A method for constructing a medicinal material fingerprint of Xiebai and determining the content of index components of Xiebai

Abstract

The present invention relates to a method for constructing a Xiebai medicinal material fingerprint and determining the content of Xiebai index components, including: taking Xiebai medicinal material to prepare a test solution; taking Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II to prepare a reference solution; injecting the test solution and the reference solution into a liquid chromatography-mass spectrometer respectively, and obtaining the fingerprint of the Xiebai medicinal material according to the liquid chromatography-mass spectrometry method, and performing content determination on the above-mentioned index components. By the method for constructing Xiebai medicinal material fingerprint and content determination provided by the present invention, the evaluation standard of Xiebai is fully improved, and the effectiveness of clinical use of Xiebai medicinal material is guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of quality analysis and detection of traditional Chinese medicines, and particularly relates to a method for constructing a Xiebai medicinal material fingerprint and determining the content of an index component of Xiebai. Background Art

[0002] Xiebai was first recorded in Shennong's Herbal Classic. It is the dried bulb of Allium macrostemon Bge. or Allium chinense G.Don, a plant of the Liliaceae family. It has the effects of promoting yang and dispersing stagnation, promoting qi and removing stagnation, and can be used to treat chest pain, abdominal distension and pain, and heaviness after diarrhea. Modern pharmacological studies believe that Xiebai has the effects of inhibiting platelet aggregation, lowering blood lipids, and resisting atherosclerosis, among which steroidal saponins are its main active ingredients. For example, 25S-Timosaponin B II and 25R-Timosaponin B II can inhibit adenosine diphosphate (ADP)-induced platelet aggregation in vitro, and 25S-Timosaponin B II also has a good inhibitory effect on arachidonic acid (AA)-induced platelet aggregation.

[0003] Xiebai is mainly produced in Shaanxi, Northeast China and other places, and is distributed in most parts of the country. It is necessary to use effective technical means to evaluate the consistency of Xiebai quality to ensure the safety, effectiveness and quality control of Xiebai medicinal materials.

[0004] However, there is no control standard for the consistency evaluation of the quality of Xiebai from different origins. The research on the fingerprint of Xiebai is currently focused on its compatibility with other medicinal materials such as Trichosanthes, Citrus aurantium, and Cinnamomum cassia. The peaks identified are mostly nucleosides with poor specificity, which makes it difficult to reflect the evaluation of the active ingredients of Xiebai. The 2020 edition of the Chinese Pharmacopoeia also lacks standards for the determination of the content of the index ingredients of Xiebai.

[0005] Therefore, there is an urgent need to develop a method for constructing the fingerprint of Xiebai medicinal materials and determining its content so as to apply it to the quality evaluation of Xiebai medicinal materials. Summary of the invention

[0006] The present invention aims to solve one of the technical problems existing in the prior art to at least a certain extent. To this end, the present invention provides a method for constructing a fingerprint of Xiebai medicinal material and determining the content of index components of Xiebai medicinal material.

[0007] According to one aspect of the present invention, a method for constructing a fingerprint of Xiebai medicinal material is provided, comprising: preparing a test solution from Xiebai medicinal material; preparing a reference solution from Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II; respectively injecting the test solution and the reference solution into a liquid chromatography-mass spectrometer, and obtaining a fingerprint of the Xiebai medicinal material according to a liquid chromatography-mass spectrometry method.

[0008] Preferably, the fingerprint of the Xiebai medicinal material contains 11 characteristic peaks, which are numbered from 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 in the LC-MS fingerprint and the identification of the characteristic peaks are as follows:

[0009]

[0010] Preferably, peak 6 is taken as the S peak, and the relative retention time ranges of peaks 1 to 5 and peaks 7 to 11 relative to the S peak are: 0.332 to 0.338, 0.362 to 0.367, 0.562 to 0.569, 0.588 to 0.593, 0.606 to 0.611, 1.036 to 1.039, 1.177 to 1.187, 1.216 to 1.229, 1.272 to 1.284, and 1.319 to 1.335, respectively.

