CN114200040A

CN114200040A - Content determination method for one-test-multiple evaluation of children-type Kaihoujian spray

Info

Publication number: CN114200040A
Application number: CN202111415900.9A
Authority: CN
Inventors: 张海; 周英; 王珏犇; 俸婷婷; 张丽艳; 石慧; 董秀
Original assignee: Guizhou Sanli Pharmaceutical Co ltd
Current assignee: Guizhou Sanli Pharmaceutical Co ltd
Priority date: 2021-11-25
Filing date: 2021-11-25
Publication date: 2022-03-18
Anticipated expiration: 2041-11-25
Also published as: CN114200040B

Abstract

The invention discloses a content determination method of Kaihoujian spray (children type), which adopts high performance liquid chromatography to establish a content determination method for determining the content of matrine, bergenin, trifolio red sandalwood glycoside and cocaine in the Kaihoujian spray (children type), and takes the bergenin as an internal standard substance for one-test multi-evaluation. The detection result is accurate and stable, and the method can be used for quality control of Kaihoujian spray (children type); meanwhile, the invention can reduce the detection cost and detection time, reduce the workload and improve the efficiency, and lays a foundation for improving the quality standard of the Kaihoujian spray (children type).

Description

Content determination method for one-test-multiple evaluation of children-type Kaihoujian spray

Technical Field

The invention relates to a detection method of a traditional Chinese medicine preparation, in particular to a content determination method for one-test-multiple evaluation of Kaihoujian spray (children type).

Background

Kaihoujian spray (for children) is a kind of single Miao medicine produced by Guizhou Sanli pharmaceutical corporation, and its prescription is formed from eight-claw gold dragon, subprostrate sophora, cicada slough and menthol, and possesses the functions of clearing away heat and toxic material, relieving swelling and stopping pain. The traditional Chinese medicine composition has obvious curative effects of clinically treating children acute pharyngitis, acute tonsillitis, herpetic stomatitis, herpangina, hand-foot-and-mouth disease, thrush and the like, and has high safety and no toxic or side effect. Currently, the throat-opening sword spray is less studied about quality control. The ingredients of the medicine are researched and analyzed, and the bergenin is the main effective chemical ingredient of the main medicine ardisia crenata willd and has good cough relieving, anti-inflammatory, antibacterial and antiviral effects. The radix Sophorae Tonkinensis mainly contains alkaloids, flavonoids, triterpenes and other components, the alkaloids are the main components of the radix Sophorae Tonkinensis, and the matrine contained in the radix Sophorae Tonkinensis can inhibit the in-vitro proliferation of pancreatic cancer cells and induce the apoptosis of the pancreatic cancer cells, and also has the effects of resisting inflammation, inhibiting bacteria, protecting liver and resisting arrhythmia. The trifoliate red sandalwood glycoside and the maka have obvious curative effect on inducing leukemia HL-60 apoptosis because of flavonoid components in the subprostrate sophora. In order to improve the quality control standard of the preparation, a multi-index content determination method taking the main drug effect components as indexes is established, meanwhile, in order to reduce the detection cost, save the detection time and improve the detection efficiency, the method takes the bergenin with high content and low price as an internal standard reference, calculates the correction factors and the content of 3 components of the matrine, the trifoliate red sandalwood glycoside and the macacaine, establishes a content determination method for one-test multi-evaluation of the Kangjian spray (children type), and lays a foundation for the improvement of the quality control standard of the Kangjian spray.

Disclosure of Invention

In order to solve the technical problems, the invention provides a content determination method for one-test-multiple evaluation of Kaihoujian spray (children type), which provides a basis for improving the quality standard of the Kaihoujian spray. In order to achieve the purpose, the invention is realized by the following technical scheme.

A content determination method for one-test-multiple evaluation of Kaihoujian spray (children type) comprises the following steps:

(1) preparing a reference substance solution;

(2) preparing a test solution;

(3) establishing high performance liquid chromatography for simultaneously determining matrine, bergenin, trifoliate red sandalwood glycoside and macaine content;

(4) taking bergenin as an internal reference substance, and determining relative correction factors of matrine, trifoliosid and malacaine;

(5) according to the measured chromatogram of the test solution, the content of bergenin is calculated, and the content of matrine, trifoliosid and cocaine is calculated respectively according to the relative correction factors.

