A detection method of Baizhu San standard decoction fingerprint combined with liquid chromatography-mass spectrometry, fingerprint and application thereof

Abstract

The application discloses a detection method of Baizhu San standard decoction fingerprint combined with liquid chromatography-mass spectrometry, the fingerprint and application thereof, and belongs to the field of traditional Chinese medicine quality standard research and application. Test sample solution and single control sample solution are injected into a high performance liquid chromatograph to obtain respective chromatograms; mass spectrometry analysis is conducted on the test sample solution to obtain mass spectrometry results; common peaks existing in the chromatogram of the test sample solution are taken as common peaks, chemical components in the Baizhu San standard decoction are identified in combination with the chromatogram of the single control sample solution and the mass spectrometry results of the test sample solution, and the Baizhu San standard decoction fingerprint is obtained. The fingerprint is constructed through liquid chromatography-mass spectrometry, 10 intersecting components are identified in combination with the mass spectrometry information of the control sample, so that the quality control of the Baizhu San standard decoction is realized, the method is simple, easy to operate, high in precision, good in stability and reproducibility, and can effectively solve the technical problem that the quality of the Baizhu San cannot be effectively evaluated.

Description

Technical Field

[0001] This invention belongs to the field of research and application technology of quality standards of traditional Chinese medicine, specifically involving a detection method for the fingerprint spectrum of standard decoction of Atractylodes macrocephala powder combined with liquid chromatography-mass spectrometry, as well as its fingerprint spectrum and application. Background Technology

[0002] Ancient classic prescriptions of traditional Chinese medicine are an important part of Han Chinese traditional medicine. They include both formulas and single-herb prescriptions, representing a summary and inheritance of medical experience in traditional Chinese medicine. They possess profound historical significance and broad clinical application value, playing a vital role in promoting the development of the traditional Chinese medicine industry. Because the effective components of traditional Chinese medicine are diverse and their interactions are complex, the site of efficacy is often not a single component, but rather the overall therapeutic effect is achieved through synergistic effects. No single component can fully reflect the overall efficacy of a traditional Chinese medicine compound; therefore, effective quality evaluation of traditional Chinese medicine compound prescriptions is necessary.

[0003] Currently, there are high performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography (UPLC) methods for determining the effective components of traditional Chinese medicine compound prescriptions. However, both methods are limited by low sensitivity and the complexity of traditional Chinese medicine compound prescriptions, resulting in many interfering peaks during separation and failing to meet the requirements of both qualitative and quantitative analysis.

[0004] The classic formula Baizhu Powder, also known as Seven-Ingredient Baizhu Powder, originates from Qian Yi's *Straightforward Explanation of Pediatric Drug Syndromes* from the Song Dynasty. This formula consists of seven herbs: ginseng, atractylodes macrocephala, poria cocos, patchouli, costus root, kudzu root, and licorice root. Ginseng, with its sweet and warm properties, tonifies qi, strengthens the spleen, and nourishes the stomach, making it the principal herb. Atractylodes macrocephala, bitter and warm, strengthens the spleen, dries dampness, and enhances qi circulation, serving as an auxiliary herb. Poria cocos, sweet and bland, strengthens the spleen and eliminates dampness; kudzu root promotes yang and generates fluids; patchouli resolves dampness and stops vomiting; costus root regulates the qi mechanism of the middle jiao. All the herbs work synergistically to primarily strengthen the spleen, eliminate dampness, and regulate qi. Licorice root, sweet and warm, tonifies qi, harmonizes the middle jiao, and balances the relationships between the herbs, acting as a moderating herb. In summary, Baizhu Powder uses ginseng, poria cocos, atractylodes macrocephala, and licorice root to tonify the spleen and harmonize the middle jiao; kudzu root to stop diarrhea, generate fluids, and reduce fever; and patchouli and costus root to invigorate the spleen and harmonize the stomach. Its effects are "strengthening the spleen and stomach, replenishing qi and stopping diarrhea", and it is mainly used to treat "children's spleen and stomach weakness, and internal heat due to fluid deficiency". However, there are currently no research reports on the quality testing of Atractylodes macrocephala powder. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a detection method for Baizhu powder standard decoction using fingerprint spectrum combined with liquid chromatography-mass spectrometry, as well as its fingerprint spectrum and application, to solve the technical problem of being unable to effectively evaluate the quality of Baizhu powder.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] In a first aspect, this invention discloses a detection method using fingerprint spectroscopy combined with liquid chromatography-mass spectrometry for a standard decoction of Atractylodes macrocephala powder, comprising the following steps:

[0008] 1) Add solvent to the freeze-dried powder of Atractylodes macrocephala powder standard decoction from different batches, dilute and make up to volume, filter, and obtain the test solution;

[0009] 2) Accurately weigh daidzein, verbascoside, isoraburoside, daidzein, ginsenoside Rb2, ginsenoside Rb1, costus lactone, atractylodes lactone II, dehydrocostus lactone and pamoate A reference standard, dissolve them to obtain a single reference standard solution.

[0010] 3) Inject the test solution and the single reference solution into the high performance liquid chromatograph for analysis to obtain chromatograms; then perform mass spectrometry analysis on the test solution to obtain mass spectrometry results;

[0011] 4) Using the chromatographic peaks present in all chromatograms of the test solution as common peaks, and combining the chromatograms of the single reference solution and the mass spectrometry results of the test solution, the chemical components in the standard decoction of Atractylodes macrocephala powder are identified, and the fingerprint spectrum of the standard decoction of Atractylodes macrocephala powder is obtained.

[0012] Preferably, step 1) specifically involves: adding 70% methanol aqueous solution to freeze-dried powder of Atractylodes macrocephala powder standard decoction in different batches, weighing, sonicating for 30 minutes, cooling, weighing again, replenishing the lost weight with 70% methanol aqueous solution, shaking well, taking the filtrate, and obtaining the test solution.

[0013] More preferably, in step 1), the ratio of the freeze-dried powder of Atractylodes macrocephala standard decoction to 70% methanol aqueous solution is 0.5g:25mL.

[0014] Preferably, in step 2), each reference standard is prepared into a single reference standard solution of 50-130 μg / mL using methanol.

[0015] Preferably, in step 3), the liquid chromatography conditions are as follows: an Accurasil C18 column, 250 mm × 4.6 mm × 5 μm; mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid aqueous solution; detection wavelength is 203 nm; column temperature is 30 °C; flow rate is 1.0 mL / min; and injection volume is 20 μL.

[0016] More preferably, the liquid chromatography elution procedure is as follows:

[0017]

[0018] Preferably, in step 3), the mass spectrometry conditions are as follows: an electrospray ionization source is used, with positive and negative ion modes for detection, a scanning range of m / z of 100–1500, a sheath gas flow rate of 40 arb, an auxiliary gas flow rate of 10 arb, an auxiliary gas temperature of 300°C, and a capillary temperature of 300°C; the spray voltage is 4000 V in positive ion mode and 3200 V in negative ion mode.

[0019] Preferably, the specific steps of step 4) are as follows: import the chromatograms of each test sample solution into the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system and perform similarity analysis. Take the chromatographic peaks that are present in the chromatograms of the test sample solutions as common peaks. Identify the chemical components in the Baizhu Powder standard decoction based on the chromatograms of the single reference solution and the mass spectrometry results of each chemical component. Calculate the relative retention time and relative peak area of ​​each common peak to obtain the fingerprint spectrum of the Baizhu Powder standard decoction.

[0020] In a second aspect, the present invention discloses a fingerprint spectrum of a standard decoction of Atractylodes macrocephala powder, consisting of 10 characteristic peaks, with peak 12 as a reference peak. The relative retention times of each characteristic peak are as follows: peak 6 is daidzein, retention time 21.788 min; peak 7 is verbascoside, retention time 24.001 min; peak 8 is isoverascoside, retention time 24.866 min; peak 11 is daidzeinogen, retention time 32.803 min. Peak 12 is ginsenoside Rb2, retention time 35.307 min; peak 13 is ginsenoside Rb1, retention time 36.264 min; peak 26 is costunolide, retention time 61.036 min; peak 27 is atractylodes lactone II, retention time 61.740 min; peak 28 is dehydrocostunolide, retention time 62.847 min; peak 30 is pamoate A, retention time 67.976 min.

