Method for constructing allium macrostemon medicinal material fingerprint spectrum and determining allium macrostemon index component content

Abstract

The invention relates to a method for constructing an allium macrostemon medicinal material fingerprint spectrum and determining the content of allium macrostemon index components. The method comprises the following steps: preparing an allium macrostemon medicinal material into a test solution; injecting the test solution into a liquid chromatograph-mass spectrometer, obtaining the fingerprint spectrum of the allium macrostemon medicinal material according to the liquid chromatograph-mass spectrometer, and determining the content of the index components. According to the method for constructing the allium macrostemon medicinal material fingerprint spectrum and measuring the content, the evaluation standard of the allium macrostemon is comprehensively improved, and the clinical use effectiveness of the allium macrostemon medicinal material is ensured.

Description

[0001] This invention is a divisional application of the invention application with the application number 202310314133.5, the application date of March 28, 2023, and the invention name of "A Method for Constructing the Fingerprint of Allium macrostemon Bge. and Determining the Content of Index Components in Allium macrostemon Bge.". Technical Field

[0002] This invention belongs to the technical field of quality analysis and detection of traditional Chinese medicine, and particularly relates to a method for constructing the fingerprint of Allium macrostemon Bge. and determining the content of index components in Allium macrostemon Bge. Background Art

[0003] Allium macrostemon Bge. was first recorded in "Shennong Ben Cao Jing". It is the dried bulb of the liliaceous plant Allium macrostemon Bge. or Allium chinense G.Don, and has the effects of promoting yang and dispersing stagnation, promoting qi and guiding stagnation, and can be used to treat chest pain, abdominal distension and pain, and tenesmus after diarrhea. Modern pharmacological research believes that Allium macrostemon Bge. has the effects of inhibiting platelet aggregation, reducing blood lipid, and anti-atherosclerosis, and steroidal saponins are its main active components. For example, 25S-Timosaponin B II and 25R-Timosaponin B II can inhibit ADP-induced platelet aggregation in vitro, and 25S-Timosaponin B II also has a good inhibitory effect on AA-induced platelet aggregation.

[0004] Allium macrostemon Bge. is mainly produced in Shaanxi, Northeast China and other places, and is distributed in most parts of the country. It is necessary to adopt effective technical means to evaluate the quality of Allium macrostemon Bge. consistently to ensure the safety, effectiveness and quality control of Allium macrostemon Bge.

[0005] However, currently, there is no control standard for evaluating the quality of Allium macrostemon Bge. from different origins consistently. The research on the fingerprint of Allium macrostemon Bge. currently only focuses on its compatibility with other herbs such as Trichosanthes kirilowii Maxim., Aurantii Fructus Immaturus, and Cinnamomi Ramulus, and the identified peaks are mostly nucleoside components, with poor specificity and difficult to reflect the evaluation of the active components of Allium macrostemon Bge. There is also a lack of a standard for determining the content of index components in Allium macrostemon Bge. in the Chinese Pharmacopoeia (2020 Edition).

[0006] Therefore, there is an urgent need to develop a method for constructing the fingerprint and content determination of Allium macrostemon Bge. for the quality evaluation of Allium macrostemon Bge. Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art to some extent. For this purpose, the present invention provides a method for constructing the fingerprint of Allium macrostemon Bge. and determining the content of index components in Allium macrostemon Bge.

[0008] According to one aspect of the present invention, a method for constructing a fingerprint of Allium macrostemon Bunge medicinal materials is provided, including: preparing a test solution from Allium macrostemon Bunge medicinal materials; preparing a reference solution from Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II; injecting the test solution and the reference solution into a liquid chromatography-mass spectrometry (LC-MS) instrument respectively, and obtaining the fingerprint of the Allium macrostemon Bunge medicinal materials according to the LC-MS method.

[0009] Preferably, in the fingerprint of the Allium macrostemon Bunge medicinal materials, there are 11 characteristic peaks, and the numbers of the characteristic peaks are Peak 1 to Peak 11; Peak 3 is Macrostemonoside I, Peak 6 is 25S-Timosaponin B II, and Peak 7 is 25R-Timosaponin B II. The characteristic peaks and the identification of the characteristic peaks in the LC-MS fingerprint are as follows:

[0010]

[0011] Preferably, taking Peak 6 as the S peak, the relative retention time ranges of Peak 1 to Peak 5 and Peak 7 to Peak 11 relative to the S peak are respectively: 0.332 - 0.338, 0.362 - 0.367, 0.562 - 0.569, 0.588 - 0.593, 0.606 - 0.611, 1.036 - 1.039, 1.177 - 1.187, 1.216 - 1.229, 1.272 - 1.284, 1.319 - 1.335.