[0011] Preferably, peak 6 is taken as the S peak, and the relative retention time RSD values ​​of peaks 1 to 5 and peaks 7 to 11 relative to the S peak are 0.46%, 0.42%, 0.32%, 0.25%, 0.22%, 0.08%, 0.24%, 0.32%, 0.27% and 0.33%, respectively.

[0012] Preferably, the chromatographic conditions of the liquid chromatography-mass spectrometry method include: stationary phase: a chromatographic column with octadecylsilane bonded silica gel as a filler; mobile phase: mobile phase A is water containing 0.1% volume concentration of formic acid, and mobile phase B is acetonitrile solution containing 0.1% volume concentration of formic acid; gradient elution is used with a flow rate of 0.3 mL / min.

[0013] Preferably, the chromatographic column is a CORTECS UPLC T3 Column (2.1×100 mm, 1.6 μm) chromatographic column.

[0014] 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%.

[0015] Preferably, the mass spectrometry conditions of the liquid chromatography-mass spectrometry method include: detection in negative ion mode; ion source is ESI; backflush nitrogen temperature is 325°C, backflush nitrogen flow rate is 11.0L / min, nebulizer gas flow rate is 45psig, auxiliary nitrogen temperature is 350°C, auxiliary nitrogen flow rate is 11.0L / min, capillary voltage is 3500V, source collision voltage is 175V, cone voltage is 65.0V; scan mode: full scan; scan range: m / z 60~1700.

[0016] Preferably, the method for preparing the test solution comprises: extracting, weighting, and filtering the Xiebai medicinal material using a first solvent to obtain the test solution.

[0017] Preferably, the preparation method of the test solution includes: taking 50 mg of the Xiebai medicinal material; adding 1 ml of the first solvent, soaking for 60 minutes and then weighing for the first time; ultrasonic extraction at 40 kHz and 30° C. for 60 minutes; weighing for the second time after cooling, and continuing to add the first solvent to make up for the weight loss; filtering to obtain the test solution, wherein the first solvent is 50%-95% ethanol.

[0018] According to another aspect of the present invention, a method for determining the content of indicative components of Xiebai medicinal material is provided, comprising: preparing a sample solution to be tested from the Xiebai medicinal material to be tested; preparing a reference solution from Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II; respectively injecting the sample solution to be tested and the reference solution into a liquid chromatography-mass spectrometer, and obtaining a test spectrum of the Xiebai medicinal material to be tested and a reference spectrum of the reference solution according to the determined liquid chromatography-mass spectrometry method; calculating 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 according to the concentration of the reference solution, the peak area of ​​the reference solution in the reference spectrum, and the peak area of ​​the components corresponding to the Xiebai medicinal material to be tested and the reference solution in the test spectrum.

[0019] Preferably, the concentration of the reference solution and the peak area of ​​the reference solution in the reference spectrum satisfy the following relationship: The first regression equation of Macrostemonoside I is y=191254.78x-6525.86(r 2 =0.9997, no weight), 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, weight 1 / x), linear range 0.25-25 μg / mL, detection limit 0.05 μg / mL (S / N≥3), quantification limit 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, weight is 1 / x), the linear range is 0.25-25μg / mL, the detection limit is 0.05μg / mL (S / N≥3), and the quantification limit is 0.25μg / 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 solution, and the ordinate is the peak area in the reference spectrum.