[0021] In a third aspect, the present invention discloses a method for establishing the fingerprint spectrum of the above-mentioned Atractylodes macrocephala powder standard decoction, and the application of the above-mentioned Atractylodes macrocephala powder standard decoction fingerprint spectrum in the production and quality control of Atractylodes macrocephala powder.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention provides a detection method combining fingerprint spectroscopy with liquid chromatography-mass spectrometry (LC-MS). Firstly, it utilizes a combination of high-performance liquid chromatography (HPLC) and mass spectrometry (MS) to construct the fingerprint spectroscopy of the standard decoction of Atractylodes macrocephala powder. LC-MS offers advantages such as high specificity, high sensitivity, and short analysis time. Secondly, using the fingerprint spectroscopy of the standard decoction of Atractylodes macrocephala powder as a quality control method avoids the bias of judging the overall quality of the preparation based on only one or two chemical components, and reduces the possibility of artificial manipulation to meet quality standards. This method was used to determine the content of the standard decoction of Atractylodes macrocephala powder. The method is simple, easy to implement, and has good adaptability. Its specificity, precision, stability, and repeatability all meet the requirements. It can provide a reference for the construction and identification of characteristic spectra of Atractylodes macrocephala powder granules, powders, and other common oral dosage forms, as well as for the application of quality standards. It enables rapid identification of Atractylodes macrocephala powder formulas and provides an objective and comprehensive evaluation of the quality of Atractylodes macrocephala powder. It fills the gap in fingerprint spectrum research in the quality control of the standard decoction of the classic formula Atractylodes macrocephala powder, provides a strong guarantee for improving the quality of the classic formula Atractylodes macrocephala powder, lays the foundation for the application of quality standards of Atractylodes macrocephala powder, and ensures its clinical efficacy.

[0024] Furthermore, by systematically analyzing multiple batches of samples during the preparation of the test solution, the quality of Atractylodes macrocephala powder can be evaluated more comprehensively and scientifically, thereby ensuring the quality and efficacy of Atractylodes macrocephala powder.

[0025] This invention provides a fingerprint spectrum of a standard decoction of Atractylodes macrocephala powder, which provides rich chemical information, excellent separation effect, high precision, good stability and reproducibility, and uses mass spectrometry to complete the qualitative identification of chemical components in Atractylodes macrocephala powder. It can comprehensively reflect the chemical component information of Atractylodes macrocephala powder, and has important reference significance for establishing a scientific and reasonable quality evaluation method for Atractylodes macrocephala powder and improving its quality control standards. Attached Figure Description

[0026] Figure 1 These are chromatograms obtained by different extraction methods during the preparation of the test sample solution according to the present invention;

[0027] Figure 2 This is a chromatogram obtained at different ultrasonic times during the preparation of the test solution according to the present invention;

[0028] Figure 3 This is a chromatogram obtained by different extraction solvents during the preparation of the test solution according to the present invention;

[0029] Figure 4 These are chromatograms obtained at different wavelengths according to the present invention;

[0030] Figure 5 These are chromatograms obtained under different mobile phases according to the present invention;

[0031] Figure 6These are chromatograms obtained at different flow rates according to the present invention;

[0032] Figure 7 These are chromatograms obtained at different column temperatures according to the present invention;

[0033] Figure 8 This is a comparison chromatogram of the Atractylodes macrocephala powder reference sample and the reference standard of the present invention;

[0034] Figure 9 This is a mass spectrum in positive ion mode of the Atractylodes macrocephala powder reference sample of the present invention;

[0035] Figure 10 This is a mass spectrum of the Atractylodes macrocephala powder reference sample in negative ion mode according to the present invention.

[0036] Figure 11 This is the mass spectrum of daidzin from the present invention;

[0037] Figure 12 The mass spectra of verbascoside and isoverascoside of this invention are shown.

[0038] Figure 13 This is the mass spectrum of daidzein of the present invention;

[0039] Figure 14 This is the mass spectrum of ginsenoside Rb2 of the present invention;

[0040] Figure 15 This is the mass spectrum of ginsenoside Rb1 of the present invention;

[0041] Figure 16 The mass spectra of atractylodes lactone II and costus lactone of the present invention are shown below.