The content determination method for the single-test multi-evaluation Kaihoujian spray (children type) comprises the following steps of (1) preparing a control solution: precisely weighing 20.26mg of a matrine reference substance, 20.06mg of a bergenin reference substance and 10.36mg of a trifoliate red sandalwood glycoside reference substance, respectively placing in a 10mL volumetric flask, placing 10.23mg of a macaine reference substance in a 25mL volumetric flask, adding methanol for ultrasonic dissolution, fixing the volume to a scale, shaking up, and taking the solution as a reference substance stock solution; then respectively and precisely measuring 3.5mL, 4mL, 0.7mL, 1.0mL and 0.6mL of the corresponding reference substance solution, placing the reference substance solution into the same 10mL volumetric flask, and metering 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.

The content determination method for the single-test multi-evaluation Kaihoujian spray (children type) comprises the following steps of (2) preparing a test solution: precisely sucking 2.5mL of Kaihoujian spray, placing in a 5mL volumetric flask, adding methanol to constant volume to scale, shaking, and filtering with 0.22 μm microporous membrane.

In the content determination method for one-test-multiple evaluation of Kaihoujian 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 percent phosphoric acid (pH is adjusted to 5.0 and B by triethylamine), and gradient elution (0-8 min, 10 percent A, 8-19 min, 10-30 percent A, 19-28 min, 30-30 percent A, 28-32 min, 30-40 percent A, 32-40 min, 40-60 percent A, 40-70 min, 60-100 percent A); the volume flow is 0.8 mL/min; the column temperature is 35 ℃; detecting wavelength of 220nm (matrine), 275nm (bergenin), and 300nm (trifoliolate pterocarpan glycoside, and cocaine); the amount of the sample was 10. mu.L.

In the one-test-and-multiple-evaluation content determination method for the sword-shaped spray (children type), in the step (4), the relative correction factors (fs/i) of 3 components including the matrine (a), the red sandalwood glycoside of trifolium bean (b) and the cocaine (c) are calculated by taking the bergenin in a sample As an internal standard(s), a multi-point correction method is adopted, fs/i obtained by calculating a plurality of mass points is taken, and an average value is taken As a quantitative fs/i (fs/i ═ fs/fi ═ As Ci/Ai Cs, wherein As is the peak area of a bergenin reference, Cs is the mass concentration of the bergenin reference, Ai is the peak area of a component to be detected, and Ci is the mass concentration of the component to be detected.

The invention has the beneficial effects that:

(1) the invention establishes a multi-index content determination method for Kaihoujian spray (children type), is based on a high performance liquid chromatography technology and combines a one-test-multiple evaluation method, is accurate and reliable, is suitable for content determination of multiple components in the Kaihoujian spray, and has the advantages of high detection sensitivity, good stability, simple and convenient operation, easy mastering and convenient further popularization.

(2) The invention relates to a method for evaluating a Kanghoujian spray (children type) by a one-test-multiple evaluation method, which takes bergenin as an internal reference substance, calculates relative correction factors of matrine, trifolio red sandalwood glycoside and cocaine in the Kanghoujian spray (children type), and calculates the contents of the matrine, the trifolio red sandalwood glycoside and the cocaine by using the correction factors. The method can reduce the detection cost and time, reduce the workload and improve the efficiency, and has important significance for the quality control and the guarantee of the clinical curative effect of the Kaihoujian spray (children type).

Drawings

FIG. 1 shows a chromatogram of a sample of bergenin with a wavelength of 275nm detected from a mobile phase of methanol-0.1% phosphoric acid solution;

FIG. 2 is a chromatogram of a matrine sample with a detection wavelength of 220nm, using mobile phase methanol-0.1% phosphoric acid water;

FIG. 3 shows a chromatogram of a test sample of trifoliolate pterocarpan glycoside and malacaine with a detection wavelength of 300nm in a mobile phase of methanol-0.1% phosphoric acid water;

FIG. 4 is a chromatogram of a sample of bergenin with a wavelength of 275nm detected with mobile phase acetonitrile-0.1% phosphoric acid water;

FIG. 5 is a chromatogram of a matrine sample with a detection wavelength of 220nm, using mobile phase acetonitrile-0.1% phosphoric acid water;

FIG. 6 is a chromatogram of a test sample of trifoliolate pterocarpan glycoside and malacaine with the detection wavelength of 300nm, using mobile phase acetonitrile-0.1% phosphoric acid water;

FIG. 7 is a chromatogram of a sample for detecting bergenin with a wavelength of 275nm, using mobile phase methanol-0.25% phosphoric acid water;