[0012] Preferably, taking Peak 6 as the S peak, the relative standard deviation (RSD) values of the relative retention times of Peak 1 to Peak 5 and Peak 7 to Peak 11 relative to the S peak are respectively: 0.46%, 0.42%, 0.32%, 0.25%, 0.22%, 0.08%, 0.24%, 0.32%, 0.27%, 0.33%.

[0013] Preferably, the chromatographic conditions of the LC-MS method include: stationary phase: a chromatographic column filled with octadecylsilane-bonded silica gel; mobile phase: mobile phase A is water containing 0.1% (v / v) formic acid, and mobile phase B is an acetonitrile solution containing 0.1% (v / v) formic acid; gradient elution is adopted, and the flow rate is 0.3 mL / min.

[0014] Preferably, the chromatographic column is a CORTECS UPLC T3 Column (2.1×100 mm, 1.6 μm) chromatographic column.

[0015] Preferably, the gradient elution is carried out according to the following procedure: 0 - 0.5 min: mobile phase A from 85% → 85%, mobile phase B from 15% → 15%; 0.5 - 3.8 min: mobile phase A from 85% → 81%, mobile phase B from 15% → 19%; 3.8 - 4.5 min: mobile phase A from 81% → 77%, mobile phase B from 19% → 23%; 4.5 - 12.5 min: mobile phase A from 77% → 77%, mobile phase B from 23% → 23%; 12.5 - 14.0 min: mobile phase A from 77% → 73%, mobile phase B from 23% → 27%.

[0016] Preferably, the mass spectrometry conditions of the liquid chromatography - mass spectrometry method include: detection in the negative ion mode; the ion source is ESI; the backflush nitrogen temperature is 325 °C, the backflush nitrogen flow rate is 11.0 L / min, the nebulizing gas flow rate is 45 psig, the auxiliary nitrogen temperature is 350 °C, the auxiliary nitrogen flow rate is 11.0 L / min, the capillary voltage is 3500 V, the in - source collision voltage is 175 V, the cone - hole voltage is 65.0 V; the scanning mode: full scan; the scanning range: m / z 60 - 1700.

[0017] Preferably, the preparation method of the test solution includes: extracting, making up the weight, and filtering the Allium macrostemon Bunge medicinal material with a first solvent to obtain the test solution.

[0018] Preferably, the preparation method of the test solution includes: taking 50 mg of the Allium macrostemon Bunge medicinal material; adding 1 ml of the first solvent, weighing for the first time after soaking for 60 minutes; ultrasonically extracting for 60 minutes at 40 kHz and 30 °C; weighing for the second time after cooling, and continuously adding the first solvent to make up for the weight loss; filtering to obtain the test solution, wherein the first solvent is 50% - 95% ethanol.

[0019] According to another aspect of the present invention, there is provided a method for determining the content of index components in Allium macrostemon Bunge medicinal materials, comprising: preparing a test sample solution from the test Allium macrostemon Bunge medicinal materials to be tested; preparing a reference substance solution from Macrostemonoside I, 25S-Timosaponin B II, and 25R-Timosaponin B II; injecting the test sample solution and the reference substance solution into a liquid chromatography-mass spectrometry instrument respectively, and obtaining a test spectrum of the test Allium macrostemon Bunge medicinal materials and a reference spectrum of the reference substance solution according to the determined liquid chromatography-mass spectrometry method; calculating the contents of the index components Macrostemonoside I, 25S-Timosaponin B II, and 25R-Timosaponin B II in the test Allium macrostemon Bunge medicinal materials according to the concentration of the reference substance solution, the peak area of the reference substance solution in the reference spectrum, and the peak area of the components corresponding to the test Allium macrostemon Bunge medicinal materials and the reference substance solution in the test spectrum.

