CN116148395A - Method for constructing allium macrostemon medicinal material fingerprint and measuring allium macrostemon index component content - Google Patents
Method for constructing allium macrostemon medicinal material fingerprint and measuring allium macrostemon index component content Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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Abstract
The invention relates to a method for constructing a fingerprint spectrum of allium macrostemon medicinal materials and measuring the content of allium macrostemon index components, which comprises the following steps: preparing Bulbus Allii Macrostemi medicinal material into sample solution; macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II are taken to prepare reference substance solutions; and respectively injecting the sample solution and the reference substance solution into a liquid chromatography-mass spectrometer, obtaining the fingerprint of the allium macrostemon medicinal material according to the liquid chromatography-mass spectrometer, and measuring the content of the index components. By the method for constructing the fingerprint spectrum and the content measurement of the allium macrostemon medicinal materials, the evaluation standard of the allium macrostemon is comprehensively perfected, and the clinical use effectiveness of the allium macrostemon medicinal materials is ensured.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine quality analysis and detection, and particularly relates to a method for constructing a fingerprint of allium macrostemon medicinal materials and measuring the content of allium macrostemon index components.
Background
The Allium macrostemon is recorded in Shennong Ben Cao Jing for the first time, is a dry bulb of Allium macrostemon Bge or Allium macrostemon G.don of the lily family plant, has the effects of activating yang, resolving masses, promoting qi circulation and removing stagnation, and can be used for treating chest stuffiness and pain, epigastric fullness and distending pain, diarrhea and diarrhea. Modern pharmacological researches suggest that allium macrostemon has the effects of inhibiting platelet aggregation, reducing blood lipid, resisting atherosclerosis and the like, wherein steroid saponin is a main active ingredient of the allium macrostemon. For example, 25S-Timosaponin B II and 25R-Timosaponin B II inhibit Adenosine Diphosphate (ADP) -induced platelet aggregation in vitro, while 25S-Timosaponin B II also has good inhibitory effect on Arachidonic Acid (AA) -induced platelet aggregation.
Allium macrostemon is mainly produced in Shaanxi and northeast, and is distributed in most areas of China. The consistency evaluation of the allium macrostemon amount is required to be carried out by adopting an effective technical means so as to ensure the safety, effectiveness and controllable quality of allium macrostemon medicinal materials.
However, control standards for consistency evaluation of allium macrostemon quantities of different places of production have not been established nowadays. The research on the allium macrostemon fingerprint is only focused on compatibility with medicinal materials such as snakegourd fruit, immature bitter orange and cassia twig at present, and the index peaks are mostly nucleoside components, so that the specificity is poor, the evaluation of the allium macrostemon active components is difficult to embody, and the standard for measuring the content of the allium macrostemon index components is also lacking in Chinese pharmacopoeia of 2020 edition.
Therefore, development of a method for constructing the fingerprint spectrum and content measurement of allium macrostemon medicinal materials is urgently needed at present so as to be applied to quality evaluation of allium macrostemon medicinal materials.
Disclosure of Invention
The invention aims to solve at least one of the technical problems existing in the prior art to a certain extent, and therefore, the invention provides a method for constructing the fingerprint spectrum of the allium macrostemon medicinal material and measuring the content of the index components of the allium macrostemon medicinal material.
According to one aspect of the invention, a method for constructing a fingerprint of allium macrostemon medicinal material is provided, which comprises the following steps: preparing Bulbus Allii Macrostemi medicinal material into sample solution; macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II are taken to prepare reference substance solutions; and respectively injecting the sample solution and the reference substance solution into a liquid chromatography-mass spectrometer, and obtaining the fingerprint of the allium macrostemon medicinal material according to the liquid chromatography-mass spectrometer.
Preferably, the fingerprint of the allium macrostemon medicinal material comprises 11 characteristic peaks, and the serial numbers of the characteristic peaks are peak 1-peak 11; the characteristic peaks in the LC-MS fingerprint are identified as follows, wherein the peak 3 is Macrostemonoside I, the peak 6 is 25S-Timosaponin B II, and the peak 7 is 25R-Timosaponin B II:
preferably, the relative retention time ranges of the peak 1 to the peak 5 and the peak 7 to the peak 11 with respect to the S peak are respectively as follows: 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, 1.319 to 1.335.