[0042] Figure 17 This is the mass spectrum of dehydroauracetam from the present invention;

[0043] Figure 18 This is the mass spectrum of pachymic acid A of the present invention;

[0044] Figure 19 The HPLC fingerprint of 15 batches of Atractylodes macrocephala powder standard decoction of this invention;

[0045] Figure 20 This is the common peak chromatogram of the standard decoction of Atractylodes macrocephala powder of the present invention. Detailed Implementation

[0046] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0047] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0048] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0049] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0050] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0051] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0052] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0053] The embodiments of the present invention will be described in detail below with reference to the examples. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0054] I. Instruments and Reagents

[0055] 1. Experimental apparatus

[0056] The instruments used in the experiment are shown in Table 1.

[0057] Table 1 Experimental Instruments

[0058]

[0059] 2. Medicines and reagents

[0060] The reference standards used in the experiment are shown in Table 2.

[0061] Table 2 Reference Standards

[0062]

[0063] The reagents used in the experiment are shown in Table 3.

[0064] Table 3 Reagents

[0065]

[0066]

[0067] The batch numbers of the 15 batches of freeze-dried powder of Atractylodes macrocephala powder standard decoction used in the experiment are shown in Table 4.

[0068] Table 4. Batch numbers of 15 batches of Atractylodes macrocephala powder standard decoction freeze-dried powder samples

[0069]

[0070] II. Exploration of fingerprint spectral detection method for standard decoction of Atractylodes macrocephala powder

[0071] 1. Investigation into the preparation of test sample solutions

[0072] The Baizhu Powder formula contains numerous medicinal materials with complex components. Different extraction methods, solvents, and times can all affect the extraction of its active ingredients. To ensure that the active ingredients are not destroyed and to extract as many as possible, this study experimentally investigated the preparation process of the Baizhu Powder test solution.

[0073] This experiment used HPLC technology to investigate the effects of different extraction methods (ultrasound, reflux, maceration), extraction times (30 min, 45 min, 60 min), and extraction solvents (pure methanol, 70% methanol aqueous solution, 50% methanol aqueous solution) on the preparation of the test solution. Peak shape, number of peaks, and resolution were used as evaluation indicators. Results are shown below. Figure 1 , Figure 2 and Figure 3 Experimental results showed that fingerprints obtained by ultrasonic extraction with 70% methanol aqueous solution as the extraction solvent for 30 min contained rich chemical information, high component content, good separation, and were easy to observe and analyze. Therefore, ultrasonic extraction with 70% methanol aqueous solution for 30 min was chosen as the final method.

[0074] 2. Exploration of chromatographic conditions

[0075] On the one hand, the Baizhu Powder formula contains many medicinal materials, making separation difficult, and the polarity of each component differs. On the other hand, the initial organic phase has a high proportion, resulting in fewer effective components. Furthermore, chromatographic conditions such as wavelength, flow rate, column temperature, and mobile phase all affect the separation of components and thus the quality of the fingerprint chromatogram. To ensure baseline stability, method feasibility, and the separation of as many effective components as possible, this method employs a gradient elution procedure for fingerprint chromatogram analysis.

[0076] 1) The effect of wavelength

[0077] This experiment used a UV-Vis detector to study chromatograms at different wavelengths (203 nm, 220 nm, 245 nm, 254 nm, 280 nm). The results are shown below. Figure 4 Experimental results show that when the detection wavelength is 203 nm, the chromatogram has more comprehensive information and the baseline is stable. Therefore, 203 nm was selected as the detection wavelength.

[0078] 2) The effect of washout time

[0079] In this experiment, chromatograms were recorded at 60 minutes when selecting the elution time for fingerprint spectroscopy. The results showed poor separation of peaks and unstable baselines. To ensure that as many active ingredients as possible could be detected in all batches of samples, the analysis time was delayed, and 80 minutes was ultimately selected as the analysis time.