[0020] Preferably, the following relationship is satisfied between the concentration of the reference substance solution and the peak area of the reference substance solution in the reference spectrum: the first regression equation of Macrostemonoside I is y = 191254.78x - 6525.86 (r 2 = 0.9997, without weighting), the linear range is 0.25 - 25 μg / mL, the detection limit is 0.05 μg / mL (S / N ≥ 3), and the quantification limit is 0.25 μg / mL (S / N ≥ 9); the second regression equation of 25S-Timosaponin B II is y = 356910.66x - 18259.39 (r 2 = 0.9996, with a weighting of 1 / x), the linear range is 0.25 - 25 μg / mL, the detection limit is 0.05 μg / mL (S / N ≥ 3), and the quantification limit is 0.25 μg / mL (S / N ≥ 9); the third regression equation of 25R-Timosaponin B II is y = 480774.43x - 17621.70 (r 2 = 0.9995, with a weighting of 1 / x), the linear range is 0.25 - 25 μg / mL, the detection limit is 0.05 μg / mL (S / N ≥ 3), and the quantification limit is 0.25 μg / mL (S / N ≥ 9); wherein, in the first regression equation, the second regression equation, and the third regression equation, the abscissa is the concentration of the reference substance solution, and the ordinate is the peak area in the reference spectrum.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for constructing a fingerprint of Allium macrostemon Bunge medicinal materials and determining the content of index components. By optimizing the liquid phase elution method, the method can effectively separate the steroidal saponin active components; using the accurate molecular weight, molecular composition and fragment information of the compounds obtained by liquid chromatography-mass spectrometry technology, the chromatographic peaks in the fingerprint can be identified and authenticated; and a quantitative analysis method for the main steroidal saponin components in Allium macrostemon Bunge is established. The present invention stably and comprehensively improves the evaluation standard of Allium macrostemon Bunge medicinal materials, ensures the effectiveness of the medicinal materials in clinical use, and provides a reference for the overall control of the quality of Allium macrostemon Bunge medicinal materials. Description of the Drawings

[0022] The drawings described herein are used to provide a further understanding of the present invention and form a part of this application. The illustrative embodiments and descriptions of the present invention are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0023] Figure 1 Shows the chromatogram of the test solution of Allium macrostemon Bunge under elution method - 1;

[0024] Figure 2 Shows the chromatogram of the test solution of Allium macrostemon Bunge under elution method - 2;

[0025] Figure 3 Shows the comparison chart of the reference solution control map and the fingerprint of Allium macrostemon Bunge medicinal materials; and

[0026] Figure 4 Shows the fingerprints of 16 batches of Allium macrostemon Bunge medicinal materials provided by the present invention, where the sample numbers are shown in Table 1 and R is the reference fingerprint. Detailed Embodiments

[0027] The following listed embodiments are to enable those skilled in the art to better understand the present invention. It should be noted that the following embodiments do not limit the scope of protection required by the present invention and are only illustrative embodiments. The raw materials, reagents or devices mentioned in the following embodiments can be obtained from commercial channels or by known existing methods without special instructions. The origin information of the Allium macrostemon Bunge medicinal materials used in the present invention is shown in Table 1.

[0028] Table 1: Origin information of 16 batches of Allium macrostemon Bunge medicinal materials

[0029]

[0030]

[0031] Example 1

[0032] Method for constructing the fingerprint of Allium macrostemon Bunge medicinal materials.

[0033] This embodiment provides a method for constructing a fingerprint of Allium macrostemon Bunge medicinal materials, including: preparing the Allium macrostemon Bunge medicinal materials into a test solution; preparing Macrostemonoside I, 25S-Timosaponin B II, and 25R-Timosaponin B II into a reference substance solution; injecting the test solution and the reference substance solution into a liquid chromatography-mass spectrometry (LC-MS) instrument respectively, and obtaining the fingerprint of Allium macrostemon Bunge medicinal materials according to the LC-MS method. The following provides a specific construction method.

[0034] 1. Preparation of the test solution

[0035] Precisely weigh 50 mg of Allium macrostemon Bunge medicinal material powder (passed through No. 3 sieve) (error less than 0.05 mg), add 1 mL of 50%-95% ethanol, soak for 60 min and then weigh, ultrasonically extract for 60 min (40 kHz, 30 °C), cool and weigh again, make up the weight loss with 50%-95% ethanol, shake well, transfer the supernatant, and obtain the test solution for the LC-MS fingerprint. Among them, ethanol is preferably 75%.

