CN114200040B - Content determination method for one-measurement-multiple-evaluation child type open throat sword spray - Google Patents

Content determination method for one-measurement-multiple-evaluation child type open throat sword spray Download PDF

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CN114200040B
CN114200040B CN202111415900.9A CN202111415900A CN114200040B CN 114200040 B CN114200040 B CN 114200040B CN 202111415900 A CN202111415900 A CN 202111415900A CN 114200040 B CN114200040 B CN 114200040B
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bergenin
matrine
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throat
pterocarpine
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CN114200040A (en
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张海
周英
王珏犇
俸婷婷
张丽艳
石慧
董秀
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Guizhou Sanli Pharmaceutical Co ltd
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Abstract

The invention discloses a content determination method of a throat-opening sword spray (children type), which adopts a high performance liquid chromatography to establish a content determination method for determining the content of matrine, bergenin, three-leaf bean pterocarpine and macadamia in the throat-opening sword spray (children type), and uses bergenin as an internal standard substance. The detection result is accurate and stable, and can be used for quality control of a throat opening sword spray (children type); meanwhile, the invention can reduce the detection cost and the detection time, reduce the workload, improve the efficiency and lay a foundation for improving the quality standard of the throat opening sword spray (children type).

Description

Content determination method for one-measurement-multiple-evaluation child type open throat sword spray
Technical Field
The invention relates to a detection method of a traditional Chinese medicine preparation, in particular to a content determination method of one-measurement-multiple-evaluation of a throat-opening sword spray (children type).
Background
The throat-opening sword spray (children type) is a large variety of single Miao medicine produced by Guizhou Sanli pharmaceutical Co., ltd, and its composition is composed of radix Ardisiae Crispae, radix Sophorae Tonkinensis, periostracum Cicadae and menthol, and has the functions of clearing heat, detoxicating, detumescence and relieving pain. Wherein, the Ardisia Crispa (Makino) which is loaded into national medicinal material quality standard (2003 edition) of Guizhou Chinese medicinal material comprises three basic sources of Ardisia Crispa, and the medicinal material used in the throat-opening sword spray formula is Ardisia Crispa. The throat-opening sword spray (children) has obvious curative effects on children acute pharyngitis, acute tonsillitis, herpetic stomatitis, herpetic angina, hand-foot-mouth disease, thrush and the like in clinic, and has high safety and no toxic or side effect. Currently, open throat sword sprays have little research on quality control. The components of the bergenin are researched and analyzed, and the bergenin is the main effective chemical component of the main medicine cinnabar root, and has good cough relieving, anti-inflammatory, antibacterial and antiviral effects. The radix Sophorae Tonkinensis mainly contains alkaloid, flavonoid, triterpenes, etc., wherein the alkaloid is the main component of radix Sophorae Tonkinensis, and the matrine can inhibit proliferation and induce apoptosis of pancreatic cancer cells in vivo and in vitro, and has antiinflammatory, antibacterial, liver protecting, and antiarrhythmic effects. The pterocarpus santalinus glycoside and the maca are significant in curative effect on inducing leukemia HL-60 apoptosis because of flavonoid components in the radix sophorae tonkinensis. In order to improve the quality control standard of the preparation, a multi-index content determination method taking the main medicinal components as indexes is established, meanwhile, in order to reduce the detection cost, save the detection time and improve the detection efficiency, the correction factors and the content of 3 components of matrine, three-leaf bean pterocarpine and macadamia are calculated by taking bergenin with high content and low price as internal standard references, and a one-measurement multi-evaluation content determination method of the open-throat sword spray (children) is established, thereby laying a foundation for improving the quality control standard of the open-throat sword spray.
Disclosure of Invention
In order to solve the technical problems, the invention provides a content measuring method for one-measurement-multiple-evaluation of a throat-opening sword spray (children type) and provides basis for improving quality standards of the throat-opening sword spray. In order to achieve the above object, the present invention is achieved by the following technical scheme.
A method for measuring the content of one-measurement-multiple-evaluation of a throat-opening sword spray (children type) specifically comprises the following steps:
(1) Preparing a reference substance solution;
(2) Preparing a sample solution;
(3) Establishing a high performance liquid chromatography for simultaneously measuring the contents of matrine, bergenin, pterocarpine and pterocarpine;
(4) Determining relative correction factors of matrine, pterocarpine and macadamia;
(5) According to the measured chromatogram of the sample solution, the bergenin content is calculated, and the matrine, the three-leaf bean pterocarpin and the marcaine content are respectively calculated according to the relative correction factors.