FIG. 8 is a chromatogram of a matrine sample with a detection wavelength of 220nm, using mobile phase methanol-0.25% phosphoric acid water;

FIG. 9 shows a chromatogram of a test sample for detecting pterocarpan glycoside and macaine with a wavelength of 300nm with mobile phase methanol-0.25% phosphoric acid water;

FIG. 10 is a chromatogram of a sample of bergenin with a wavelength of 275nm detected by mobile phase acetonitrile-0.25% phosphoric acid water;

FIG. 11 is a chromatogram of a matrine sample with a detection wavelength of 220nm, using mobile phase acetonitrile-0.25% phosphoric acid water;

FIG. 12 is a chromatogram of a sample for detecting pterocarpan bean pterocarpan glycoside and maca factor with a wavelength of 300nm, using mobile phase acetonitrile-0.25% phosphoric acid water;

FIG. 13 is a chromatogram of a sample of bergenin with a wavelength of 275nm detected from a mobile phase of methanol-0.1% phosphoric acid-triethylamine water;

FIG. 14 is a chromatogram of a sample of matrine with a detection wavelength of 220nm, using mobile phase methanol-0.1% phosphoric acid-triethylamine water;

FIG. 15 shows a chromatogram of a test sample of trifoliolate pterocarpan glycoside and cocaine with a detection wavelength of 300nm in a mobile phase of methanol-0.1% phosphoric acid-triethylamine water;

FIG. 16 elution method-1, detecting chromatogram of bergenin test sample with wavelength of 275 nm;

FIG. 17 shows an elution method-1, in which a chromatogram of a sample of matrine having a wavelength of 220nm is detected;

FIG. 18 shows the chromatogram of the test sample of trifoliolate pterocarpan glycoside and malacaine with the wavelength of 300nm detected by the elution method-1;

FIG. 19 elution method-2, detecting chromatogram of bergenin test sample with wavelength of 275 nm;

FIG. 20 shows an elution method-2, in which a chromatogram of a matrine sample having a detection wavelength of 220nm is obtained;

FIG. 21 shows the chromatogram of the test sample of trifoliolate pterocarpin and malacaine with the wavelength of 300nm detected by the elution method-2;

FIG. 22 elution method-3, detecting chromatogram of bergenin test sample with wavelength of 275 nm;

FIG. 23 shows elution method-3, in which a chromatogram of a sample of matrine having a wavelength of 220nm is detected;

FIG. 24 shows the chromatogram of the test sample of trifoliolate pterocarpan glycoside and malacaine with the detection wavelength of 300nm in the elution method-3;

FIG. 25 elution method-4, detecting chromatogram of bergenin test sample with wavelength of 275 nm;

FIG. 26 elution method-4, detecting chromatogram of matrine sample with wavelength of 220 nm;

FIG. 27 shows the chromatogram of the test sample for detecting pterocarpan glycoside and macaine with the wavelength of 300nm according to elution method-4;

FIG. 28 is a chromatogram of a sample for detecting bergenin with a wavelength of 275nm at a flow rate of 0.8 mL/min;

FIG. 29 is a chromatogram of a sample for detecting bergenin with a wavelength of 275nm at a flow rate of 1.0 mL/min;

FIG. 30 shows a chromatogram of a sample of bergenin with a flow rate of 1.2mL/min and a detection wavelength of 275 nm;

FIG. 31 is a chromatogram of a test sample for detecting matrine with a wavelength of 220nm at a flow rate of 0.8 mL/min;

FIG. 32 is a chromatogram of a test sample for detecting matrine with a wavelength of 220nm at a flow rate of 1.0 mL/min;

FIG. 33 is a chromatogram of a test sample for detecting matrine with a wavelength of 220nm at a flow rate of 1.2 mL/min;

FIG. 34 is a chromatogram of a test sample for detecting pterocarpan santalin and cocaine with a wavelength of 300nm at a flow rate of 0.8 mL/min;

FIG. 35 is a chromatogram of a test sample for detecting pterocarpan santalin and cocaine with a wavelength of 300nm at a flow rate of 1.0 mL/min;

FIG. 36 shows a chromatogram of a test sample for detecting pterocarpan santalin and cocaine with a wavelength of 300nm at a flow rate of 1.2 mL/min;

FIG. 37 is a chromatogram of a sample of bergenin with a wavelength of 275nm detected at a column temperature of 25 ℃;

FIG. 38 is a chromatogram of a sample of bergenin with a wavelength of 275nm detected at a column temperature of 30 ℃;