[0036] 2. Preparation of the reference substance solution

[0037] Precisely weigh 1 mg each of Macrostemonoside I, 25S-Timosaponin B II, and 25R-Timosaponin B II, add a methanol solution with a volume concentration of 50% to prepare a reference substance stock solution of 1 mg / mL, and then take an appropriate amount of the reference substance stock solution and mix it to prepare a mixed reference substance solution with a concentration of 25 μg / mL.

[0038] 3. Optimization of the LC-MS method conditions

[0039] Take a preset amount of the reference substance solution and the test solution, inject them into the LC-MS instrument, use octadecylsilane chemically bonded silica as the filler, use a formic acid aqueous solution with a volume concentration of 0.1% as mobile phase A, and a formic acid acetonitrile solution with a volume concentration of 0.1% as mobile phase B for gradient elution, with a flow rate of 0.3 mL / min, and construct the LC-MS fingerprint of Allium macrostemon Bunge medicinal materials.

[0040] The high-resolution mass spectrometry detection conditions are: detect in the negative ion mode; the ion source is ESI; the backflush nitrogen temperature is 325 °C, the backflush nitrogen flow rate is 11.0 L / min, the nebulizing gas flow rate is 45 psig, the auxiliary nitrogen temperature is 350 °C, the auxiliary nitrogen flow rate is 11.0 L / min, the capillary voltage is 3500 V, the in-source collision voltage is 175 V, the cone voltage is 65.0 V; the scanning mode: full scan; the scanning range: m / z 60 - 1700.

[0041] 3.1 Optimization of the LC elution method

[0042] Based on the previous research on the components of Allium macrostemon Bunge, under the above liquid chromatography-mass spectrometry conditions, the steroidal saponin components mainly elute in the linear range where mobile phase B linearly increases from 15% to 40% (using an aqueous solution of formic acid with a volume concentration of 0.1% as mobile phase A and an acetonitrile solution of formic acid with a volume concentration of 0.1% as mobile phase B). Based on the above experimental results, the gradient elution method -1 for the LC-MS fingerprint was optimized, and the elution gradient is shown in Table 2.

[0043] Table 2: Gradient elution table for LC-MS fingerprint

[0044]

[0045] Based on the experiment of the LC-MS fingerprint gradient elution method -1, the results show that 11 compound peaks need to be concerned, among which there are 3 pairs of isomers expected to achieve baseline separation, namely: peak 6 and peak 7, peak 8 and peak 9, peak 10 and peak 11. The chromatogram of the test solution of Allium macrostemon Bunge under elution method -1 is shown in Figure 1 . Therefore, based on the LC-MS fingerprint gradient elution method -1, the LC-MS fingerprint gradient elution method -2 ( Figure 2 ) was optimized, and the elution gradient is shown in Table 3.

[0046] Table 3: Gradient elution table for LC-MS fingerprint

[0047]

[0048] Based on the experiment of the LC-MS fingerprint gradient elution method -2, the results show that the retention times of peaks 1 - 5 are significantly shifted forward as a whole, the peak shapes are complete, and the separation effect is good, which is beneficial to shortening the detection time; under the isocratic elution condition where mobile phase B maintains 23% within 4.5 min - 12.5 min, the 3 pairs of isomeric compound peaks can all achieve baseline separation, which is beneficial to the establishment of the subsequent quantitative method for steroidal saponins. The chromatogram of the test solution of Allium macrostemon Bunge under elution method -2 is shown in Figure 2 .

[0049] 3.2 Determination of liquid chromatography conditions

[0050] Using a CORTECS UPLC T3 Column column (2.1×100 mm, 1.6 μm) as the elution column, an aqueous solution of formic acid with a volume concentration of 0.1% as mobile phase A, an acetonitrile solution of formic acid with a volume concentration of 0.1% as mobile phase B, the flow rate is 0.3 mL / min, the column temperature is 30 °C, and the gradient elution is carried out according to the following procedure.