In the method for measuring the content of one test and multiple evaluations of the throat-opening sword spray (children), in the step (1), the reference substance solution is prepared: precisely weighing 20.26mg of matrine reference substance, 20.06mg of bergenin reference substance and 10.36mg of three-leaf bean pterocarpan reference substance, respectively placing into 10mL volumetric flask, placing 10.23mg of cocaine reference substance into 25mL volumetric flask, adding methanol, ultrasonically dissolving, and fixing volume to scale, shaking to obtain reference substance stock solution; and 3.5mL, 4mL, 1.0mL and 0.6mL of the corresponding reference substance solutions are respectively measured precisely, placed in the same 10mL volumetric flask and fixed in volume by methanol to prepare mixed reference substance solutions with the concentrations of 0.7091mg/mL, 0.8024mg/mL, 0.1036mg/mL and 0.02456mg/mL in sequence.
In the above-mentioned method for measuring the content of one test and multiple evaluations of the open throat sword spray (children type), in the step (2), the preparation method of the sample solution is as follows: precisely sucking 2.5mL of the throat-opening sword spray, placing in a 5mL volumetric flask, adding methanol to a fixed volume to a scale, shaking uniformly, and filtering with a 0.22 μm microporous filter membrane to obtain the final product.
In the method for measuring the content of one test and multiple evaluations of the open throat sword spray (children type), in the step (3), the chromatographic conditions of the high performance liquid chromatography are as follows: a Platisil-ODS column (250 mm. Times.4.6 mm,5 μm) was used; the mobile phase is methanol (A) -0.1% phosphoric acid aqueous solution (the pH is regulated to 5.0 by triethylamine, B), gradient elution is carried out (0-8 min,10% A; 8-19 min, 10-30% A; 19-28 min, 30-30% A; 28-32 min, 30-40% A; 32-40 min, 40-60% A; 40-70 min, 60-100% A); the volume flow is 0.8mL/min; column temperature is 35 ℃; detection wavelength 220nm (matrine), 275nm (bergenin), 300nm (Trifolium pratense pterocarpine glycoside); the sample injection amount was 10. Mu.L.
In the method for measuring the content of one or more tests of the open throat sword spray (children), in the step (4), bergenin in a sample is used as an internal standard(s), and relative correction factors (f) of 3 components of matrine (a), red sandalwood glycoside (b) and macadamia (c) are calculated s/i ) Taking f calculated by a plurality of quality points by adopting a multipoint correction method s/i Taking the average value as f for quantification s/i [f s/i ＝f s /f i ＝A s C i /A i C s Wherein A is s Peak area, C, of bergenin control s A is the mass concentration of bergenin reference substance i For the peak area of the component to be measured, C i For the mass concentration of the component to be measured]。
The invention has the beneficial effects that:
(1) The invention establishes a multi-index content determination method of a throat-opening sword spray (children), which is based on a high-performance liquid chromatography technology and combines a multi-evaluation method, and the method is accurate and reliable, is suitable for the content determination of multiple components in the throat-opening sword spray, has high detection sensitivity, good stability, simple and convenient operation, is easy to master, and is convenient to popularize further.
(2) The invention relates to a method for evaluating a throat-opening sword spray (children type) by a one-test-multiple-evaluation method, which uses bergenin as an internal reference, calculates relative correction factors of matrine, three-leaf bean pterocarpine and Ma Kayin in the throat-opening sword spray (children type), and calculates the contents of matrine, three-leaf bean pterocarpine and Ma Kayin by using the correction factors. The method can reduce the detection cost and detection time, reduce the workload, improve the efficiency, and has important significance for the quality control of the throat-opening sword spray (children) and the guarantee of clinical curative effect.