FIG. 39 is a chromatogram of a sample of bergenin with a detection wavelength of 275nm at a column temperature of 35 ℃;

FIG. 40 is a chromatogram of a sample of matrine with a detection wavelength of 220nm at a column temperature of 25 ℃;

FIG. 41 is a chromatogram of a sample of matrine with a detection wavelength of 220nm at a column temperature of 30 ℃;

FIG. 42 is a chromatogram of a matrine sample with a detection wavelength of 220nm at a column temperature of 35 ℃;

FIG. 43 shows a chromatogram of a test sample for detecting pterocarpan bean glycoside and macane with a wavelength of 300nm at a column temperature of 25 deg.C;

FIG. 44 is a chromatogram of a test sample for detecting pterocarpan bean pterocarpan glycoside and cocaine with a wavelength of 300nm at a column temperature of 30 ℃;

FIG. 45 shows a chromatogram of a test sample for detecting pterocarpan bean pterocarpan glycoside and cocaine with a wavelength of 300nm at a column temperature of 35 ℃;

FIG. 46 shows a chromatogram of a bergenin control with a wavelength of 275 nm;

FIG. 47 is a chromatogram of a test sample of bergenin with a detection wavelength of 275 nm;

FIG. 48 is a chromatogram of a matrine control with a detection wavelength of 220 nm;

FIG. 49 chromatogram of matrine test sample with detection wavelength of 220 nm;

FIG. 50 is a chromatogram of a control for detecting pterocarpan bean pterocarpan glycoside and cocaine with a wavelength of 300 nm;

FIG. 51 is a chromatogram of a test sample for detecting pterocarpan bean pterocarpan glycoside and cocaine with a wavelength of 300 nm;

FIG. 52 is a chromatogram of a southern star gold negative test sample with a detection wavelength of 220 nm;

FIG. 53 is a chromatogram of a negative test sample lacking Ardisia disiacta Makino with a detection wavelength of 300 nm;

FIG. 54 is a chromatogram of a negative test sample of absent Sophora subprostrata with a detection wavelength of 275 nm;

in order to make the technical solutions of the present invention better understood and enable those skilled in the art to practice the present invention, the following embodiments are further described, but the present invention is not limited to the following embodiments.

Detailed Description

Example 1: method for simultaneously determining 4 components in Kaihoujian spray by multiple evaluation

(1) Preparing a reference substance solution: precisely weighing 20.26mg of a matrine reference substance, 20.06mg of a bergenin reference substance and 10.36mg of a trifoliate red sandalwood glycoside reference substance in 10mL volumetric flasks respectively, putting 10.23mg of a macaine reference substance in 25mL volumetric flasks, adding methanol for ultrasonic dissolution, fixing the volume to a scale, shaking up to obtain a reference substance stock solution; respectively and precisely measuring 3.5mL, 4mL, 0.7mL, 1.0mL and 0.6mL of the corresponding reference substance solution, putting the reference substance solution into the same 10mL volumetric flask, and metering 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) preparation of a test solution: precisely sucking 2.5mL of Kaihoujian spray, placing in a 5mL volumetric flask, adding methanol to constant volume to scale, shaking, and filtering with 0.22 μm microporous membrane;

(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 percent of phosphoric acid (the pH value is adjusted to 5.0 and B by triethylamine), and the gradient elution is carried out (0-8 min, 10 percent of A, 8-19 min, 10-30 percent of A, 19-28 min, 30-30 percent of A, 28-32 min, 30-40 percent of A, 32-40 min, 40-60 percent of A, 40-70 min and 60-100 percent of A); the volume flow is 0.8 mL/min; the column temperature is 35 ℃; detecting wavelength of 220nm (matrine), 275nm (bergenin), and 300nm (pterocarpan glycoside, and malacaine); the sample volume is 10 mu L;

(4) taking bergenin in a sample As an internal standard(s), calculating a relative correction factor (fs/i) of 3 components of the matrine (a), the triallate pterocarpan glycoside (b) and the tetracaine (c), adopting a multi-point correction method, taking fs/i obtained by calculating a plurality of mass points, taking an average value As fs/i for quantification, wherein fs/i is fs/fi is As Ci/Ai Cs, As is the peak area of a bergenin reference substance, Cs is the mass concentration of the bergenin reference substance, Ai is the peak area of a component to be detected, and Ci is the mass concentration of the component to be detected; the results show that fs/i between matrine, pterocarpan trifoliate, malacaine and the internal reference bergenin are 1.6370, 1.8639 and 1.1217 respectively.