[0051] 0 - 0.5 min, mobile phase A from 85% → 85%, mobile phase B from 15% → 15%;

[0052] 0.5 - 3.8 min, mobile phase A from 85% → 81%, mobile phase B from 15% → 19%;

[0053] 3.8 - 4.5 min, mobile phase A from 81% → 77%, mobile phase B from 19% → 23%;

[0054] 4.5 - 12.5 min, mobile phase A from 77% → 77%, mobile phase B from 23% → 23%;

[0055] 12.5 - 14 min, mobile phase A from 77% → 73%, mobile phase B from 23% → 27%.

[0056] 4. Chemical Constituent Identification of LC-MS Fingerprint

[0057] Using LC-MS method, the reference solution and test solution were detected. By comparing the retention time of chromatographic peaks of compounds, accurate molecular weight and the fragmentation rules of secondary spectra, a total of 3 compounds corresponding to chromatographic peaks were determined, namely Macrostemonoside I, 25S-Timosaponin B II, and 25R-Timosaponin B II. Using LC-MS method, by comparing with the structures reported in the literature, retention time and combining the fragmentation rules of secondary spectra, a total of 8 compounds corresponding to chromatographic peaks were identified, namely Macrostemonoside G or isomers, Elephanoside E or isomers, 25R-Macrostemonoside I or isomers, (25R)-12α-OH-timosaponin B II or isomers, 5(6)-ene-25S-Macrostemonoside B or isomers, 5(6)-ene-25R-Macrostemonoside B or isomers, 25S-Macrostemonoside B or isomers, 25R-Macrostemonoside B or isomers.

[0058] The fingerprint of Allium macrostemon Bunge medicinal materials contains 11 characteristic peaks, and the numbers of the characteristic peaks are Peak 1 - Peak 11; Peak 3 is Macrostemonoside I, Peak 6 is 25S-Timosaponin B II, and Peak 7 is 25R-Timosaponin B II, as shown in Table 4 for details.

[0059] Table 4: LC-MS Characteristic Peaks and Identification of Characteristic Peaks

[0060]

[0061] Taking peak 6 as the S peak, the relative retention time ranges of peaks 1-5 and peaks 7-11 relative to the S peak are respectively: 0.332-0.338, 0.362-0.367, 0.562-0.569, 0.588-0.593, 0.606-0.611, 1.036-1.039, 1.177-1.187, 1.216-1.229, 1.272-1.284, 1.319-1.335.

[0062] Taking peak 6 as the S peak, the RSD values of the relative retention times of peaks 1-5 and peaks 7-11 relative to the S peak are respectively: 0.46%, 0.42%, 0.32%, 0.25%, 0.22%, 0.08%, 0.24%, 0.32%, 0.27%, 0.33%.

[0063] Figure 3 The comparison diagrams of the reference substance solution control chromatogram and the fingerprint chromatogram of Allium macrostemon Bunge are shown.

[0064] 5. Fingerprint similarity evaluation

[0065] Import the LC-MS fingerprint chromatogram txt format of the test solution of 16 batches of Allium macrostemon Bunge into the software of "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version), calculate the similarity of each fingerprint chromatogram, and the results are shown in Table 5.

[0066] The results show that taking the 25S-Timosaponin BⅡ chromatographic peak (peak 6) as the reference peak S peak, generating a control chromatogram by the average method, establishing a control fingerprint chromatogram of Allium macrostemon Bunge, obtaining the superimposed chromatogram of the fingerprint chromatogram of Allium macrostemon Bunge, calculating the similarity, and the similarities of the fingerprint chromatograms of 16 batches of Allium macrostemon Bunge are between 0.985 and 0.999, indicating that this fingerprint chromatogram can better evaluate the quality of Allium macrostemon Bunge.

[0067] Figure 4 This is the fingerprint chromatogram of 16 batches of Allium macrostemon Bunge provided by the present invention; for the sample numbers, please refer to Table 1, and R is the control fingerprint chromatogram.