Preferably, the relative retention time RSD values for peak 1 to peak 5 and peak 7 to peak 11 with respect 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%.
Preferably, the chromatographic conditions of the liquid chromatography-mass spectrometry include: stationary phase: chromatographic column with octadecylsilane chemically bonded silica as filler; mobile phase: mobile phase A is formic acid water with 0.1% volume concentration, mobile phase B is formic acid acetonitrile solution with 0.1% volume concentration; gradient elution was used at a flow rate of 0.3mL/min.
Preferably, the chromatographic Column is a CORTECS UPLC T3 Column (2.1X100 mm,1.6 μm) chromatographic Column.
Preferably, the gradient elution is performed according to the following procedure: 0 to 0.5min: mobile phase a from 85% to 85%, mobile phase B from 15% to 15%;0.5 to 3.8min: mobile phase a from 85% to 81% and mobile phase B from 15% to 19%;3.8 to 4.5 minutes: mobile phase a from 81% to 77% and mobile phase B from 19% to 23%;4.5 to 12.5 minutes: mobile phase a from 77% to 77% and mobile phase B from 23% to 23%;12.5 to 14.0min: mobile phase a from 77% to 73% and mobile phase B from 23% to 27%.
Preferably, the mass spectrometry conditions of the liquid chromatography-mass spectrometry comprise: detecting in a negative ion mode; the ion source is ESI; the temperature of back-blowing nitrogen is 325 ℃, the flow rate of back-blowing nitrogen is 11.0L/min, the flow rate of atomization gas is 45psig, the temperature of auxiliary nitrogen is 350 ℃, the flow rate of auxiliary nitrogen is 11.0L/min, the capillary voltage is 3500V, the collision voltage in a source is 175V, and the taper hole voltage is 65.0V; scanning mode: full scanning; scanning range: m/z is 60-1700.
Preferably, the preparation method of the sample solution comprises the following steps: extracting, supplementing weight and filtering the allium macrostemon medicinal material by adopting a first solvent to obtain the sample solution.
Preferably, the preparation method of the sample solution comprises the following steps: taking 50mg of allium macrostemon medicinal material; 1ml of the first solvent is added, and the first weighing is carried out after soaking for 60 minutes; ultrasonic extracting at 40kHz and 30deg.C for 60 min; after cooling, weighing for the second time, and continuing to add the first solvent to complement the weightlessness; filtering to obtain the sample solution, wherein the first solvent is 50% -95% ethanol.
According to another aspect of the invention, a method for measuring the content of index components of allium macrostemon medicinal materials is provided, which comprises the following steps: preparing a to-be-detected allium macrostemon medicinal material into a to-be-detected sample solution; macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II are taken to prepare reference substance solutions; respectively injecting the sample solution to be detected and the reference substance solution into a liquid chromatography-mass spectrometer, and obtaining a detected spectrum of the allium macrostemon medicinal material to be detected and a reference spectrum of the reference substance solution according to the determined liquid chromatography-mass spectrometer; calculating the contents of index components Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II in the allium macrostemon medicinal material to be tested according to the concentration of the reference solution, the peak area of the reference solution in the reference map and the peak area of the components corresponding to the reference solution in the reference map.
Preferably, the concentration of the control solutionThe following relationship is satisfied between the degree and the peak area of the control solution in the control profile: the first regression equation of Macrostemonoside I is y= 191254.78x-6525.86 (r 2 =0.9997, no weight), linear range 0.25-25 μg/mL, detection limit 0.05 μg/mL (S/N. Gtoreq.3), quantitative limit 0.25 μg/mL (S/N. Gtoreq.9); the second regression equation of the 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), quantitative limit 0.25 μg/mL (S/N > 9); the third regression equation for 25R-Timosaponin B II is y= 480774.43x-17621.70 (R 2 =0.9995, weight 1/x), linear range 0.25-25 μg/mL, detection limit 0.05 μg/mL (S/N. Gtoreq.3), quantitative limit 0.25 μg/mL (S/N. Gtoreq.9); and 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 map.