[0080] 3) The impact of the elution system

[0081] This experiment compared the elution effects of several different elution systems under different gradients, including water-pure methanol, water-pure acetonitrile, 0.1% phosphoric acid aqueous solution-pure methanol, 0.1% phosphoric acid aqueous solution-pure acetonitrile, 0.05% formic acid aqueous solution-pure acetonitrile, 0.1% formic acid aqueous solution-pure acetonitrile, different flow rates (0.5 mL / min, 0.8 mL / min, 1.0 mL / min), and different column temperatures (25℃, 30℃, 35℃). The results are shown below. Figure 5 , Figure 6 and Figure 7 Experimental results showed that the mobile phase of 0.1% phosphoric acid aqueous solution-pure acetonitrile, the flow rate of 1.0 mL / min, and the column temperature of 30℃ resulted in better peak elution, a stable baseline, and better separation of the components. Therefore, 0.1% phosphoric acid aqueous solution-pure acetonitrile was selected as the mobile phase, with a flow rate of 1.0 mL / min and a column temperature of 30℃ for detection.

[0082] 4) The impact of the elution process

[0083] After determining the above detection conditions, the optimal gradient elution program was screened through a large number of experiments. The experiments showed that when the gradient elution program in Table 5 was used, good separation of each chromatographic peak in the fingerprint spectrum could be achieved.

[0084] Table 5 Gradient elution program

[0085]

[0086] III. Fingerprint Spectrum Detection Method for Standard Decoction of Atractylodes macrocephala Powder

[0087] S1. Preparation of the test solution of the standard decoction of Atractylodes macrocephala powder: Accurately weigh 0.5 g of the freeze-dried powder of the standard decoction of Atractylodes macrocephala powder of 15 batches shown in Table 4, place them in a stoppered conical flask, add 25 mL of 70% methanol aqueous solution, weigh, extract by ultrasonication (ultrasonic power 250 W, frequency 40 kHz) for 30 min, cool, weigh again, make up the lost weight with 70% methanol aqueous solution, shake well, filter, take the filtrate, filter through a 0.45 μm microporous membrane to obtain the test solution.

[0088] S2. Preparation of reference solutions: Accurately weigh the reference standards daidzein, verbascoside, isoverascoside, daidzein, ginsenoside Rb2, ginsenoside Rb1, costunolide, atractylodes lactone II, dehydrocostunolide, and pamoate A. Using methanol as solvent, prepare single reference solutions with mass concentrations of 106 μg / mL, 88 μg / mL, 90 μg / mL, 110 μg / mL, 78 μg / mL, 63 μg / mL, 97 μg / mL, 124 μg / mL, 101 μg / mL, and 45 μg / mL for each component. Store the above reference solutions at 4℃ for later use.

[0089] S3. Accurately pipette 15 batches of the standard decoction of Atractylodes macrocephala powder and inject them into the high-performance liquid chromatograph (HPLC). Record the chromatograms of the fingerprints of the 15 batches of standard decoction of Atractylodes macrocephala powder. Inject the prepared single reference solution and methanol solvent into the HPLC and record the chromatograms of the reference solution and blank solvent. Figure 8 The liquid chromatography conditions were as follows: column: Accurasil C18 (250 mm × 4.6 mm × 5 μm); mobile phase: acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B); gradient elution; UV-Vis detector; detection wavelength: 203 nm; column temperature: 30 ℃; flow rate: 1.0 mL / min; injection volume: 20 μL; elution program is shown in Table 5.

[0090] The test solution was then subjected to high-resolution mass spectrometry analysis. The detection data was imported into Xcalibur 4.1 software, and the Qual Browser interface was used to analyze the data based on the peaks of the chemical components, resulting in a total ion chromatogram and mass spectrometry results of the chemical components. Figures 9-18 The identification error (δ) was selected to be within 5 ppm (1 ppm = 1 × 10⁻⁶). -6 The chemical components of Atractylodes macrocephala were determined within a certain range, and the source of the components was confirmed by comparing the chromatograms with those of the reference standard.

[0091] The mass spectrometry testing conditions were as follows: an electrospray ionization source was used, with positive and negative ion modes for detection, a scanning range of m / z of 100–1500, a sheath gas flow rate of 40 arb, an auxiliary gas flow rate of 10 arb, an auxiliary gas temperature of 300℃, and a capillary temperature of 300℃; the spray voltage was 4000V in positive ion mode and 3200V in negative ion mode.