[0068] Table 5: Similarities of LC-MS fingerprint chromatograms of 16 batches of Allium macrostemon Bunge

[0069]

[0070] Example 2

[0071] This embodiment provides a method for determining the content of the index components of Allium macrostemon Bunge medicinal materials, including: preparing a test sample solution from the test Allium macrostemon Bunge medicinal materials; preparing Macrostemonoside I, 25S-Timosaponin B II, and 25R-Timosaponin B II into a reference substance stock solution, and mixing the reference substance stock solutions to obtain a reference substance solution; injecting the test sample solution and the reference substance solution into a liquid chromatography-mass spectrometry instrument respectively, and obtaining the test spectrum of the test Allium macrostemon Bunge medicinal materials and the reference spectrum of the reference substance solution according to the liquid chromatography-mass spectrometry method determined in Example 2; calculating the contents of the index components Macrostemonoside I, 25S-Timosaponin B II, and 25R-Timosaponin B II in the test Allium macrostemon Bunge medicinal materials based on the concentration of the reference substance solution, the peak area of the reference substance solution in the reference spectrum, and the peak area of the components corresponding to the test Allium macrostemon Bunge medicinal materials and the reference substance solution in the test spectrum. The following provides a specific determination method.

[0072] 1. Selection of experimental conditions for the determination method

[0073] The UHPLC-Q-TOF-MS conditions are as follows: the chromatographic column is a CORTECS UPLC T3 Column column (2.1×100 mm, 1.6 μm), the flow rate is 0.3 mL / min, and the column temperature is 30 °C. The eluent is an aqueous solution of formic acid with a volume concentration of 0.1% (mobile phase A) and an acetonitrile solution of formic acid with a volume concentration of 0.1% (mobile phase B). The injection volume is 1 μL, and the gradient elution is carried out according to the following procedure.

[0074] 0 - 0.5 min, mobile phase A from 85% → 85%, mobile phase B from 15% → 15%;

[0075] 0.5 - 3.8 min, mobile phase A from 85% → 81%, mobile phase B from 15% → 19%;

[0076] 3.8 - 4.5 min, mobile phase A from 81% → 77%, mobile phase B from 19% → 23%;

[0077] 4.5 - 12.5 min, mobile phase A from 77% → 77%, mobile phase B from 23% → 23%;

[0078] 12.5 - 14 min, mobile phase A from 77% → 73%, mobile phase B from 23% → 27%.

[0079] Mass spectrometry detection was performed in the ESI negative ion mode, and the ion source was ESI; the temperature of the backflush nitrogen was 325 °C, the flow rate of the backflush nitrogen was 11.0 L / min, the flow rate of the nebulizing gas was 45 psig, the temperature of the auxiliary nitrogen was 350 °C, the flow rate of the auxiliary nitrogen was 11.0 L / min, the capillary voltage was 3500 V, the in-source collision voltage was 175 V, and the cone voltage was 65.0 V; scanning mode: full scan; scanning range: m / z 60 - 1700.

[0080] 2. Methodological verification

[0081] The items of methodological verification include detection limit, quantitation limit, linear range, precision, accuracy, recovery rate, matrix effect and stability.

[0082] 2.1 Detection limit, quantitation limit, linear range

[0083] Preparation of the standard solution: Weigh accurately 1 mg each of Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II, add methanol solution with a volume concentration of 50% to prepare a reference stock solution of 1 mg / mL, and then take an appropriate amount of the reference stock solution and mix them to prepare a mixed reference solution with a concentration of 25 μg / mL.

[0084] Dilute the mixed reference solution to a concentration of 0.05 - 25 μg / mL to prepare a series of working solutions. Dilute the stock solution to three concentrations of low (1 μg / mL), medium (5 μg / mL) and high (10 μg / mL) as the quality control samples of the mixed reference. The standard curve graph takes the concentration as the abscissa and the peak area as the ordinate, performs linear regression, and obtains the standard curve: The standard curve of Macrostemonoside I is y = 191254.78x - 6525.86 (r 2 = 0.9997, without weighting), the linear range is 0.25 - 25 μg / mL, the detection limit is 0.05 μg / mL (S / N ≥ 3), and the quantitation limit is 0.25 μg / mL (S / N ≥ 9); the standard curve of 25S-Timosaponin B II is y = 356910.66x - 18259.39 (r 2 = 0.9996, with a weighting of 1 / x), the linear range is 0.25 - 25 μg / mL, the detection limit is 0.05 μg / mL (S / N ≥ 3), and the quantitation limit is 0.25 μg / mL (S / N ≥ 9); the standard curve of 25R-Timosaponin B II is y = 480774.43x - 17621.70 (r 2= 0.9995, with a weight of 1 / x), the linear range is 0.25 - 25 μg / mL, the detection limit is 0.05 μg / mL (S / N ≥ 3), and the quantitation limit is 0.25 μg / mL (S / N ≥ 9).