[0020] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a method for constructing a fingerprint of Xiebai medicinal material and determining the content of index components, the method realizes effective separation of steroidal saponin active components by optimizing the liquid phase elution method; the precise molecular weight, molecular composition and fragment information of the compound obtained by liquid chromatography-mass spectrometry are used to realize the identification and appraisal of the chromatographic peaks in the fingerprint; and a quantitative analysis method for the main steroidal saponin components in Xiebai is established. The present invention stably and comprehensively improves the evaluation standard of Xiebai medicinal material, ensures the effectiveness of clinical use of medicinal materials, and provides a reference for overall control of the quality of Xiebai medicinal material. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0022] Figure 1 The chromatogram of the Xiebai test solution under elution method-1 is shown;

[0023] Figure 2 The chromatogram of the Xiebai test solution under elution method-2 is shown;

[0024] Figure 3 A comparison diagram of the reference solution reference spectrum and the Xiebai medicinal material fingerprint spectrum is shown; and

[0025] Figure 4 The fingerprints of 16 batches of Xiebai medicinal materials provided by the present invention are shown, wherein the sample numbers are shown in Table 1, and R is the control fingerprint. DETAILED DESCRIPTION

[0026] The following examples are provided to allow those skilled in the art to more clearly understand the present invention. It should be noted that the following examples do not limit the scope of protection claimed in the present invention and are only illustrative examples. The raw materials, reagents or devices mentioned in the following examples, unless otherwise specified, can be obtained from commercial sources or by known existing methods. The origin information of the Radix Scutellariae used in the present invention is shown in Table 1.

[0027] Table 1: Origin information of 16 batches of Xiebai medicinal materials

[0028]

[0029]

[0030] Embodiment 1

[0031] Method for constructing fingerprint of Xiebai medicinal material.

[0032] This embodiment provides a method for constructing a fingerprint of Xiebai medicinal material, comprising: preparing a test solution from Xiebai medicinal material; preparing a reference solution from Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II; respectively injecting the test solution and the reference solution into a liquid chromatography-mass spectrometer, and obtaining a fingerprint of Xiebai medicinal material according to a liquid chromatography-mass spectrometry method. The specific construction method is provided below.

[0033] 1. Preparation of test solution

[0034] Accurately weigh 50 mg of Xiebai medicinal material powder (passed through No. 3 sieve) (error is less than 0.05 mg), add 1 mL of 50%-95% ethanol, soak for 60 minutes and then weigh, ultrasonically extract for 60 minutes (40kHz, 30°C), weigh again after cooling, make up for the weight loss with 50%-95% ethanol, shake well, transfer the supernatant, and obtain the LC-MS fingerprint sample solution, wherein the ethanol is preferably 75%.

[0035] 2. Preparation of reference solution

[0036] Accurately weigh 1 mg each of Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II, add 50% volume concentration methanol solution to prepare 1 mg / mL reference substance stock solution, and then take appropriate amount of reference substance stock solution and mix them to prepare mixed reference substance solutions with a concentration of 25 μg / mL.

[0037] 3. Optimization of HPLC-MS Conditions

[0038] Take a preset amount of reference solution and test solution, inject them into the LC-MS liquid spectrometer, use octadecylsilane bonded silica gel as filler, 0.1% volume concentration of formic acid aqueous solution as mobile phase A, 0.1% volume concentration of formic acid acetonitrile solution as mobile phase B for gradient elution, the flow rate is 0.3mL / min, and the LC-MS fingerprint of Xiebai medicinal material is constructed.

[0039] High-resolution mass spectrometry detection conditions are as follows: detection in negative ion mode; ion source is ESI; backflush nitrogen temperature is 325°C, backflush nitrogen flow rate is 11.0 L / min, nebulizer gas flow rate is 45 psig, auxiliary nitrogen temperature is 350°C, auxiliary nitrogen flow rate is 11.0 L / min, capillary voltage is 3500 V, source collision voltage is 175 V, cone voltage is 65.0 V; scan mode: full scan; scan range: m / z 60-1700.

[0040] 3.1 Optimization of LC elution method

[0041] Based on the previous study on the components of Xiebai, under the above liquid chromatography-mass spectrometry conditions, the steroidal saponin components mainly peaked in the linear range of mobile phase B from 15% to 40% (with a 0.1% volume concentration of formic acid aqueous solution as mobile phase A and a 0.1% volume concentration of formic acid acetonitrile solution as mobile phase B). Based on the above experimental results, the LC-MS fingerprint gradient elution method-1 was optimized, and the elution gradient is shown in Table 2.

