make pills, which are then coated with black iron oxide, dried, and polished to obtain the muscle-relaxing and anti-inflammatory preparation (YX01).

[0145] The Yin-nourishing preparation for strengthening the waist (YX02) is prepared by omitting Cibotium barometz, and using the remaining ingredients according to the prescription for the Yin-nourishing and Kidney-strengthening Pills listed in standard WS3-B-0538-91 attached to the document Pharmacopoeia

[2001] No. 578. The preparation method is the same as the self-made reference preparation. Specifically, the woody part of *Gnaphalium affine*, *Spatholobus suberectus*, *Rosa laevigata*, *Millettia speciosa*, and *Achyranthes bidentata* are decocted twice with water (excluding Cibotium barometz), the decoctions are combined, filtered, and the filtrate is concentrated into a thick paste. 1000g of the bark of *Gnaphalium affine*, *Cuscuta chinensis*, *Ligustrum lucidum*, *Taxillus chinensis*, and Cibotium barometz are pulverized into coarse powder, added to the above thick paste, mixed well, dried, pulverized into fine powder, sieved, and mixed well. For every 100g of powder, 50-60g of refined honey and an appropriate amount of water are used to make pills, which are then coated with medicinal charcoal, dried, and polished to obtain the Yin-nourishing preparation for strengthening the waist (YX02).

[0146] Table 24 Information on Cibotium barometz preparations

[0147] The samples in Table 24 were prepared according to "Example 7 1.5 Test Solution" to obtain the Cibotium barometz preparation solution, and were tested on the instrument according to "2.2 Instrument Conditions" to obtain the determination results of ginsenoside-2'-O-glucoside and ginsenoside in the Cibotium barometz preparation sample, as shown in Table 25.

[0148] Table 25. Determination results of the content of fern-2'-O-glucoside and fern extract in Cibotium barometz preparations.

[0149] Note: ND indicates not detected.

[0150] As shown in Table 25, the target compound was not detected in any of the negative control preparations, but its content could be detected in the medicinal materials and related preparations, which confirms that the established method has good specificity.

[0151] Among them, the contents of Jinfenting fern-2'-O-glucoside and Jinfenting fern in three batches of Shujin Jianyao Pills (ZJ01, ZJ02, ZJ03) ranged from 20.05 to 23.17 mg / kg and 10.10 to 11.60 mg / kg, respectively. The contents of Jinfenting fern-2'-O-glucoside and Jinfenting fern in four batches of Zhuangyao Jianshen Pills (ZJ04, ZJ05, ZJ06, ZJ07) ranged from 16.26 to 21.38 mg / kg and 8.36 to 17.72 mg / kg, respectively.

[0152] In summary, this invention establishes a liquid chromatography-tandem mass spectrometry (LC-MS / MS) method for the determination of the active ingredients, ginsenoside-2'-O-glucoside and ginsenoside, in Cibotium barometz samples. Methodological validation of the method's specificity, system suitability, limits of detection and quantitation, linearity and range, recovery, repeatability, intermediate precision, solution stability, and robustness all meet the requirements of the "Guiding Principles for Validation of Analytical Methods" in Part IV, 9101 of the 2025 edition of the Pharmacopoeia of the People's Republic of China. This demonstrates that the method of this invention can accurately detect the content of ginsenoside-2'-O-glucoside and ginsenoside in Cibotium barometz, achieving comprehensive and specific control over the intrinsic quality of Cibotium barometz medicinal materials and their preparations.

[0153] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for determining the content of active ingredients in Cibotium barometz and its preparations, characterized in that, The triple quadrupole liquid chromatography-mass spectrometry method was used to simultaneously detect the active ingredients, ginsenoside and ginsenoside-2'-O-glucoside, in the tested sample of Cibotium barometz and its preparations. The liquid chromatography conditions include: Mobile phase composition: Mobile phase A is methanol, and mobile phase B is water; The gradient elution procedure is as follows: 0-2 min, 20% mobile phase A, 80% mobile phase B; 2-2.1 min, 20%→60% mobile phase A, 80%→40% mobile phase B; 2.1-6 min, 60% mobile phase A, 40% mobile phase B; 6-6.1 min, 60%→100% mobile phase A, 40%→0% mobile phase B; 6.1-8 min, 100% mobile phase A; 8-8.1 min, 100%→20% mobile phase A, 0%→80% mobile phase B; 8.1-12 min, 20% mobile phase A, 80% mobile phase B.

2. The method for determining the content of active ingredients according to claim 1, characterized in that, The pretreatment of the test sample, Cibotium barometz and its preparations, includes: adding the test sample to the extraction solvent for ultrasonic extraction, then centrifuging, and filtering the supernatant to obtain the final product.

3. The method for determining the content of active ingredients according to claim 2, characterized in that, The ultrasonic extraction frequency is 16-32 kHz; the ultrasonic extraction time is 15-30 min.

4. The method for determining the content of active ingredients according to claim 3, characterized in that, The ultrasonic extraction frequency is 32 kHz; the ultrasonic extraction time is 30 min.

5. The method for determining the content of active ingredients according to claim 1, characterized in that, The liquid chromatography conditions also include: column temperature of 25-35℃; flow rate of 0.25-0.35 mL / min.

6. The method for determining the content of active ingredients according to claim 5, characterized in that, The column temperature is 30°C; the flow rate is 0.3 mL / min.

7. The method for determining the content of active ingredients according to claim 1, characterized in that, The chromatographic column used in the liquid chromatography is selected from any one of Poroshell 120 EC-C18, Shim-pack Velox C18, and Kinetex C18.

8. The method for determining the content of active ingredients according to claim 1, characterized in that, The mass spectrometry conditions for the triple quadrupole liquid chromatography-mass spectrometry method are as follows: ESI ion source, positive ion mode; capillary voltage: 4000 V; nozzle voltage: 400 V.

9. The method for determining the content of active ingredients according to claim 1, characterized in that, The mass spectrometry conditions for the triple quadrupole liquid chromatography-mass spectrometry method also include: a drying gas temperature of 315-385℃ and a drying gas flow rate of 4-6 L / min.

10. The method for determining the content of active ingredients according to claim 9, characterized in that, The temperature of the drying gas is 350℃; the flow rate of the drying gas is 5 L / min.

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