[0085] 2.2 Precision, accuracy, recovery rate, matrix effect, stability

[0086] The low, medium, and high concentration quality control samples were each detected 3 times on the same day, and the relative standard deviation (RSD) values of the peak areas of the 3 analytes to be detected were calculated to obtain the within-day precision; the low, medium, and high concentration quality control samples were injected and detected 3 times every day for three consecutive days to obtain the between-day precision. Accuracy was evaluated by the percentage ratio of the detected concentration to the actual concentration of the quality control sample. The experimental results showed that the within-day and between-day precisions of the low, medium, and high concentration quality control samples were within ±15%, and the within-day and between-day accuracies were between 85% - 115%, meeting the requirements.

[0087] The recovery experiment and matrix effect experiment were carried out by adding low, medium, and high concentration quality control samples to the samples. The recovery rate and matrix effect were obtained according to the following formula. The results showed that the recovery rates and matrix effects of the low, medium, and high concentration quality control samples were all between 85% - 115%, meeting the requirements.

[0088]

[0089] Stability was calculated from the peak areas of the same low, medium, and high concentration quality control samples injected within 24 hours at 4°C. The results showed that the stability RSD of the low, medium, and high concentration quality control samples was within ±15%, meeting the requirements.

[0090] The above results (Tables 6 and 7) indicate that the established quantitative method can stably and accurately quantify the steroidal saponin compounds in the Allium macrostemon Bunge samples.

[0091] Table 6: Experimental results of precision and accuracy of steroidal saponin reference standards

[0092]

[0093]

[0094] Table 7: Experimental results of stability, recovery rate, and matrix effect of steroidal saponin reference standards

[0095]

[0096] 2.3 Determination of the contents of the index components in 16 batches of Allium macrostemon Bunge samples

[0097] Precisely weigh 50 mg of the powder of 16 batches of Allium macrostemon Bunge medicinal materials respectively, prepare the test solution according to the method in Example 2, inject and detect according to the UHPLC-Q-TOF-MS conditions in this example, record the peak area, and calculate that the contents of Macrostemonoside I, 25S-Timosaponin B II, and 25R-Timosaponin B II in the Allium macrostemon Bunge samples from different origins are 0.012% ± 0.003%, 0.011% ± 0.003%, and 0.034% ± 0.006% respectively. The specific determination results are shown in Table 8.

[0098] Table 8: Quantitative results of steroidal saponins in 16 batches of Allium macrostemon Bunge

[0099]

[0100]

[0101] * The content calculation formula is: the content in the Allium macrostemon Bunge sample = m / M × 100% (where m is the content of the compound to be measured detected in the sample through linear detection, and M is the initial mass of the Allium macrostemon Bunge powder)

[0102] In summary, the present invention provides a method for constructing the fingerprint of Allium macrostemon Bunge medicinal materials and determining the content of index components. This method makes up for the problem of poor specificity of the previous fingerprint of Allium macrostemon Bunge medicinal materials, optimizes the liquid-phase elution method, effectively separates the compound peaks of steroidal saponin active components, and on this basis, establishes an LC-MS fingerprint. The similarity of 16 batches of Allium macrostemon Bunge samples is between 0.985 and 0.999, indicating that this fingerprint can better evaluate the quality of Allium macrostemon Bunge medicinal materials. Based on this liquid-phase elution method, a UHPLC-Q-TOF-MS quantitative detection method is established to determine the contents of the main steroidal saponin components (Macrostemonoside I, 25S-Timosaponin B II, and 25R-Timosaponin B II) in Allium macrostemon Bunge. The results show that the contents of Macrostemonoside I, 25S-Timosaponin B II, and 25R-Timosaponin B II in the Allium macrostemon Bunge samples from different origins are 0.012% ± 0.003%, 0.011% ± 0.003%, and 0.034% ± 0.006% respectively. This method improves the evaluation standard of Allium macrostemon Bunge and provides a reference for the overall and comprehensive quality control of Allium macrostemon Bunge.