Drawings
FIG. 1 is a chromatogram of a bergenin test sample with a detection wavelength of 275nm from mobile phase methanol-0.1% phosphoric acid water;
FIG. 2 is a chromatogram of a sample of matrine with a detection wavelength of 220nm from mobile phase methanol-0.1% phosphoric acid water;
FIG. 3 is a chromatogram of a mobile phase methanol-0.1% phosphoric acid water, and a test sample of pterocarpin and Ma Kayin with a detection wavelength of 300nm;
FIG. 4 is a chromatogram of a bergenin test sample at a detection wavelength of 275nm with mobile phase acetonitrile-0.1% phosphoric acid water;
FIG. 5 chromatogram of a sample of matrine with a wavelength of 220nm under detection of acetonitrile-0.1% phosphoric acid water as mobile phase;
FIG. 6 chromatogram of mobile phase acetonitrile-0.1% phosphoric acid water, and detecting pterocarpan trilobate glycoside and Ma Kayin sample with wavelength of 300nm;
FIG. 7 is a chromatogram of a bergenin test sample at a detection wavelength of 275nm with mobile phase methanol-0.25% phosphoric acid water;
FIG. 8 is a chromatogram of a sample of matrine with a detection wavelength of 220nm with methanol-0.25% phosphoric acid water as mobile phase;
FIG. 9 is a chromatogram of a mobile phase methanol-0.25% phosphoric acid water, and a test sample of pterocarpin and Ma Kayin with a detection wavelength of 300nm;
FIG. 10 is a sample chromatogram of bergenin with a mobile phase acetonitrile-0.25% phosphoric acid water detection wavelength of 275 nm;
FIG. 11 chromatogram of a sample of matrine with a wavelength of 220nm with acetonitrile-0.25% phosphoric acid water as mobile phase;
FIG. 12 chromatogram of mobile phase acetonitrile-0.25% phosphoric acid water, and test sample containing pterocarpan side and Ma Kayin with wavelength of 300nm;
FIG. 13 is a chromatogram of a bergenin test sample at a detection wavelength of 275nm with mobile phase methanol-0.1% phosphoric acid-triethylamine water;
FIG. 14 chromatogram of a sample of matrine with a wavelength of 220nm with mobile phase methanol-0.1% phosphoric acid-triethylamine water;
FIG. 15 chromatogram of mobile phase methanol-0.1% phosphoric acid-triethylamine water, and detecting pterocarpan trilobate glycoside and Ma Kayin sample with wavelength of 300nm;
FIG. 16 elution method-1, detecting a bergenin test sample chromatogram at a wavelength of 275 nm;
FIG. 17 elution method-1, detecting a sample chromatogram of matrine at 220 nm;
FIG. 18 elution method-1, detecting the chromatograms of pterocarpin and Ma Kayin samples of three-leaf beans with the wavelength of 300nm;
FIG. 19 elution method-2, detecting a bergenin test sample chromatogram at a wavelength of 275 nm;
FIG. 20 elution method-2, detecting a sample chromatogram of matrine at 220 nm;
FIG. 21 elution method-2, detecting the chromatograms of the test sample of pterocarpin and Ma Kayin with 300nm wavelength;
FIG. 22 elution method-3, detecting a bergenin test sample chromatogram at a wavelength of 275 nm;
FIG. 23 elution method-3, detecting a sample chromatogram of matrine at 220 nm;
FIG. 24 elution method-3, detecting the chromatograms of the test sample of pterocarpin and Ma Kayin with 300nm wavelength;
FIG. 25 elution method-4, detecting a bergenin test sample chromatogram at a wavelength of 275 nm;
FIG. 26 elution method-4, detecting a sample chromatogram of matrine at 220 nm;
FIG. 27 elution method-4, detecting the chromatograms of pterocarpin and Ma Kayin samples of Trifolium pratense at 300nm;
FIG. 28 is a sample chromatogram of bergenin at a detection wavelength of 275nm at a flow rate of 0.8mL/min;
FIG. 29 is a sample chromatogram of bergenin at a detection wavelength of 275nm at a flow rate of 1.0 mL/min;
FIG. 30 is a sample chromatogram of bergenin at a detection wavelength of 275nm at a flow rate of 1.2 mL/min;
FIG. 31 is a chromatogram of a sample of matrine with a flow rate of 0.8mL/min and a detection wavelength of 220 nm;
FIG. 32 is a chromatogram of a sample of matrine with a detection wavelength of 220nm at a flow rate of 1.0 mL/min;
FIG. 33 is a chromatogram of a sample of matrine with a detection wavelength of 220nm at a flow rate of 1.2 mL/min;
FIG. 34 is a chromatogram of a sample of pterocarside and Ma Kayin with a flow rate of 0.8mL/min and a detection wavelength of 300nm;
FIG. 35 is a chromatogram of a sample of pterocarside and Ma Kayin with a flow rate of 1.0mL/min and a detection wavelength of 300nm;
FIG. 36 is a chromatogram of a sample of pterocarside and Ma Kayin with a flow rate of 1.2mL/min and a detection wavelength of 300nm;
FIG. 37 is a chromatogram of a bergenin test sample at a detection wavelength of 275nm at a column temperature of 25 ℃;
FIG. 38 is a chromatogram of a bergenin test sample at a column temperature of 30℃and a detection wavelength of 275 nm;