The inventors carried out a number of experiments and the following were studies of the detection method of the present invention:

methodology study

1.1 materials

1.1.1 instruments

An Agilent model 1260 high performance liquid chromatograph (Agilent corporation, usa); one ten-thousandth analytical balance model XS-205 (Mettler-Toledo, Switzerland).

1.1.2 Agents and drugs

Matrine (batch number: PS011495, purity not less than 98.5%), bergenin (batch number: PS010520, purity not less than 98.0%), trifoliosid (batch number PS012410, purity not less than 98.0%), cocaine (batch number PS210802-06, purity not less than 98.0%) reference substances are purchased from Kyoto Biotech limited; methanol, phosphoric acid and triethylamine are all chromatographically pure (Tianjin, Kemiou chemical reagent Co., Ltd.); purified water (guangzhou drochen food and beverage limited); kaihoujian spray (Guizhou Sanli pharmacy Co., Ltd., batch No. 20200706, 20200720, 20200730, 20200733, 20200736, 20200748, 20200804, 20200805, 20200812, 20200814, respectively).

1.2 analytical method optimization

1.2.1 selection of elution System

In the experiment, 4 elution solvent systems of methanol-0.1% phosphoric acid aqueous solution, acetonitrile-0.1% phosphoric acid aqueous solution, methanol-0.25% phosphoric acid aqueous solution and acetonitrile-0.25% phosphoric acid aqueous solution are firstly examined to determine an elution solvent system suitable for the study. As shown in fig. 1 to 15, it was found that the concentration of the phosphoric acid solution had no significant difference in the separation effect, and a methanol-0.1% phosphoric acid aqueous solution was selected as the mobile phase, using as an index that the detected components were more and the chromatographic peak separation effect was better. Meanwhile, in order to improve the peak shape of the component to be detected and inhibit the tailing of the peak, a triethylamine solution is added into a mobile phase water phase, and finally a methanol-0.1% phosphoric acid (pH is adjusted to 5.0 by triethylamine) solution is determined as the mobile phase.

1.2.2 elution gradient

In the experiment, 4-elution gradient method inspection is carried out on index components on the basis of a preset gradient to determine a proper gradient condition, a method-4 is determined as a final elution gradient condition according to separation effect and elution time, and the result is shown in tables 1-4 and chromatograms 16-27.

TABLE 1 gradient elution method for determining index component content of Kaihoujian

TABLE 2 gradient elution method for determining index component content of Kaihoujian-2

TABLE 3 gradient elution method for determining index component content of Kaihoujian-3

TABLE 4 gradient elution method for determining index component content of Kaihoujian

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 optimum flow rate was determined to be 0.8ml/min depending on the peak separation condition of each measurement index chromatogram.

1.2.4 selection of column temperature

Since different column temperatures also have a certain influence on chromatographic separation, different column temperatures of 25 ℃, 30 ℃ and 35 ℃ are selected for investigation to determine the optimal column temperature. As shown in fig. 37-45, the optimum column temperature was determined to be 30 ℃.

1.2.5 selection of detection wavelength

The full-wavelength scanning (190-400 nm) is carried out on the mixed reference solution by using a Diode Array Detector (DAD) detector, and the result shows that the matrine and the bergenin have larger absorption at 220nm and 275 nm; the trifolium pterocarpus santalin and the malacaine have larger absorption at 300nm and better separation degree with nearby chromatographic peaks, so that the 3 absorption wavelengths are selected as the detection wavelengths, the peak areas of the three wavelengths are good, the detection sensitivity is improved, and the results are 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 percent phosphoric acid (pH is adjusted to 5.0 and B by triethylamine), and gradient elution (0-8 min, 10 percent A, 8-19 min, 10-30 percent A, 19-28 min, 30-30 percent A, 28-32 min, 30-40 percent A, 32-40 min, 40-60 percent A, 40-70 min, 60-100 percent A); the volume flow is 0.8 mL/min; the column temperature is 35 ℃; detecting wavelength of 220nm (matrine), 275nm (bergenin), and 300nm (trifoliolate pterocarpan glycoside, and cocaine); the amount of the sample was 10. mu.L.