[0092] S4. The chromatograms of the 15 batches of Atractylodes macrocephala powder standard decoction test solutions obtained in S3 were converted into AIA format and imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2004A). Using the characteristic chromatogram of sample S1 as the reference chromatogram, the mean method was used with a time window set to 0.1 min for multi-point correction to obtain the superimposed fingerprint chromatograms of the 15 batches of material references corresponding to the actual substances. Figure 19 ), and selected the chromatographic peaks that were present in chromatograms from different batches as common peaks. Figure 20 The similarity results of chromatograms of test solutions of different batches of Atractylodes macrocephala powder standard decoction were exported.

[0093] Based on the chromatograms of the single reference standard and the mass spectrometry information of the test solution obtained in S3, identify the chemical components of the Baizhu Powder standard decoction in the similarity results table, and calculate the relative retention times and relative peak areas of each common peak. The reliability of the results is confirmed based on the similarity results and methodological investigation results.

[0094] After comparison, a total of 10 cross-linked components were identified, namely: daidzein, verbascoside, isoruascoside, daidzein, ginsenoside Rb2, ginsenoside Rb1, costunolide, atractylodes lactone II, dehydrocostunolide, and pamoate A.

[0095] Chromatograms of 15 batches of Atractylodes macrocephala powder standard decoction were obtained by analysis and calculation. The common chromatographic peaks showed good similarity, indicating that the fingerprint spectrum established by this method can effectively detect the quality of Atractylodes macrocephala powder. The results are shown in Table 6.

[0096] Table 6. Similarity evaluation results of fingerprint spectra of 15 batches of Atractylodes macrocephala powder standard decoction

[0097]

[0098] IV. Methodological Study on Fingerprint Construction Based on Standard Decoction of Atractylodes macrocephala Powder

[0099] 1. Precision Study

[0100] The prepared standard decoction of Atractylodes macrocephala powder was used as the test solution and analyzed according to the fingerprint chromatogram detection method for the standard decoction of Atractylodes macrocephala powder. Six parallel injections were performed, and the chromatograms were recorded. Using peak 12 as the reference peak (S), the relative retention times of each common peak were calculated. The results are shown in Table 7. It was found that the RSD of the relative retention times of each common peak was less than 1.10%, and the RSD of the relative peak areas was less than 2.56%, indicating that the precision of the equipment is good.

[0101] Table 7 Peak area and retention time in precision studies

[0102]

[0103]

[0104] 2. Reproducibility studies

[0105] Six parallel solutions of the Atractylodes macrocephala powder standard decoction were prepared according to the aforementioned method. The solutions were analyzed using the fingerprint chromatogram detection method for the Atractylodes macrocephala powder standard decoction, and the chromatograms were recorded. Using peak 12 as the reference peak (S), the relative retention times of each common peak were calculated. The results are shown in Table 8. It was found that the RSD of the relative retention times of each common peak was less than 0.86%, and the RSD of the relative peak areas was less than 2.70%, indicating that the method has good repeatability and can be used for the detection of the fingerprint chromatogram of Atractylodes macrocephala powder.

[0106] Table 8 Peak area and retention time in repeatability studies

[0107]

[0108] 3. Stability Study

[0109] The test solution of the standard decoction of Atractylodes macrocephala powder prepared by the above method was analyzed at different times of 0, 6, 12, 18 and 24 h according to the detection method of fingerprint spectrum of standard decoction of Atractylodes macrocephala powder. The relative retention time of each common peak was obtained with chromatographic peak No. 12 as reference peak (S). The results are shown in Table 9. The RSD of the relative retention time of each common peak is less than 0.76%, and the RSD of the relative peak area is less than 2.58%, indicating that the stability of the test solution is good within 24 h.

[0110] Table 9 Peak area and retention time in stability studies

[0111]

[0112] The above experimental results show that the fingerprint spectrum combined with liquid chromatography-mass spectrometry (LC-MS) detection method for the standard decoction of Atractylodes macrocephala powder provided by this invention exhibits good stability, reproducibility, and high precision, meeting the requirements for characteristic spectrum construction methods. This method can be used for the qualitative identification of Atractylodes macrocephala powder, enabling a scientific, comprehensive, and objective evaluation of its quality and ensuring its clinical efficacy.