Compared with the prior art, the invention has the following beneficial effects: the invention provides a method for constructing a fingerprint spectrum of allium macrostemon medicinal materials and measuring the content of index components, which realizes effective separation of steroid saponin active components by optimizing a liquid phase elution method; the accurate molecular weight, molecular composition and fragment information of the compound obtained by utilizing the liquid chromatography-mass spectrometry technology are utilized to realize identification and authentication of chromatographic peaks in the fingerprint; and a quantitative analysis method of main steroid saponin components in the allium macrostemon is established, the invention stably and comprehensively perfects the evaluation standard of allium macrostemon medicinal materials, ensures the effectiveness of clinical use of medicinal materials, and provides a reference for integrally controlling the quality of allium macrostemon medicinal materials.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation on the invention. In the drawings:
FIG. 1 shows a chromatogram of a test solution of allium macrostemon under elution method-1;
FIG. 2 shows a chromatogram of a test solution of allium macrostemon under elution method-2;
FIG. 3 shows a comparison of the reference solution reference pattern and the allium macrostemon drug fingerprint pattern; and
fig. 4 shows fingerprint patterns of 16 batches of allium macrostemon medicinal materials provided by the invention, wherein the sample numbers are shown in table 1, and R is a control fingerprint pattern.
Detailed Description
The examples set forth below are presented to provide those skilled in the art with a more clear understanding of the present invention. The following examples are not intended to limit the scope of the invention, but are merely illustrative examples. The starting materials, reagents or apparatus mentioned in the examples below are all available commercially, or are obtained by known existing means, unless otherwise specified. The information of the producing places of allium macrostemon medicinal materials adopted in the invention is shown in table 1.
Table 1:16 batches of allium macrostemon medicinal material producing area information
Example 1
A method for constructing fingerprint of Bulbus Allii Macrostemi.
The embodiment provides a method for constructing a fingerprint of allium macrostemon medicinal material, which comprises the following steps: preparing Bulbus Allii Macrostemi medicinal material into sample solution; macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II are taken to prepare reference substance solutions; and respectively injecting the sample solution and the reference substance solution into a liquid chromatography-mass spectrometer, and obtaining the fingerprint of the allium macrostemon medicinal material according to the liquid chromatography-mass spectrometer. Specific construction methods are provided below.
1. Preparation of test solutions
Accurately weighing 50mg of allium macrostemon medicinal material powder (sieving with a third sieve) (error is less than 0.05 mg), adding 1mL of 50% -95% ethanol, soaking for 60min, weighing, ultrasonic extracting for 60min (40 kHz,30 ℃), cooling, weighing again, supplementing weight loss with 50% -95% ethanol, shaking, and transferring supernatant to obtain LC-MS fingerprint sample solution, wherein ethanol is preferably 75%.
2. Preparation of control solution
Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II are precisely weighed 1mg each, 50% by volume of methanol solution is added to prepare 1mg/mL of reference stock solution, and then a proper amount of reference stock solution is taken and mixed to prepare a mixed reference solution with the concentration of 25 mug/mL.
3. Condition optimization for liquid chromatography-mass spectrometry
Taking a preset amount of reference substance solution and test substance solution, injecting into an LC-MS liquid chromatography-mass spectrometer, taking octadecylsilane chemically bonded silica as a filler, taking formic acid aqueous solution with 0.1% volume concentration as a mobile phase A, taking formic acid acetonitrile solution with 0.1% volume concentration as a mobile phase B, performing gradient elution, and constructing an LC-MS fingerprint of the allium macrostemon medicinal material at the flow rate of 0.3mL/min.
The high-resolution mass spectrum detection conditions are as follows: detecting in a negative ion mode; the ion source is ESI; the temperature of back-blowing nitrogen is 325 ℃, the flow rate of back-blowing nitrogen is 11.0L/min, the flow rate of atomization gas is 45psig, the temperature of auxiliary nitrogen is 350 ℃, the flow rate of auxiliary nitrogen is 11.0L/min, the capillary voltage is 3500V, the collision voltage in a source is 175V, and the taper hole voltage is 65.0V; scanning mode: full scanning; scanning range: m/z is 60-1700.
3.1 optimization of liquid chromatography elution methods
Based on the earlier study of the allium macrostemon component, under the condition of the liquid chromatography-mass spectrometry, the steroid saponin component mainly shows a peak in the linear range of the linear increase of the mobile phase B from 15% to 40% (taking an aqueous solution containing 0.1% by volume of formic acid as a mobile phase A and an acetonitrile solution containing 0.1% by volume of formic acid as a mobile phase B). Based on the experimental results, an LC-MS fingerprint gradient elution method-1 is optimized, and the elution gradient is shown in Table 2.