FIG. 39 is a chromatogram of a bergenin test sample at a column temperature of 35℃and a detection wavelength of 275 nm;
FIG. 40 is a chromatogram of matrine test sample with a detection wavelength of 220nm at a column temperature of 25deg.C;
FIG. 41 is a chromatogram of a matrine sample with a detection wavelength of 220nm at 30℃for the column temperature;
FIG. 42 is a chromatogram of matrine test sample at a column temperature of 35℃and a detection wavelength of 220 nm;
FIG. 43 is a chromatogram of a sample of pterocarpin and Ma Kayin with a detection wavelength of 300nm at a column temperature of 25 ℃;
FIG. 44 is a chromatogram of a sample of pterocarpin and Ma Kayin with a detection wavelength of 300nm at 30℃for a column temperature;
FIG. 45 is a chromatogram of a sample of pterocarpin and Ma Kayin with a column temperature of 35 ℃ and a detection wavelength of 300nm;
FIG. 46 shows a bergenin control chromatogram at a detection wavelength of 275 nm;
FIG. 47 shows a bergenin test sample chromatogram at a wavelength of 275 nm;
FIG. 48 is a chromatogram of a matrine control for detection at 220 nm;
FIG. 49 is a chromatogram of a test matrine sample with a wavelength of 220 nm;
FIG. 50 is a chromatogram of a control of pterocarpine, of three-leaf beans at 300nm;
FIG. 51 is a chromatogram of a test sample of pterocarpin, ma Kayin, detected at 300nm;
FIG. 52 is a chromatogram of a test sample negative for the detection of cinnabar deficiency at a wavelength of 220 nm;
FIG. 53 is a chromatogram of a test sample negative for cinnabar deficiency at a wavelength of 300nm;
FIG. 54 depicts a chromatogram of a negative test sample from Sophora hypogaea having a wavelength of 275 nm;
in order that those skilled in the art will better understand the technical solution of the present invention, the present invention will be further described with reference to specific examples, but the examples are not intended to limit the present invention.
Detailed Description
Example 1: one-test-multiple-evaluation method for simultaneously measuring 4 components in open throat sword spray
(1) Preparing a reference substance solution: precisely weighing 20.26mg of matrine reference substance, 20.06mg of bergenin reference substance and 10.36mg of three-leaf bean pterocarpan reference substance, respectively placing into 10mL volumetric flask, placing 10.23mg of cocaine reference substance into 25mL volumetric flask, adding methanol, ultrasonically dissolving, and fixing volume to scale, shaking to obtain reference substance stock solution; respectively measuring 3.5mL, 4mL, 1.0mL and 0.6mL of the corresponding reference substance solutions precisely, placing the solutions into the same 10mL volumetric flask, and fixing the volume by methanol to prepare mixed reference substance solutions with the concentrations of 0.7091mg/mL, 0.8024mg/mL, 0.1036mg/mL and 0.02456mg/mL in sequence;
(2) Preparation of test solution: precisely sucking 2.5mL of the throat-opening sword spray, placing in a 5mL volumetric flask, adding methanol to a fixed volume to a scale, shaking uniformly, and filtering with a 0.22 μm microporous filter membrane to obtain the final product;
(3) The chromatographic conditions of the high performance liquid chromatography are as follows: a Platisil-ODS column (250 mm. Times.4.6 mm,5 μm) was used; the mobile phase is methanol (A) -0.1% phosphoric acid aqueous solution (the pH is regulated to 5.0 by triethylamine, B), gradient elution is carried out (0-8 min,10% A; 8-19 min, 10-30% A; 19-28 min, 30-30% A; 28-32 min, 30-40% A; 32-40 min, 40-60% A; 40-70 min, 60-100% A); the volume flow is 0.8mL/min; column temperature is 35 ℃; detection wavelength 220nm (matrine), 275nm (bergenin), 300nm (Trifolium pratense pterocarpine glycoside); the sample injection amount is 10 mu L;
(4) Calculating matrine (a), trifolium pratense pterocarpine (b) and macadamia by taking bergenin in sample as internal standard(s)Due to the relative correction factor (f) of (c) 3 components s/i ) Taking f calculated by a plurality of quality points by adopting a multipoint correction method s/i Taking the average value as f for quantification s/i [f s/i ＝f s /f i ＝A s C i /A i C s Wherein A is s Peak area, C, of bergenin control s A is the mass concentration of bergenin reference substance i For the peak area of the component to be measured, C i For the mass concentration of the component to be measured]The method comprises the steps of carrying out a first treatment on the surface of the The results show that f between matrine, pterocarpin, ma Kayin and bergenin as internal reference s/i 1.6370, 1.8639, 1.1217, respectively.
(5) According to the measured chromatogram of the sample solution, the bergenin content is calculated, and the matrine, the three-leaf bean pterocarpin and the marcaine content are respectively calculated according to the relative correction factors.
