1. A quality detection method for Xintong granules is characterized in that the method adopts ultra-high performance liquid chromatography to simultaneously determine the contents of puerarin, daidzin, 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside, naringin, salvianolic acid B, icariin and tanshinone IIA in the preparation.

2. The Xintong granule quality detection method according to claim 1, characterized by comprising the following steps:

A. preparation of a test solution: taking a proper amount of Xintong granules, grinding into fine powder, precisely weighing 1g, placing in a 50ml volumetric flask, adding 50ml of 70-90% methanol, carrying out ultrasonic treatment for 30min, cooling, fixing the volume to a scale with 70-90% methanol, shaking up, filtering to obtain a test solution;

B. preparation of control solutions: accurately weighing appropriate amount of puerarin, daidzin, 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside, naringin, salvianolic acid B, icariin and tanshinone IIA reference substances, placing in a volumetric flask, adding 70-90% methanol for dissolving and diluting to scale, and shaking;

C. UPLC chromatographic conditions: performing gradient elution by using acetonitrile A-0.1% phosphoric acid solution B as a mobile phase; the detection wavelength is 250nm-320 nm; the sample volume is 2 mu L;

D. the determination method comprises the following steps: and D, precisely sucking 2 mu L of the reference solution and the test solution respectively, injecting into a liquid chromatograph, and measuring according to the chromatographic conditions in the step C to obtain the test solution.

3. The quality detection method of Xintong granules according to claim 2, wherein 50ml of 80% methanol is added in the step A, ultrasonic treatment is carried out for 30min, cooling is carried out, 80% methanol is used for fixing the volume to a scale, and shaking is carried out uniformly.

4. The quality detection method for Xintong granules according to claim 2, wherein in the step B, a proper amount of puerarin, daidzin, 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside, naringin, salvianolic acid B, icariin and tanshinone IIA reference substances are precisely weighed, placed in a volumetric flask, added with 80% methanol for dissolving and diluting to scale, and shaken up.

5. The Xintong granule quality detection method according to claim 2, wherein the wavelength switching procedure of the step C is as follows:

time, min Detection wavelength, nm 0 250 15 320 23 283 36 270 56 270

。

6. The Xintong particle quality detection method according to any one of claims 2 to 5, wherein the step C mobile phase gradient content and flow rate gradient change elution conditions are as follows:

time, min Flow rate, ml/min Mobile phase A,% Mobile phase B,% 0-27 0.5→0.3 5％→20％ 95％→80％ 27-40 0.3 20％→35％ 80％→65％ 40-45 0.3→0.5 35％→60％ 65％→40％ 45-55 0.5 60％ 40％ 55-56 0.5 60％→5％ 40％→95％

。

Background

The Xintong granules are prepared from thirteen traditional Chinese medicine raw materials including astragalus, codonopsis pilosula, radix ophiopogonis, polygonum multiflorum, epimedium, radix puerariae, angelica, salvia miltiorrhiza, spina gleditsiae, seaweed, kelp, oyster and immature bitter orange, have the effects of tonifying qi, activating blood, reducing phlegm and dredging collaterals, and are used for treating chest obstruction such as coronary heart disease and angina pectoris caused by deficiency of both qi and yin and phlegm stasis obstruction, and the symptoms are as follows: heartache, chest distress, short breath, nausea and vomiting and anorexia, and has obvious clinical effect.

At present, the current inspection items about Xintong granules in the national ministerial standard YBZ16392009-2017 mainly comprise thin-layer identification of astragalus, fleece-flower root, epimedium and red sage root and content determination of kudzu root (HPLC method for determining puerarin). The traditional Chinese medicine compound preparation has complex components, only uses thin-layer chromatography to carry out qualitative identification of the components, and uses ultra-high performance liquid chromatography to carry out content determination of single component puerarin in content determination of drug effect components, which is not enough to comprehensively reflect the quality of Xintong granules.

Ultra Performance Liquid Chromatography (UPLC) covers brand new technologies such as small particle packing, very low system volume, rapid detection means and the like by means of the theory and principle of HPLC, increases the flux of analysis and chromatographic peak capacity, has the characteristics of simplicity, accuracy, high sensitivity, good repeatability, multiple detector types and the like, and is one of important methods for constructing determination of the content of various components of a preparation. At present, no patent publication and literature report exists for the determination of the content of 7 components in the Xintong granules by using the UPLC method. The invention discloses a UPLC method for quantifying 7 components of Xintong granules, which comprehensively evaluates and controls the quality of the Xintong granules, thereby ensuring the stability of the product quality and the safety and the effectiveness of clinical medication. Meanwhile, the UPLC is adopted to simultaneously determine multiple components in the Xintong granules, the detection of 7 components is completed within 56min, the test result shows that the UPLC has high separation efficiency, the analysis cost is saved, the usage amount of a solvent is reduced, the separation method is more environment-friendly, and the separation method established by the test provides data reference for improving the quality standard of the Xintong granules.