Table 2: LC-MS fingerprint gradient elution table
Based on the LC-MS fingerprint gradient elution method-1 experiment, the results show that 11 compound peaks need to be focused, wherein baseline separation is expected for 3 pairs of isomers, respectively: peak 6 and peak 7, peak 8 and peak 9, peak 10 and peak 11. The elution method-1 is shown in figure 1. Therefore, the LC-MS fingerprint gradient elution method-2 (figure 2) is optimized on the basis of the LC-MS fingerprint gradient elution method-1, and the elution gradient is shown in table 3.
Table 3: LC-MS fingerprint gradient elution table
The result of the LC-MS fingerprint gradient elution method-2 experiment shows that the retention time of the peaks 1 to 5 is obviously advanced, the peak shape is complete, the separation effect is good, and the detection time is shortened; under the condition that the mobile phase B maintains 23% of isocratic elution within 4.5-12.5 min, the peak-to-average energy baseline separation of 3 pairs of isomer compounds is beneficial to the establishment of a subsequent steroid saponin quantitative method, and the chromatogram of the allium macrostemon sample solution under the elution method-2 is shown in figure 2.
3.2 determination of liquid chromatography conditions
The gradient elution was carried out using a CORTECS UPLC T3 Column (2.1X100 mm,1.6 μm) as the elution Column, an aqueous formic acid solution containing 0.1% by volume as mobile phase A, an acetonitrile formic acid solution containing 0.1% by volume as mobile phase B, a flow rate of 0.3mL/min, a Column temperature of 30℃and the like.
0 to 0.5min, the mobile phase A is from 85 percent to 85 percent, and the mobile phase B is from 15 percent to 15 percent;
0.5-3.8 min, mobile phase A from 85% to 81%, mobile phase B from 15% to 19%;
3.8-4.5 min, the mobile phase A is from 81% to 77%, and the mobile phase B is from 19% to 23%;
4.5-12.5 min, the mobile phase A is from 77% to 77%, and the mobile phase B is from 23% to 23%;
12.5-14 min, the mobile phase A is from 77% to 73%, and the mobile phase B is from 23% to 27%.
Identification of chemical Components of LC-MS finger print
The LC-MS method is adopted to detect the reference substance solution and the sample solution, and the compounds corresponding to 3 chromatographic peaks are determined by comparing the retention time of the chromatographic peaks, the accurate molecular weight and the secondary spectrum splitting law of the compounds, namely Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II. By adopting an LC-MS method, a total of 8 chromatographic peak corresponding compounds are identified by comparing a reported structure with a reported literature and a retention time and combining a secondary spectrum cleavage rule, wherein the corresponding compounds are Macrostemonoside G or an isomer, elethanoside E or an isomer, 25R-Macrostemonoside I or an isomer, (25R) -12 alpha-OH-timosaponin B II or an isomer, 5 (6) -ene-25S-Macrostemonoside B or an isomer, 5 (6) -ene-25R-Macrostemonoside B or an isomer, 25S-Macrostemonoside B or an isomer, and 25R-Macrostemonoside B or an isomer respectively.
The fingerprint of the allium macrostemon medicinal material comprises 11 characteristic peaks, and the serial numbers of the characteristic peaks are peak 1-peak 11; peak 3 is Macrostemonoside I, peak 6 is 25S-Timosaponin B II, peak 7 is 25R-Timosaponin B II, see Table 4 for details.
Table 4: identification of LC-MS characteristic peaks and characteristic peaks
The relative retention time ranges of peak 6 as an S peak, peak 1 to peak 5 and peak 7 to peak 11 relative to the S peak are respectively: 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, 1.319 to 1.335.
The relative retention time RSD values for peak 1 to peak 5 and peak 7 to peak 11 with respect to the S peak were respectively: 0.46%, 0.42%, 0.32%, 0.25%, 0.22%, 0.08%, 0.24%, 0.32%, 0.27%, 0.33%.
Fig. 3 shows a comparison of the reference solution reference pattern and the allium macrostemon medicinal material fingerprint pattern.