The inventors conducted a large number of experiments, and the following are studies of the detection method of the present invention:
methodology study
1.1 materials
1.1.1 instruments
Agilent 1260 type high performance liquid chromatograph (Agilent inc., usa); model XS-205 analytical balance in ten thousandth (Mettler-Toledo company, switzerland).
1.1.2 Agents and medicaments
Matrine (lot number: PS011495, purity not less than 98.5%), bergenin (lot number: PS010520, purity not less than 98.0%), trifolin (lot number PS012410, purity not less than 98.0%), and macaine (lot number PS210802-06, purity not less than 98.0%) controls were all purchased from Chengdu Poisson biotechnology Co., ltd; methanol, phosphoric acid, and triethylamine are all chromatographic purities (Tianjin, miou chemical reagent Co., ltd.); purified water (drohent food and beverage limited, guangzhou); open throat sword spray (Guizhou Sanli pharmaceutical Co., ltd., lot numbers 20200706, 20200720, 20200730, 20200733, 20200736, 20200748, 20200804, 20200805, 20200812, 20200814, respectively).
1.2 analytical method optimization
1.2.1 selection of elution System
The experiment first examined 4 eluting solvent systems of methanol-0.1% phosphoric acid aqueous solution, acetonitrile-0.1% phosphoric acid aqueous solution, methanol-0.25% phosphoric acid aqueous solution, acetonitrile-0.25% phosphoric acid aqueous solution to determine the eluting solvent system suitable for the study. As shown in FIGS. 1 to 15, the concentration of the phosphoric acid solution was found to have no significant difference in separation effect, and methanol-0.1% phosphoric acid aqueous solution was selected as the mobile phase with the detection component being more and the chromatographic peak separation effect being better as an index. Meanwhile, in order to improve the peak shape of the component to be detected, the peak tailing is restrained, a triethylamine solution is added into the mobile phase water phase, and finally, a methanol-0.1% phosphoric acid (the pH value is adjusted to 5.0 by using the triethylamine) solution is determined to be the mobile phase.
1.2.2 elution gradient
The experiment carries out method investigation of 4 elution gradients on the index components on the basis of preset gradients to determine proper gradient conditions, and determines a method-4 as a final elution gradient condition according to the separation effect and the elution time, and the results are shown in tables 1-4 and chromatograms 16-27.
TABLE 1 gradient elution method for determining content of index components of open throat sisal
TABLE 2 gradient elution method for determining content of index components of open throat sisal
TABLE 3 gradient elution method for determining content of index components of open throat sisal
TABLE 4 gradient elution method for determining content of index components of open throat sisal
1.2.3 selection of flow Rate
Since different flow rates also have some effect on chromatographic separation, flow rates of 0.8mL/min, 1.0mL/min, 1.2mL/min were selected for investigation to determine the optimal flow rate. As shown in FIGS. 28 to 36, the optimal flow rate was determined to be 0.8ml/min based on the separation of the chromatographic peaks of each detection index.
1.2.4 selection of column temperature
Since different column temperatures also have a certain influence on the chromatographic separation, different column temperatures of 25 ℃,30 ℃ and 35 ℃ are selected for investigation to determine the optimal column temperature. As shown in FIGS. 37-45, the optimum column temperature was determined to be 30 ℃.
1.2.5 selection of detection wavelength
The mixed reference substance solution is subjected to full-wavelength scanning (190-400 nm) by using a Diode Array Detector (DAD) detector, and the result shows that matrine and bergenin are absorbed more greatly at 220 and 275 nm; the pterocarpan trilobate glycoside and Ma Kayin have larger absorption at 300nm and better separation from nearby chromatographic peaks, so that the 3 absorption wavelengths are selected as detection wavelengths, the peak area is good, the detection sensitivity is improved, and the result is more accurate.
1.2.6 determination of chromatographic conditions
A Platisil-ODS column (250 mm. Times.4.6 mm,5 μm) was used; the mobile phase is methanol (A) -0.1% phosphoric acid (the pH value is regulated to 5.0 by triethylamine, B), gradient elution is carried out (0-8 min,10% A, 8-19 min, 10-30% A, 19-28 min, 30-30% A, 28-32 min, 30-40% A, 32-40 min, 40-60% A, 40-70 min, 60-100% A); the volume flow is 0.8mL/min; column temperature is 35 ℃; detection wavelength 220nm (matrine), 275nm (bergenin), 300nm (Trifolium pratense pterocarpine glycoside); the sample injection amount was 10. Mu.L.