1.3 solution preparation

1.3.1 control solutions

Precisely weighing 20.26mg of a matrine reference substance, 20.06mg of a bergenin reference substance and 10.36mg of a trifoliate red sandalwood glycoside reference substance, respectively placing in a 10mL volumetric flask, placing 10.23mg of a macaine reference substance in a 25mL volumetric flask, adding methanol for ultrasonic dissolution, fixing the volume to a scale, shaking up, and taking the solution as a reference substance stock solution; then respectively and precisely measuring 3.5mL, 4mL, 0.7mL, 1.0mL and 0.6mL of the corresponding reference substance solution, placing the reference substance solution into the same 10mL volumetric flask, and metering 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.

1.3.2 test article solution

Precisely sucking 2.5mL of Kaihoujian spray, placing in a 5mL volumetric flask, adding methanol to constant volume to scale, shaking, and filtering with 0.22 μm microporous membrane.

1.3.3 negative test solution

Preparing negative Kaihoujian spray without radix Ardisiae Japonicae and radix Sophorae Tonkinensis according to the method of Kaihoujian spray, precisely sucking each 2.5mL of negative Kaihoujian spray, and preparing negative test solution according to the method of '1.3.2'.

1.4, carrying out content measurement according to the determined chromatographic conditions to obtain HPLC chromatograms of the negative samples with different wavelengths of the components.

2. Methodology investigation

2.1 specificity test

Taking appropriate amount of reference substance, test sample, and negative test sample solution, performing sample injection determination under the condition of "1.2.6" chromatography, and recording chromatogram, wherein the result is shown in FIGS. 46-54 (note: chromatographic peaks in chromatogram are respectively expressed as 1. bergenin, 2. matrine, 3. trifolium pterocarpum glycoside, and 4. malacaine). Therefore, the chromatographic peaks of the components to be detected have good separation degree, the retention time of the chromatographic peaks of the sample is consistent with that of the chromatographic peaks of the reference substance, and the components to be detected are not detected in the corresponding negative test samples, so that the specificity of the method is good.

2.2 Linear relationship investigation

Precisely sucking 0.4 mL, 1.2mL, 2.0 mL, 2.5mL and 3.5mL of reference substance solution under the item of '1.3.1', respectively placing in a 5mL volumetric flask, adding methanol to fix the volume to the scale, shaking up to prepare a series of mixed reference substance solutions, carrying out sample injection measurement under the chromatographic condition of the item of '1.2.6', taking the concentration of the reference substance solution as an abscissa and the area of a target peak as an ordinate, calculating a regression equation, and finding a result in Table 5, wherein the linear relationship of each component in each range is good.

TABLE 5 Linear relationship of the ingredients

2.3 precision test

Precisely absorbing the mixed reference substance solution of item 1.3.1, continuously injecting sample for 6 times according to the chromatographic condition of item 1.2.6, recording peak areas, and calculating to obtain RSD of peak areas of the matrine, the bergenin, the pterocarpus santalinus glycoside and the cocaine which are 1.09%, 1.68%, 1.24% and 1.16% respectively, which indicates that the precision of the instrument is good, and the result is shown in Table 6.

TABLE 6 area test results of precision peak of each component

2.4 stability test

Taking the Kaihoujian spray, preparing a test solution according to the method under the item '1.3.2', respectively injecting samples for 0, 2, 4, 6, 8 and 12 hours after preparation according to the chromatographic condition under the item '1.2.6', recording peak areas, and calculating to obtain the RSD of the peak areas of the matrine, the bergenin, the trifoliate red sandalwood glycoside and the cocaine to be 1.99%, 0.80%, 1.81% and 0.87%, which indicates that the prepared test solution has good stability in 12 hours, and the result is shown in the table 7.

TABLE 7 peak area test results for stability of each component

2.5 repeatability test

6 parts of Kaihoujian spray is taken, a test 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', and the RSD of the content of the matrine, the bergenin, the trifoliate red sandalwood glycoside and the macaine are respectively 1.37%, 1.17%, 1.50% and 1.54%, which shows that the repeatability of the preparation method of the test solution is good, and the result is shown in a table 8.

TABLE 8 test results of the content of each component in terms of repeatability

2.6 sample recovery test

Precisely sucking 1.25mL of each Kaihoujian spray with known content, respectively taking 6 parts, respectively placing in a 5mL volumetric flask, respectively adding 1.25mL of mixed reference substance solution (containing 0.3526mg.mL-1 matrine, 0.6452 mg.mL-1 bergenin, 0.0564mg.mL-1 trifoliate red sandalwood glycoside and 0.01350mg.mL-1), respectively, preparing 6 parts of test substance solution according to the method under the item of '1.3.2', performing sample injection measurement under the chromatographic condition of '1.2.6', recording peak area, and calculating sample injection recovery (%), wherein the results are shown in Table 9, which indicates that the sample injection recovery of the test substance preparation method is good.