[0042] Table 2: LC-MS fingerprint gradient elution table

[0043]

[0044] Based on the LC-MS fingerprint gradient elution method-1 experiment, the results show that there are 11 compound peaks that need to be paid attention to, among which there are 3 pairs of isomers that are expected to be baseline separated, namely: peak 6 and peak 7, peak 8 and peak 9, peak 10 and peak 11. The chromatogram of the Xiebai test solution 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 was optimized ( Figure 2 ), the elution gradient is shown in Table 3.

[0045] Table 3: LC-MS fingerprint gradient elution table

[0046]

[0047]

[0048] Based on the LC-MS fingerprint gradient elution method-2 experiment, the results showed that the retention time of peaks 1 to 5 was significantly shifted forward as a whole, the peak shape was complete, the separation effect was good, and it was beneficial to shorten the detection time; under the condition of 23% isocratic elution of mobile phase B within 4.5min to 12.5min, the three pairs of isomeric compound peaks could be baseline separated, which was beneficial to the establishment of the subsequent steroidal saponin quantitative method. The chromatogram of the Xiebai test sample solution under elution method-2 is shown in Figure 2 .

[0049] 3.2 Determination of liquid chromatography conditions

[0050] A CORTECS UPLC T3 Column (2.1×100 mm, 1.6 μm) was used as the elution column, an aqueous solution containing 0.1% by volume formic acid was used as the mobile phase A, an acetonitrile solution containing 0.1% by volume formic acid was used as the mobile phase B, the flow rate was 0.3 mL / min, the column temperature was 30° C., and gradient elution was performed according to the following procedure.

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

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

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

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

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

[0056] 4. Chemical component identification by LC-MS fingerprint

[0057] The reference solution and the test solution were detected by LC-MS. By comparing the chromatographic peak retention time, accurate molecular weight and secondary spectrum fragmentation rules of the compounds, a total of three chromatographic peaks corresponding to the compounds were determined, namely Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II. By using the LC-MS method, a total of 8 compounds corresponding to the chromatographic peaks were identified by comparing the structures and retention times reported in the literature and combining with the fragmentation rules of the secondary spectrum. They are 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, and 25R-Macrostemonoside B or isomers.

[0058] The fingerprint of Xiebai medicinal material contains 11 characteristic peaks, which are numbered from 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, as shown in Table 4 for details.

[0059] Table 4: LC-MS characteristic peaks and their identification

[0060]

[0061] Taking peak 6 as the S peak, the relative retention time ranges of peaks 1 to 5 and peaks 7 to 11 relative to the S peak are 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, and 1.319-1.335, respectively.

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

[0063] Figure 3 A comparison chart of the reference solution reference spectrum and the Xiebai medicinal material fingerprint spectrum is shown.

[0064] 5. Fingerprint similarity evaluation

[0065] The LC-MS fingerprints of 16 batches of Xiebai medicinal material solution were imported into the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System” (2012 version) software in txt format to calculate the similarity of each fingerprint. The results are shown in Table 5.

[0066] The results showed that the 25S-Timosaponin BⅡ chromatographic peak (peak 6) was used as the reference peak S, and the reference spectrum was generated by the average method. The reference fingerprint of Xiebai medicinal material was established, and the superposition spectrum of the fingerprint spectrum of Xiebai medicinal material was obtained. The similarity was calculated, and the similarity of the 16 Xiebai medicinal material fingerprints was between 0.985 and 0.999, indicating that the fingerprint spectrum can be used to evaluate the quality of Xiebai medicinal material relatively well.

[0067] Figure 4 These are the fingerprints of 16 batches of Xiebai medicinal materials provided by the present invention; the sample numbers can be found in Table 1, and R is the control fingerprint.