1.3 preparation of solutions
1.3.1 control solutions
Precisely weighing 20.26mg of matrine reference substance, 20.06mg of bergenin reference substance and 10.36mg of three-leaf bean pterocarpan reference substance, respectively placing into 10mL volumetric flask, placing 10.23mg of cocaine reference substance into 25mL volumetric flask, adding methanol, ultrasonically dissolving, and fixing volume to scale, shaking to obtain reference substance stock solution; and 3.5mL, 4mL, 1.0mL and 0.6mL of the corresponding reference substance solutions are respectively measured precisely, placed in the same 10mL volumetric flask and fixed in volume by methanol to prepare mixed reference substance solutions with the concentrations of 0.7091mg/mL, 0.8024mg/mL, 0.1036mg/mL and 0.02456mg/mL in sequence.
1.3.2 sample solutions
Precisely sucking 2.5mL of the throat-opening sword spray, placing in a 5mL volumetric flask, adding methanol to a fixed volume to a scale, shaking uniformly, and filtering with a 0.22 μm microporous filter membrane to obtain the final product.
1.3.3 negative test sample solution
Preparing a negative open throat sword spray of cinnabar-lack roots and subprostrate sophora according to the process method of the open throat sword spray, precisely sucking 2.5mL of each negative open throat sword spray, and preparing a negative test sample solution according to the method under item 1.3.2.
1.4, content determination is carried out according to the determined chromatographic conditions, and HPLC chromatograms of different wavelengths and negative samples of each component are obtained.
2. Methodology investigation
2.1 specificity test
Taking appropriate amounts of control, test and negative test solutions, and performing sample injection measurement under the condition of 1.2.6, and recording chromatograms, wherein the results are shown in fig. 46-54 (the chromatographic peaks in the chromatograms are respectively expressed as 1. Bergenin, 2. Matrine, 3. Trifolium prandial, and 4. Macadamia). Therefore, the chromatographic peak of each component to be detected has good separation degree, the retention time of the chromatographic peak of the sample is consistent with that of the chromatographic peak of the reference substance, and the components to be detected are not detected in the corresponding negative test sample, so that the method has good specificity.
2.2 linear relationship investigation
Precisely sucking 0.4, 1.2, 2.0, 2.5 and 3.5mL of the reference substance solution under the item "1.3.1", respectively, placing the reference substance solutions into 5mL volumetric flasks, adding methanol to fix the volume to the scale, shaking uniformly to prepare a series of mixed reference substance solutions, carrying out sample injection measurement under the chromatographic condition of the item "1.2.6", taking the concentration of the reference substance solutions as the abscissa and the target peak area as the ordinate, and calculating a regression equation, wherein the result is shown in Table 5, and shows that the components have good linear relation in the respective ranges.
TABLE 5 Linear relationship of the components
2.3 precision test
The mixed reference substance solution of item 1.3.1 is precisely sucked, the sample is continuously injected and measured for 6 times according to the chromatographic condition of item 1.2.6, the peak area is recorded, and the RSD of the peak areas of matrine, bergenin, pterocarpine and Ma Kayin are calculated to be 1.09%, 1.68%, 1.24% and 1.16%, respectively, which shows that the instrument precision is good, and the result is shown in Table 6.
TABLE 6 results of precision peak area test for each component
2.4 stability test
Taking a throat-opening sword spray, preparing a test sample solution according to the method under the item "1.3.2", then injecting samples for 0, 2, 4, 6, 8 and 12 hours according to the chromatographic conditions under the item "1.2.6", recording peak areas, and calculating to obtain RSD (reactive species decomposition) of matrine, bergenin, pterocarpan henryi and Ma Kayin peak areas of 2.00%, 0.81%, 1.81% and 0.88%, wherein the prepared test sample solution is good in stability within 12 hours, and the results are shown in Table 7.
TABLE 7 stability peak area test results for each component
2.5 repeatability test
6 parts of a throat-opening sword spray is taken, a test sample solution is prepared according to the method under the item "1.3.2", and then sample injection measurement is carried out according to the chromatographic condition under the item "1.2.6", so that RSD (reactive species decomposition) of matrine, bergenin, pterocarpine content are respectively 1.38%, 1.18%, 1.50% and 1.55%, which shows that the test sample preparation method has good repeatability, and the result is shown in Table 8.