TABLE 9 recovery test results for each component (n ═ 6)

2.7 relative correction factor determination

Precisely absorbing the mixed reference solution of item 1.3.1, performing sample injection measurement under the chromatographic condition of item 1.2.6, taking bergenin in a sample As an internal standard(s), calculating relative correction factors (fs/i) of 3 components of matrine (a), trifoliolate pterocarpan glycoside (b) and macaine (c), adopting a multi-point correction method, taking a plurality of mass points to calculate the obtained fs/i, taking an average value As fs/i [ fs/fi is As/Ai Cs for quantification, wherein As is the peak area of the bergenin reference, Cs is the mass concentration of the bergenin reference, Ai is the peak area of the component to be measured, and Ci is the mass concentration of the component to be measured ], and obtaining the result shown in table 10. The results show that fs/i between matrine, pterocarpan trifoliate, macacaine and the internal reference bergenin are 1.6370, 1.8639 and 1.1217 respectively, and RSD values are 0.78%, 0.17% and 0.98% respectively.

TABLE 10 relative correction factor for each component

Note that s is bergenin, and a-c are matrine, trifolio red sandalwood glycoside and malacaine respectively

2.8 Effect of different instruments and columns on the relative correction factor

In the experiment, 3 chromatographic columns of Agilent 1260, Waters e2695 high performance liquid chromatograph, Platisil-ODS, Diamonsil-C18 and Agilent 5TC-C18 (the specifications are 250mm multiplied by 4.6mm and 5 mu m) are compared, and the influence of the chromatographic columns on relative correction factors is examined. The results are shown in FIG. 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 Effect of different instruments and columns on relative correction factors

2.9 Effect of different column temperatures on the relative correction factor

The Agilent 1260 high performance liquid chromatograph and the Platisil-ODS chromatographic column are adopted in the experiment, the influence of the column temperatures of 30 ℃, 35 and 40 ℃ on the relative correction factors is respectively inspected, the results are shown in a table 12, and the results show that different column temperatures have no obvious influence on the relative correction factors of all the components.

TABLE 12 Effect of different column temperatures on relative correction factors

2.10 Effect of different volumetric flows on the relative correction factor

In the experiment, an Agilent 1260 high performance liquid chromatograph and a Platisil-ODS chromatographic column are adopted, the influence of the volume flow of 0.7, 0.8 and 0.9mL/min on the relative correction factor is respectively inspected, the result is shown in a table 13, and the result shows that different volume flows have no obvious influence on the relative correction factor of each component.

TABLE 13 Effect of different volumetric flows on relative correction factor

2.11 chromatographic Peak localization

The method for locating the chromatographic peaks of the components to be detected in the literature is a relative retention time locating method or a retention time difference locating method, and the relative retention time (ti/s) and the retention time difference (delta ti/s) of 3 components of matrine (a), pterocarpus santalinus glycoside (b) and macaque (c) under a 2.8-item instrument and a chromatographic column are calculated by taking bergenin as an internal standard(s). The results are shown in tables 14 and 15, and the experimental results show that the RSD values of the trifolium pterocarpus santaline and the maka positioned by adopting the relative retention time and the retention time difference are both less than 5 percent and can be positioned 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 both more than 5 percent, so that the positioning of the matrine can only be positioned by adopting a qualitative reference substance.

TABLE 14 relative Retention time of ingredients

TABLE 15 difference in retention time of ingredients

2.12 sample assay

10 batches of spray samples of Kaihoujian spray with different batches are taken, a test solution is prepared according to the method under the item '1.3.2', then sample injection and measurement are carried out according to the chromatographic condition under the item '1.2.6', the content is respectively calculated by adopting an External Standard Method (ESM) and a one-test-multiple evaluation method (QAMS), the results obtained by the 2 methods are compared by taking a Relative Error (RE) as a parameter, and the formula is that RE is [ (QAMS measured value-ESM measured value)/ESM measured value ] × 100%, and the results are shown in a table 16. The relative error of the mass fraction value calculated by the results measured by the conventional external standard method and the one-test-multiple-evaluation method is less than 3 percent, and the result shows that the results measured by the one-test-multiple-evaluation method and the external standard method have no obvious difference. Therefore, the application of the one-test-multiple-evaluation method in the quality evaluation of the multiple-index components of the Kaihoujian spray is feasible.