[0068] Table 5: Similarity of LC-MS fingerprints of 16 batches of Xiebai medicinal materials

[0069]

[0070] Embodiment 2

[0071] The present embodiment provides a method for determining the content of an index component of a Xiebai medicinal material, including: taking a Xiebai medicinal material to be tested and preparing it as a sample solution to be tested; taking Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II to prepare a reference substance stock solution, mixing the reference substance stock solution to obtain a reference substance solution; injecting the sample solution to be tested and the reference substance solution into a liquid chromatography-mass spectrometer respectively, and obtaining a reference spectrum of the Xiebai medicinal material to be tested and a reference spectrum of the reference substance solution according to the liquid chromatography-mass spectrometry method determined in Example 2; 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 Xiebai medicinal material to be tested and the reference substance solution in the spectrum to be tested, calculate the content of the index components Macrostemonoside I, 25S-Timosaponin BII and 25R-Timosaponin B II in the Xiebai medicinal material to be tested. A specific determination method is provided below.

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

[0073] UHPLC-Q-TOF-MS conditions are as follows: the chromatographic column is CORTECS UPLC T3 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 a 0.1% volume concentration of formic acid in water (mobile phase A) and a 0.1% volume concentration of formic acid in acetonitrile (mobile phase B). The injection volume is 1 μL, and the gradient elution is performed according to the following procedure.

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

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

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

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

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

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

[0080] 2. Methodological Validation

[0081] Method validation items include detection limit, quantification limit, linear range, precision, accuracy, recovery, matrix effect and stability.

[0082] 2.1 Detection limit, quantification limit, and linear range

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

[0084] The mixed reference solution was diluted to a concentration of 0.05–25 μg / mL to prepare a series of working solutions. The stock solution was diluted to three concentrations of low (1 μg / mL), medium (5 μg / mL), and high (10 μg / mL) as quality control samples for the mixed reference. The standard curve was plotted with concentration as the horizontal axis and peak area as the vertical axis. Linear regression was performed to obtain the standard curve: The standard curve of Macrostemonoside I was y=191254.78x-6525.86(r 2 =0.9997, no weight), the linear range was 0.25-25 μg / mL, the detection limit was 0.05 μg / mL (S / N≥3), and the quantification limit was 0.25 μg / mL (S / N≥9); the standard curve of 25S-Timosaponin B II was y=356910.66x-18259.39 (r 2 =0.9996, weight 1 / x), linear range 0.25-25μg / mL, detection limit 0.05μg / mL (S / N≥3), quantification limit 0.25μg / mL (S / N≥9); the standard curve of 25R-Timosaponin B II is y=480774.43x-17621.70 (r 2=0.9995, weight 1 / x), the linear range was 0.25-25 μg / mL, the detection limit was 0.05 μg / mL (S / N≥3), and the quantification limit was 0.25 μg / mL (S / N≥9).

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

[0086] The low, medium and high concentration quality control samples were tested three times on the same day, and the relative standard deviation (RSD) values ​​of the peak areas of the three test substances were calculated to obtain the intra-day precision; the low, medium and high concentration quality control samples were injected and tested three times a day for three consecutive days to obtain the inter-day precision. The accuracy was evaluated by the percentage ratio of the test concentration to the actual concentration of the quality control sample. The experimental results showed that the intra-day and inter-day precision of the low, medium and high concentration quality control samples were within ±15%, and the intra-day and inter-day accuracy was between 85% and 115%, which met the requirements.

[0087] Recovery rate experiments and matrix effect experiments 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 rate and matrix effect of the low, medium and high concentration quality control samples were between 85% and 115%, which met the requirements.

[0088]

[0089]

[0090] The stability is calculated by the peak area of ​​the same low, medium and high concentration quality control samples placed at 4°C for 24 hours. The results show that the stability RSD of the three concentrations of quality control samples is within ±15%, which meets the requirements.

[0091] The above results (Tables 6 and 7) indicate that the established quantitative method can stably and accurately quantify steroidal saponin compounds in Xiebai medicinal material samples.