TABLE 8 results of the repeatability content test of the components
2.6 sample recovery test
Precisely sucking 1.25mL of each throat-opening sword spray with known content, respectively placing 6 parts into 5mL volumetric flasks, and respectively adding mixed reference solution (containing matrine 0.3526mg. ML) -1 Bergenin 0.6452mg.mL -1 Trifolium pratense pterocarpine glycoside 0.05645mg.mL -1 Marcaine 0.013350 mg.mL -1 ) 1.25mL, 6 parts of sample solution is prepared according to the method under the item "1.3.2", then the sample is introduced and measured according to the chromatographic condition under the item "1.2.6", the peak area is recorded, the sample recovery (%) is calculated, and the result is shown in Table 9, so that the sample recovery is good in the sample preparation method.
Table 9 results of the sample recovery test of each component (n=6)
2.7 relative correction factor determination
Precisely sucking the mixed reference solution of item 1.3.1, performing sample injection measurement according to chromatographic conditions of item 1.2.6, taking bergenin in the sample as internal standard(s), and calculating relative correction factors (f) of 3 components of matrine (a), three-leaf bean pterocarpine (b) and macadamia (c) s/i ) Taking f calculated by a plurality of quality points by adopting a multipoint correction method s/i Taking the average value as f for quantification s/i [f s/i ＝f s /f i ＝A s C i /A i C s ]Wherein A is s Peak area, C, of bergenin control s A is the mass concentration of bergenin reference substance i For the peak area of the component to be measured, C i For the mass concentration of the component to be measured]The results are shown in Table 10. The results show that f between matrine, pterocarpin, ma Kayin and bergenin as internal reference s/i 1.6370, 1.8639 and 1.1217, respectively, and rsd values of 0.78%, 0.17% and 0.98%, respectively.
Table 10 relative correction factors for each component
S is bergenin, and a-c are matrine, pterocarpine, and marcaine respectively
2.8 Effect of different instruments and columns on the relative correction factor
The experiment compares 3 chromatographic columns of Agilent 1260, waters e2695 high performance liquid chromatograph and Platisil-ODS, diamonsil-C18, agilent 5TC-C18 (specification is 250mm×4.6mm,5 μm), and examines the effect on the relative correction factor. The results are shown in Table 11. The results show that the chromatographic columns of different chromatographic instruments and different manufacturers have no obvious influence on the relative correction factors.
TABLE 11 influence of different instruments and columns on the relative correction factors
S is bergenin, and a-c are matrine, pterocarpine, and marcaine respectively
2.9 influence of different column temperatures on the relative correction factor
The experiment adopts an Agilent 1260 high performance liquid chromatograph and a Platisil-ODS chromatographic column to respectively examine the influence of column temperatures of 30, 35 and 40 ℃ on relative correction factors, the results are shown in Table 12, and the results show that different column temperatures have no obvious influence on the relative correction factors of all components.
TABLE 12 influence of different column temperatures on the relative correction factors
S is bergenin, and a-c are matrine, pterocarpine, and marcaine respectively
2.10 influence of different volume flows on the relative correction factor
The experiment adopts an Agilent 1260 high performance liquid chromatograph and a Platisil-ODS chromatographic column to respectively examine the influences of the volume flow rates of 0.7, 0.8 and 0.9mL/min on the relative correction factors, the results are shown in Table 13, and the results show that the different volume flow rates have no obvious influence on the relative correction factors of all components.
TABLE 13 influence of different volume flows on the relative correction factor
S is bergenin, and a-c are matrine, pterocarpine, and marcaine respectively
2.11 chromatographic Peak positioning
The method for locating the chromatographic peaks of each component to be detected in the literature is a relative retention time locating method or a retention time difference locating method, bergenin is used as an internal standard(s), and 3 components of the matrine (a), the pterocarpan-side (b) and the cocaine (c) under a 2.8-item instrument and a chromatographic column are calculated to have relative retention time (ti/s) and retention time difference (delta ti/s). The results are shown in Table 14 and Table 15, and the experimental results show that the RSD values of the pterocarpine and the marcaine positioned by adopting the relative retention time and the retention time difference are less than 5%, and the positioning can be carried out by adopting the relative retention time and the retention time difference, but the RSD values of the matrine positioned by adopting the relative retention time and the retention time difference are more than 5%, so that the positioning of the matrine can only be carried out by adopting the qualitative reference substance.
TABLE 14 relative Retention time of the ingredients
S is bergenin, and a-c are matrine, pterocarpine, and marcaine respectively
TABLE 15 retention time differences for each component
S is bergenin, and a-c are matrine, pterocarpine, and marcaine respectively
2.12 sample content determination
10 batches of open throat sword spray samples with different batch numbers are taken, a test solution is prepared according to the method under the item of 1.3.2, then the sample is sampled and measured according to the chromatographic condition under the item of 1.2.6, the content is calculated by an External Standard Method (ESM) and a multi-evaluation method (QAMS) respectively, and the results obtained by the 2 methods are compared by taking the Relative Error (RE) as a parameter, wherein the formula is RE= [ (QAMS measured value-ESM measured value)/ESM measured value ] ×100%, and the results are shown in Table 16. The relative error of the mass fraction value calculated by the conventional external standard method and the result measured by the one-test multi-evaluation method is less than 3%, and the result shows that the results measured by the one-test multi-evaluation method and the external standard method have no obvious difference. Therefore, the method is applicable to the quality evaluation of multiple index components of the open throat sword spray.