[0103] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing a fingerprint of Allium macrostemon Bunge medicinal materials, characterized in that, Comprising: Preparing the Allium macrostemon Bunge medicinal material into a test solution; Injecting the test solution into a liquid chromatography-mass spectrometry (LC-MS) instrument, and obtaining the fingerprint of the Allium macrostemon Bunge medicinal material according to the LC-MS method.

2. The construction method according to claim 1, characterized in that, In the fingerprint of the Allium macrostemon Bunge medicinal material, 11 characteristic peaks are included, and the numbers of the characteristic peaks are Peak 1 to Peak 11; The identification of the characteristic peaks and the characteristic peaks is as follows:

3. The construction method according to claim 2, characterized in that, Taking Peak 6 as the S peak, the relative retention time ranges of Peak 1 to Peak 5 and Peak 7 to Peak 11 relative to the S peak are respectively: 0.332～0.338、0.362～0.367、0.562～0.569、0.588～0.593、0.606～0.611、1.036～1.039、1.177～1.187、1.216～1.229、1.272～1.284、1.319～1.335。 4. The construction method according to claim 3, characterized in that, Taking Peak 6 as the S peak, the relative standard deviation (RSD) values of the relative retention times of Peak 1 to Peak 5 and Peak 7 to Peak 11 relative to the S peak are respectively: 0.46％、0.42％、0.32％、0.25％、0.22％、0.08％、0.24％、0.32％、0.27％、0.33％。 5. The construction method according to claim 1, characterized in that, The chromatographic conditions of the LC-MS method include: Stationary phase: a chromatographic column filled with octadecylsilyl-bonded silica gel; Mobile phase: Mobile phase A is water containing 0.1% (v / v) formic acid, and mobile phase B is an acetonitrile solution containing 0.1% (v / v) formic acid; Gradient elution is adopted, and the flow rate is 0.3 mL / min.

6. The construction method according to claim 5, characterized in that, The chromatographic column is a CORTECS UPLC T3 Column (2.1×100 mm, 1.6 μm) chromatographic column.

7. The construction method according to claim 5, characterized in that, The gradient elution is carried out according to the following procedure: 0 - 0.5 min: Mobile phase A changes from 85% → 85%, and mobile phase B changes from 15% → 15%; 0.5 - 3.8 min: Mobile phase A changes from 85% → 81%, and mobile phase B changes from 15% → 19%; 3.8 - 4.5 min: Mobile phase A changes from 81% → 77%, and mobile phase B changes from 19% → 23%; 4.5 - 12.5 min: Mobile phase A changes from 77% → 77%, and mobile phase B changes from 23% → 23%; 12.5 - 14.0 min: Mobile phase A changes from 77% → 73%, and mobile phase B changes from 23% → 27%.

8. The construction method according to claim 1, characterized in that, The mass spectrometry conditions of the LC-MS method include: Detecting in the negative ion mode; Ion source: ESI; Backflush nitrogen temperature 325 °C, backflush nitrogen flow rate 11.0 L / min, nebulizing gas flow rate 45 psig, auxiliary nitrogen temperature 350 °C, auxiliary nitrogen flow rate 11.0 L / min, capillary voltage 3500 V, in-source collision voltage 175 V, cone voltage 65.0 V; Scanning mode: full scan; Scanning range: m / z 60 - 1700.

9. The construction method according to claim 1, characterized in that, The preparation method of the test solution includes: Extracting, reweighing, and filtering the Allium macrostemon Bunge medicinal material with a first solvent to obtain the test solution.

10. A method for determining the content of index components in Allium macrostemon Bunge medicinal materials, characterized in that,Including: Preparing the test Allium macrostemon Bunge medicinal material into a test sample solution; Preparing Macrostemonoside I, 25S-Timosaponin B II, and 25R-Timosaponin B II into a reference solution; Injecting the test sample solution and the reference solution into a liquid chromatography-mass spectrometry (LC-MS) instrument respectively, and obtaining the test spectrum of the test Allium macrostemon Bunge medicinal material and the reference spectrum of the reference solution according to the LC-MS method determined in any one of Claims 1 to 10. According to the concentration of the reference solution, the peak area of ​​the reference solution in the reference spectrum, and the peak areas of the components corresponding to the reference solution in the tested spectrum of the Xiebai medicinal material to be tested, the contents of the indicative components Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II in the Xiebai medicinal material to be tested are calculated.

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