TABLE 16 measurement results (mg/mL) of the contents of the respective components

Discussion of 3

The experiment selects bergenin as an internal reference substance because of the high content, stable peak area and retention time, low price and easy acquisition of the bergenin in the test sample.

The invention establishes a multi-index content determination method for Kaihoujian spray (children type), is based on a high performance liquid chromatography technology and combines a multi-evaluation method, is accurate and reliable, is suitable for determining the content of multiple components in the Kaihoujian spray, and has the advantages of high detection sensitivity, good stability, simple operation, easy mastering and convenient further popularization. Meanwhile, the bergenin is used as an internal reference material, relative correction factors of the picrophenine, the trifolirhizin and the macacaine in the Kaihoujian spray (children type) are calculated, and the contents of the matrine, the trifolirhizin and the macaine are calculated by using the correction factors. The method can reduce the detection cost and time, reduce the workload and improve the efficiency, and has important significance for the quality control and the guarantee of the clinical curative effect of the Kaihoujian spray (children type).

Claims

1. A content determination method for one-test-multiple evaluation of children-type Kaihoujian spray is characterized by comprising the following steps of: the content determination method comprises the following steps:

(1) preparing a reference substance solution;

(2) preparing a test solution;

(3) establishing high performance liquid chromatography for simultaneously determining the content of matrine, bergenin, trifoliate red sandalwood glycoside and macaine;

(5) according to the measured chromatogram of the test solution, the content of bergenin is calculated, and the content of matrine, trifolium pterocarpus santaline and cocaine are respectively calculated according to the relative correction factors.

2. The method for determining the content of a child-type kaihoujian spray with one test and multiple comments as claimed in claim 1, wherein the method comprises the following steps: in the step (1), preparing a reference substance solution: precisely weighing 20.26mg of a matrine reference substance, 20.06mg of a bergenin reference substance and 10.36mg of a trifoliate red sandalwood glycoside reference substance, respectively placing in a 10mL volumetric flask, placing 10.23mg of a macaine reference substance in a 25mL volumetric flask, adding methanol for ultrasonic dissolution, fixing the volume to a scale, shaking up, and taking the solution as a reference substance stock solution; then respectively and precisely measuring 3.5mL, 4mL, 0.7mL, 1.0mL and 0.6mL of the corresponding reference substance solution, placing the reference substance solution into the same 10mL volumetric flask, and metering 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.

3. The method for determining the content of a child-type kaihoujian spray with one test and multiple comments as claimed in claim 1, wherein the method comprises the following steps: in the step (2), the preparation method of the test solution comprises the following steps: precisely sucking 2.5mL of Kaihoujian spray, placing in a 5mL volumetric flask, adding methanol to constant volume to scale, shaking, and filtering with 0.22 μm microporous membrane.

4. The method for determining the content of a child-type kaihoujian spray with one test and multiple comments as claimed in claim 1, wherein the method comprises the following steps: in the step (3), the chromatographic conditions of the high performance liquid chromatography are as follows: adopting a Platisil-ODS chromatographic column of 250mm multiplied by 4.6mm and 5 mu m; the mobile phase is methanol A-0.1% phosphoric acid B, and the gradient elution is as follows: 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; 60-100% A for 40-70 min; the volume flow is 0.8 mL/min; the column temperature is 35 ℃; detection wavelength: matrine 220nm, bergenin 275nm, and trifolium pterocarpus santaline and macaine 300 nm; the amount of the sample was 10. mu.L.

5. The method for determining the one-test-multiple-evaluation content of the childhood Kaihoujian spray according to claim 4, wherein the method comprises the following steps: in the step (3), the pH of the 0.1% phosphoric acid is firstly adjusted to 5.0 by triethylamine.

6. The method for determining the content of a child-type kaihoujian spray with one test and multiple comments as claimed in claim 1, wherein the method comprises the following steps: in the step (4), the bergenin in the sample is taken as an internal standard s, the relative correction factor fs/i of 3 components of the matrine a, the trifolium pterocarpus indicus glycoside b and the cocaine c is calculated, a multi-point correction method is adopted, fs/i obtained by calculating a plurality of mass points is taken, and the average value is taken as the fs/i for quantification; and fs/i f s/fi As Ci/Ai Cs, wherein As is the peak area of the bergenin reference substance, Cs is the mass concentration of the bergenin reference substance, Ai is the peak area of the component to be detected, and Ci is the mass concentration of the component to be detected.