Table 16 results of measuring the contents of the components (mg/mL)
Discussion 3
The bergenin content in the sample is high, the peak area and the retention time are stable, and the price is low and the bergenin is easy to obtain, so the bergenin is selected as an internal reference in the experiment.
The invention establishes a multi-index content determination method of a throat-opening sword spray (children), which is based on a high-performance liquid chromatography technology and combines a multi-evaluation method, and the method is accurate and reliable, is suitable for the content determination of multiple components in the throat-opening sword spray, has high detection sensitivity, good stability, simple and convenient operation, is easy to master, and is convenient to popularize further. Meanwhile, bergenin is used as an internal reference, relative correction factors of matrine, pterocarpine and Ma Kayin in the throat-opening sword spray (children) are calculated, and the contents of matrine, pterocarpine and Ma Kayin are calculated by using the correction factors. The method can reduce the detection cost and detection time, reduce the workload, improve the efficiency, and has important significance for the quality control of the throat-opening sword spray (children) and the guarantee of clinical curative effect.

Claims (2)

1. A content determination method for one-measurement-multiple-evaluation of children type open throat sword spray is characterized by comprising the following steps of: the content determination method comprises the following steps:
(1) Preparing a reference substance solution: precisely weighing matrine reference 20.26mg, bergenin reference 20.06mg and Trifolium pratense pterocarpine reference 10.36mg, respectively placing in 10mL volumetric flask, placing in 25mL volumetric flask with 10.23mg of macadamia reference, adding methanol, ultrasonically dissolving, fixing volume to scale, shaking, and collecting as reference stock solution; respectively measuring 3.5mL, 4mL, 1.0mL and 0.6mL of the corresponding reference substance solutions precisely, placing the solutions into the same 10mL volumetric flask, and fixing the volume by using methanol to prepare mixed reference substance solutions with the concentrations of 0.7091mg/mL, 0.8024mg/mL, 0.1036mg/mL and 0.02456mg/mL in sequence;
(2) The preparation method of the sample solution comprises the following steps: precisely sucking 2.5. 2.5mL of the open throat sword spray, placing in a 5mL volumetric flask, adding methanol to a certain volume to scale, shaking uniformly, and filtering with 0.22 μm microporous membrane to obtain the final product;
(3) Establishing a high performance liquid chromatography for simultaneously measuring the contents of matrine, bergenin, pterocarpine and pterocarpine;
chromatographic conditions of high performance liquid chromatography: using Platisil-ODS column 250mm ×4.6mm,5 μm; the mobile phase is methanol A-0.1% phosphoric acid aqueous solution B, and gradient elution is carried out: 0-8 min,10% A; 8-19 min, 10-30% of A; 19-28 min, 30-30% of A; 28-32 min, 30-40% of A; 32-40 min, 40-60% of A; 40-70 min, 60-100% of A; the volume flow is 0.8mL/min; column temperature is 35 ℃; detection wavelength: matrine 220nm, bergenin 275nm, pterocarpan trilobate glycoside and macadamia 300nm; the sample injection amount is 10 mu L;
the pH of the 0.1% phosphoric acid aqueous solution is firstly regulated to 5.0 by triethylamine;
(4) Determining relative correction factors of matrine, pterocarpine and macadamia;
(5) According to the measured chromatogram of the sample solution, the bergenin content is calculated, and the matrine, the three-leaf bean pterocarpin and the marcaine content are respectively calculated according to the relative correction factors.
2. The method for measuring the content of one-test-multiple-evaluation child open-throat sword spray according to claim 1, which is characterized in that: in the step (4), bergenin in a sample is used as an internal standard s, and relative correction factors f of 3 components of matrine a, pterocarpan-side b and macadamia c are calculated s/i Taking f calculated by a plurality of quality points by adopting a multipoint correction method s/i Taking the average value as f for quantification s/i ；f s/i ＝f s / f i ＝A s C i /A i C s Wherein A is s Peak area, C, of bergenin control s A is the mass concentration of bergenin reference substance i For the peak area of the component to be measured, C i The mass concentration of the components to be measured.
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