5. Fingerprint similarity evaluation
The LC-MS fingerprint txt format of 16 batches of allium macrostemon medicinal material sample solution is imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) software, and the similarity of each fingerprint is calculated, and the result is shown in Table 5.
The result shows that the 25S-Timosaponin BII chromatographic peak (peak 6) is taken as a reference peak S-peak, a reference spectrum is generated according to an average method, the allium macrostemon medicinal material reference fingerprint spectrum is established, the allium macrostemon medicinal material fingerprint spectrum superposition spectrum is obtained, the similarity is calculated, and the 16 parts of allium macrostemon medicinal material fingerprint spectrum similarity is between 0.985 and 0.999, so that the quality of allium macrostemon medicinal material can be better evaluated by the fingerprint spectrum.
FIG. 4 is a fingerprint of 16 batches of allium macrostemon medicinal materials provided by the invention; wherein the sample numbers can be seen in table 1, and r is a control fingerprint.
Table 5: LC-MS fingerprint similarity of 16 batches of allium macrostemon medicinal materials
Example two
The embodiment provides a method for measuring the content of index components of allium macrostemon medicinal materials, which comprises the following steps: preparing a to-be-detected allium macrostemon medicinal material into a to-be-detected sample solution; macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II are taken to prepare a reference substance stock solution, and the reference substance stock solution is mixed to obtain a reference substance solution; respectively injecting a sample solution to be detected and a reference substance solution into a liquid chromatography-mass spectrometer, and obtaining a to-be-detected spectrum of the allium macrostemon medicinal material to be detected and a reference spectrum of the reference substance solution according to the liquid chromatography-mass spectrometer method determined in the second embodiment; calculating the contents of index components Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II in the allium macrostemon medicinal material to be detected 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 allium macrostemon medicinal material to be detected and the reference substance solution in the spectrogram to be detected. Specific measurement methods are provided below.
1. Selection of experimental conditions for assay methods
UHPLC-Q-TOF-MS conditions were: the Column was a CORTECS UPLC T3 Column (2.1X100 mm,1.6 μm) with a flow rate of 0.3mL/min and a Column temperature of 30 ℃. The eluent is an aqueous formic acid solution (mobile phase A) containing 0.1% by volume and an acetonitrile formic acid solution (mobile phase B) containing 0.1% by volume. The sample loading was 1. Mu.L, and the gradient elution was performed according to the following procedure.
0 to 0.5min, the mobile phase A is from 85 percent to 85 percent, and the mobile phase B is from 15 percent to 15 percent;
0.5-3.8 min, mobile phase A from 85% to 81%, mobile phase B from 15% to 19%;
3.8-4.5 min, the mobile phase A is from 81% to 77%, and the mobile phase B is from 19% to 23%;
4.5-12.5 min, the mobile phase A is from 77% to 77%, and the mobile phase B is from 23% to 23%;
12.5-14 min, the mobile phase A is from 77% to 73%, and the mobile phase B is from 23% to 27%.
The mass spectrum detection adopts an ESI negative ion mode, and an ion source is ESI; the temperature of back-blowing nitrogen is 325 ℃, the flow rate of back-blowing nitrogen is 11.0L/min, the flow rate of atomization gas is 45psig, the temperature of auxiliary nitrogen is 350 ℃, the flow rate of auxiliary nitrogen is 11.0L/min, the capillary voltage is 3500V, the collision voltage in a source is 175V, and the taper hole voltage is 65.0V; scanning mode: full scanning; scanning range: m/z is 60-1700.
2. Methodological verification
Methodological validation projects include detection limits, quantification limits, linear ranges, precision, accuracy, recovery, matrix effects, and stability.
2.1 detection limit, quantitative limit, linear Range
Preparing a standard solution: macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II are precisely weighed 1mg each, 50% by volume of methanol solution is added to prepare 1mg/mL of reference stock solution, and then a proper amount of reference stock solution is taken and mixed to prepare a mixed reference solution with the concentration of 25 mug/mL.