[0113] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A detection method for the standard decoction of Qiwei Baizhu Powder using fingerprint spectroscopy combined with liquid chromatography-mass spectrometry, characterized in that, The steps are as follows: 1) Add 70% methanol aqueous solution to freeze-dried powder of Qiwei Baizhu powder standard decoction in different batches, dilute and make up to volume, filter, and obtain test solution; 2) Accurately weigh daidzein, verbascoside, isoraburoside, daidzein, ginsenoside Rb2, ginsenoside Rb1, costus lactone, atractylodes lactone II, dehydrocostus lactone and pamoate A reference standards respectively, dissolve them to obtain single reference standard solutions. 3) Inject the test solution and the single reference solution into the high performance liquid chromatograph for analysis to obtain chromatograms; then perform mass spectrometry analysis on the test solution to obtain mass spectrometry results; The liquid chromatography conditions were as follows: an Accurasil C18 column (250 mm × 4.6 mm × 5 μm); mobile phase A was acetonitrile, and mobile phase B was 0.1% phosphoric acid aqueous solution; the detection wavelength was 203 nm; the column temperature was 30 ℃; the flow rate was 1.0 mL / min; the injection volume was 20 μL; and the liquid chromatography elution program was as follows: 4) Using the chromatographic peaks present in all the chromatograms of the test solution as common peaks, and combining the chromatograms of the single reference solution and the mass spectrometry results of the test solution, the chemical components in the Qiwei Baizhu San standard decoction were identified, and the fingerprint chromatogram of the Qiwei Baizhu San standard decoction was obtained.

2. The detection method of the standard decoction of Qiwei Baizhu Powder combined with liquid chromatography-mass spectrometry according to claim 1, characterized in that, Step 1) Specifically: Add 70% methanol aqueous solution to the freeze-dried powder of Qiwei Baizhu San standard decoction in different batches, weigh the powder, sonicate for 30 min, cool, weigh again, replenish the lost weight with 70% methanol aqueous solution, shake well, and take the filtrate to obtain the test solution.

3. The detection method for the standard decoction of Qiwei Baizhu Powder combined with liquid chromatography-mass spectrometry according to claim 2, characterized in that, In step 1), the ratio of freeze-dried powder of Qiwei Baizhu powder standard decoction to 70% methanol aqueous solution is 0.5g:25mL.

4. The detection method of the standard decoction of Qiwei Baizhu Powder combined with liquid chromatography-mass spectrometry according to claim 1, characterized in that, In step 2), each reference standard is prepared into a single reference standard solution of 50~130 μg / mL using methanol.

5. The detection method for the standard decoction of Qiwei Baizhu Powder using fingerprint spectroscopy combined with liquid chromatography-mass spectrometry according to claim 1, characterized in that, In step 3), the mass spectrometry conditions are: electrospray ionization source, positive and negative ion modes for detection, and scanning range. m / z The range is 100~1500, the sheath gas flow rate is 40 arb, the auxiliary gas flow rate is 10 arb, the auxiliary gas temperature is 300℃, the capillary temperature is 300℃; the spray voltage is 4000 V in positive ion mode and 3200 V in negative ion mode.

6. The detection method of the standard decoction of Qiwei Baizhu Powder combined with liquid chromatography-mass spectrometry according to claim 1, characterized in that, Step 4) involves importing the chromatograms of each test solution into the Chinese herbal chromatographic fingerprint similarity evaluation system and performing similarity analysis. Chromatographic peaks present in all test solution chromatograms are taken as common peaks. Based on the chromatograms of a single reference solution and the mass spectrometry results of each chemical component, the chemical components in the Qiwei Baizhu San standard decoction are identified. The relative retention time and relative peak area of ​​each common peak are calculated to obtain the fingerprint spectrum of the Qiwei Baizhu San standard decoction.

7. The application of the fingerprint spectrum combined with liquid chromatography-mass spectrometry detection method of the standard decoction of Qiwei Baizhu Powder as described in any one of claims 1 to 6 in the production and quality control of Qiwei Baizhu Powder.

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