[0092] Table 6: Experimental results of precision and accuracy of steroidal saponin standards

[0093]

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

[0095]

[0096] 2.3 Determination of the content of index components in 16 batches of Xiebai medicinal materials

[0097] 16 batches of Xiebai medicinal material powder were accurately weighed, each with 50 mg, and the test solution was prepared according to the method in Example 2. The sample was injected and detected according to the UHPLC-Q-TOF-MS conditions in this example, and the peak area was recorded. The content of Macrostemonoside I in Xiebai samples from different origins was calculated to be 0.012% ± 0.003%, the content of 25S-Timosaponin B II was 0.011% ± 0.003%, and the content of 25R-Timosaponin B II was 0.034% ± 0.006%. The specific measurement results are shown in Table 8.

[0098] Table 8: Quantitative results of steroidal saponins in 16 batches of Xiebai

[0099]

[0100]

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

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

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. 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 a medicinal material of Radix Xiebai, characterized in that: include: Prepare the test solution from Xiebai medicinal material; Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II were prepared as reference solutions; The test solution and the reference solution are respectively injected into a liquid chromatography-mass spectrometer, and a fingerprint of the Xiebai medicinal material is obtained according to the liquid chromatography-mass spectrometry method; The chromatographic conditions of the liquid chromatography-mass spectrometry method include: Stationary phase: CORTECS UPLC T3 Column, 2.1×100 mm, 1.6 μm, elution column; Mobile phase: Mobile phase A is water containing 0.1% volume concentration of formic acid, and mobile phase B is acetonitrile solution containing 0.1% volume concentration of formic acid; Gradient elution was used with a flow rate of 0.3 mL / min and a column temperature of 30 °C; The gradient elution was performed according to the following procedure: 0~0.5min: mobile phase A from 85%→85%, mobile phase B from 15%→15%; 0.5~3.8min: mobile phase A from 85% to 81%, mobile phase B from 15% to 19%; 3.8~4.5min: Mobile phase A from 81% to 77%, mobile phase B from 19% to 23%; 4.5-12.5min: Mobile phase A from 77% to 77%, mobile phase B from 23% to 23%; 12.5-14.0min: mobile phase A from 77% to 73%, mobile phase B from 23% to 27%; The mass spectrometry conditions of the liquid chromatography-mass spectrometry method include: Detection in negative ion mode; The ion source is ESI; Backflush nitrogen temperature 325°C, backflush nitrogen flow 11.0 L / min, nebulizer gas flow 45 psig, auxiliary nitrogen temperature 350°C, auxiliary nitrogen flow 11.0 L / min, capillary voltage 3500 V, source collision voltage 175 V, cone voltage 65.0 V; Scan mode: full scan; Scan range: m / z 60~1700; The fingerprint of the Xiebai medicinal material contains 11 characteristic peaks, which are numbered from peak 1 to peak 11; The characteristic peaks and the identification of the characteristic peaks are as follows: 。 2. The construction method according to claim 1, characterized in that: Taking peak 6 as the S peak, the relative retention time ranges of peaks 1 to 5 and peaks 7 to 11 relative to the S peak are: 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。 3. The construction method according to claim 2, characterized in that: Taking peak 6 as the S peak, the relative retention time RSD values ​​of peaks 1 to 5 and peaks 7 to 11 relative to the S peak are: 0.46%、0.42%、0.32%、0.25%、0.22%、0.08%、0.24%、0.32%、0.27%、0.33%。 4. The construction method according to claim 1, characterized in that: The preparation method of the test solution comprises: The first solvent is used to extract, weigh and filter the Xiebai medicinal material to obtain the test solution.

5. A method for determining the content of indicative components of Xiebai medicinal material, characterized in that: include: Taking the Xiebai medicinal material to be tested to prepare a sample solution to be tested; Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II were prepared as reference solutions; Injecting the sample solution to be tested and the reference solution into a liquid chromatography-mass spectrometer respectively, and obtaining a test spectrum of the tested Xiebai medicinal material and a reference spectrum of the reference solution according to the liquid chromatography-mass spectrometry method determined according to any one of claims 1 to 4; 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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