And diluting the mixed reference substance solution to the concentration of 0.05-25 mug/mL to prepare a series of working solutions. Stock solutions were diluted to three concentrations of low (1. Mu.g/mL), medium (5. Mu.g/mL) and high (10. Mu.g/mL) as quality control samples for the mixed control. The standard curve graph is subjected to linear regression by taking the concentration as an abscissa and the peak area as an ordinate, so as to obtain the standard curve: the standard curve for Macrostemonoside I is y= 191254.78x-6525.86 (r 2 =0.9997, no weight), linear range 0.25-25 μg/mL, detection limit 0.05 μg/mL (S/N. Gtoreq.3), quantitative limit 0.25 μg/mL (S/N. Gtoreq.9); the standard curve for 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), quantitative limit 0.25 μg/mL (S/N > 9); the standard curve for 25R-Timosaponin B II is y= 480774.43x-17621.70 (R 2 =0.9995, weight 1/x), linear range 0.25-25 μg/mL, detection limit 0.05 μg/mL (S/N. Gtoreq.3), quantitative limit 0.25 μg/mL (S/N. Gtoreq.9).
2.2 precision, accuracy, recovery, matrix Effect, stability
Detecting low, medium and high concentration quality control samples 3 times a day respectively, and calculating Relative Standard Deviation (RSD) values of peak areas of 3 substances to be detected to obtain the daily precision; the quality control samples with low, medium and high concentrations are continuously sampled and detected for 3 times daily for three days to obtain the daytime precision. Accuracy is assessed by detecting the percentage ratio of the concentration to the actual concentration of the quality control sample. Experimental results show that the precision of the quality control samples with low, medium and high concentrations in the measurement day and the precision of the quality control samples in the measurement day are within +/-15%, and the accuracy of the quality control samples in the measurement day and the quality control samples in the measurement day are between 85% and 115%, and meet the requirements.
The recovery rate experiment and the matrix effect experiment are carried out by adding low, medium and high concentration quality control samples into the samples, the recovery rate and the matrix effect are obtained according to the following formulas, and the result shows that the recovery rate and the matrix effect measured by the low, medium and high concentration quality control samples are between 85% and 115%, and meet the requirements.
Stability is calculated by the peak area of sample injection detection of the same low, medium and high concentration quality control samples at 4 ℃ for 24 hours. The result shows that the stability RSD of the quality control samples with the low, medium and high concentrations is within +/-15 percent, which meets the requirements.
The results (tables 6 and 7) show that the established quantitative method can stably and accurately quantify the steroid saponin compounds in the allium macrostemon medicinal material sample.
Table 6: precision and accuracy experimental result of steroid saponin standard
Table 7: stability, recovery rate and matrix effect experimental result of steroid saponin standard substance
2.3 Measurement of index component content of 16 batches of allium macrostemon medicinal materials
50mg of each of 16 batches of allium macrostemon medicinal material powder is precisely weighed, a sample solution is prepared according to the method in the second embodiment, sample injection detection is carried out according to UHPLC-Q-TOF-MS conditions in the second embodiment, peak areas are recorded, the Macrostemonoside I content in allium macrostemon samples in different producing areas is calculated to be 0.012+/-0.003%, the 25S-Timosaponin B II content is 0.011+/-0.003%, the 25R-Timosaponin B II content is 0.034+/-0.006%, and specific measurement results are shown in Table 8.
Table 8: quantitative results of steroid saponins in 16 Xiebai
* The content calculation formula is: content in allium macrostemon sample = M/M x 100% (where M is the content of the compound to be tested in the sample detected by linearity, M is the initial mass of allium macrostemon powder)
In summary, the invention provides a method for constructing the fingerprint of the allium macrostemon medicinal material and measuring the content of index components, which solves the problem of poor specificity of the fingerprint of the allium macrostemon medicinal material before, optimizes a liquid phase elution method, effectively separates steroid saponin active ingredient compound peaks, establishes an LC-MS fingerprint on the basis, and has the similarity of 16 batches of allium macrostemon samples of 0.985-0.999, thus indicating that the fingerprint can evaluate the quality of the allium macrostemon medicinal material better. Based on the liquid phase elution method, a UHPLC-Q-TOF-MS quantitative detection method is established, and content determination is carried out on main steroid saponin components (Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II) in allium macrostemon, so that the result shows that the content of Macrostemonoside I in allium macrostemon samples of different production places is 0.012% +/-0.003%, the content of 25S-Timosaponin B II is 0.011% +/-0.003% and the content of 25R-Timosaponin B II is 0.034% +/-0.006%. The method improves evaluation standard of Bulbus Allii Macrostemi, and provides reference for overall quality control of Bulbus Allii Macrostemi.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The method for constructing the allium macrostemon medicinal material fingerprint is characterized by comprising the following steps of:
preparing Bulbus Allii Macrostemi medicinal material into sample solution;
preparing a control solution from macromonosideI, 25S-Timosaponin B II and 25R-Timosaponin B II;
and respectively injecting the sample solution and the reference substance solution into a liquid chromatography-mass spectrometer, and obtaining the fingerprint of the allium macrostemon medicinal material according to the liquid chromatography-mass spectrometer.
2. The construction method according to claim 1, wherein the fingerprint of the allium macrostemon medicinal material comprises 11 characteristic peaks, and the characteristic peaks are numbered from peak 1 to peak 11;
the characteristic peaks and the identification of the characteristic peaks are as follows:
3. the construction method according to claim 2, wherein the relative retention time ranges of the peak 6 as an S peak, the peak 1 to the peak 5, and the peak 7 to the peak 11 with respect 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, wherein the relative retention time RSD values of the S peak with respect to the peaks 1 to 5 and the peak 7 to 11 are respectively:
0.46％、0.42％、0.32％、0.25％、0.22％、0.08％、0.24％、0.32％、0.27％、0.33％。
5. the method of claim 1, wherein the chromatographic conditions of the liquid chromatography-mass spectrometry include:
stationary phase: chromatographic column with octadecylsilane chemically bonded silica as filler;
mobile phase: mobile phase A is formic acid water with 0.1% volume concentration, mobile phase B is formic acid acetonitrile solution with 0.1% volume concentration;
gradient elution was used at a flow rate of 0.3mL/min.
6. The method according to claim 5, wherein the chromatographic Column is a CORTECS UPLC T3 Column (2.1X100 mm,1.6 μm).
7. The method of claim 5, wherein the gradient elution is performed according to the following procedure:
0 to 0.5min: mobile phase a from 85% to 85%, mobile phase B from 15% to 15%;
0.5 to 3.8min: mobile phase a from 85% to 81% and mobile phase B from 15% to 19%;
3.8 to 4.5 minutes: mobile phase a from 81% to 77% and mobile phase B from 19% to 23%;
4.5 to 12.5 minutes: mobile phase a from 77% to 77% and mobile phase B from 23% to 23%;
12.5 to 14.0min: mobile phase a from 77% to 73% and mobile phase B from 23% to 27%.
8. The method of claim 1, wherein the mass spectrometry conditions of the liquid chromatography-mass spectrometry comprise:
detecting in a negative ion mode;
the ion source is ESI;
the temperature of back-blowing nitrogen is 325 ℃, the flow rate of back-blowing nitrogen is 11.0L/min, the flow rate of atomization gas is 45psig, the temperature of auxiliary nitrogen is 350 ℃, the flow rate of auxiliary nitrogen is 11.0L/min, the capillary voltage is 3500V, the collision voltage in a source is 175V, and the taper hole voltage is 65.0V;
scanning mode: full scanning;
scanning range: m/z is 60-1700.
9. The method of claim 1, wherein the method of preparing the test solution comprises:
extracting, supplementing weight and filtering the allium macrostemon medicinal material by adopting a first solvent to obtain the sample solution.
10. A method for measuring index component content of allium macrostemon medicinal material is characterized by comprising the following steps:
preparing a to-be-detected allium macrostemon medicinal material into a to-be-detected sample solution;
preparing a control solution from macromonosideI, 25S-Timosaponin B II and 25R-Timosaponin B II;
injecting the sample solution to be tested and the reference solution into a liquid chromatography-mass spectrometer respectively, and obtaining a test pattern of the allium macrostemon medicinal material to be tested and a reference pattern of the reference solution according to the liquid chromatography-mass spectrometer method determined by any one of claims 1 to 10;
calculating the contents of index components Macrostemonoside I, 25S-Timosaponin B II and 25R-Timosaponin B II in the allium macrostemon medicinal material to be tested according to the concentration of the reference solution, the peak area of the reference solution in the reference map and the peak area of the components corresponding to the reference solution in the reference map.
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