Disclosure of Invention

The invention aims to solve the technical problem that a preparation detection method is further optimized on the basis of the standard YBZ16392009-2017 issued by the ministry of China, so that the operation method is simplified, and the detection efficiency is improved. The inventor successfully integrates UPLC content determination of puerarin, daidzin, 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside, naringin, salvianolic acid B, icariin and tanshinone IIA 7 in the preparation into a whole through long-time exploration and repeated tests, and provides a novel method for more comprehensively and accurately detecting Xintong granules.

The invention aims to provide a quality detection method of Chinese patent medicine Xintong granules for treating thoracic obstruction, and the Xintong granules are prepared by extracting and purifying thirteen traditional Chinese medicine compositions of astragalus, codonopsis pilosula, dwarf lilyturf tuber, tuber fleeceflower root, epimedium herb, kudzuvine root, Chinese angelica, red-rooted salvia root, spina gleditsiae, seaweed, kelp, oyster and immature bitter orange. Wherein puerarin and daidzin belong to the components of radix Puerariae, 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside is the component of radix Polygoni Multiflori, naringin is the component of fructus Aurantii Immaturus, icariin is the component of herba Epimedii, and salvianolic acid B and tanshinone IIA are the components of radix Salviae Miltiorrhizae.

The invention adopts UPLC to simultaneously determine the contents of puerarin, daidzin, 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside, naringin, salvianolic acid B, icariin and tanshinone IIA in the preparation, realizes comprehensive evaluation and control of the quality of Xintong granules, and thus provides accurate basis for true and false identification and internal quality detection of the Xintong granules.

A quality detection method of Xintong granules comprises the following steps:

A. preparation of a test solution: taking a proper amount of Xintong granules, grinding into fine powder, precisely weighing 1g, placing in a 50ml volumetric flask, adding 50ml of 70-90% methanol, carrying out ultrasonic treatment for 30min, cooling, fixing the volume to a scale with 70-90% methanol, shaking up, filtering to obtain a test solution;

B. preparation of control solutions: accurately weighing appropriate amount of puerarin, daidzin, 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside, naringin, salvianolic acid B, icariin and tanshinone IIA reference substances, placing in a volumetric flask, adding 70-90% methanol for dissolving and diluting to scale, and shaking;

C. UPLC chromatographic conditions: performing gradient elution by using acetonitrile A-0.1% phosphoric acid solution B as a mobile phase; the detection wavelength is 250nm-320 nm; the sample volume is 2 mu L;

D. the determination method comprises the following steps: and D, precisely sucking 2 mu L of the reference solution and the test solution respectively, injecting into a liquid chromatograph, and measuring according to the chromatographic conditions in the step C to obtain the test solution.

Preferably, 50ml of 80% methanol is added in the step A, ultrasonic treatment is carried out for 30min, cooling is carried out, volume is determined to the scale with 80% methanol, and shaking is carried out uniformly.

Preferably, in the step B, a proper amount of puerarin, daidzin, 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside, naringin, salvianolic acid B, icariin and tanshinone IIA reference substances are precisely weighed, placed in a volumetric flask, added with 80% methanol for dissolving and diluting to the scale, and shaken uniformly.

The wavelength switching procedure of the step C is as follows:

time, min	Detection wavelength, nm
		0	250
15	320
		23	283
36	270
		56	270

Switching wavelength, detecting puerarin and daidzin at 0-15min and 250nm for 15-23min and detecting 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside at 320 nm; detecting naringin and salvianolic acid B at 283nm within 23-36 min; detecting icariin and tanshinone IIA at 270nm for 36-56 min.

Preferably, the gradient content and flow rate gradient change elution conditions of the mobile phase in the step C are as follows:

time, min	Flow rate, ml/min	Mobile phase A,%	Mobile phase B,%
				0-27	0.5→0.3	5％→20％	95％→80％
27-40	0.3	20％→35％	80％→65％
				40-45	0.3→0.5	35％→60％	65％→40％
45-55	0.5	60％	40％
				55-56	0.5	60％→5％	40％→95％

。

Compared with the prior art, the invention has the following beneficial technical effects:

1) the invention establishes an upgraded quality detection method for Xintong granules, adopts UPLC to quantitatively and simultaneously determine the contents of 7 components of puerarin, daidzin, 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside, naringin, salvianolic acid B, icariin and tanshinone IIA in a preparation, can further effectively control the quality of the Xintong granules, and realizes the comprehensive evaluation of the quality of the preparation.

2) The invention successfully integrates UPLC content determination of 7 components of puerarin, daidzin, 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside, naringin, salvianolic acid B, icariin and tanshinone IIA in the preparation into a whole by a gradient elution procedure and a wavelength switching method, and provides a novel method for more comprehensively and accurately detecting Xintong granules.

3) The method provided by the invention can be used for simultaneously detecting 7 components in the Xintong particles by one-time multi-evaluation, so that the synchronous detection of the multiple components is realized, the operation method is simplified, the detection efficiency is improved, the consumables and the labor cost are saved, and the detection method which is more rapid, convenient, energy-saving and environment-friendly is provided.

4) Compared with the existing quality detection method for Xintong particles, the method disclosed by the invention has the advantages of rapidness, sensitivity, good repeatability and the like, can accurately detect 7 components in the Xintong particles, is strong in specificity and good in reproducibility, can comprehensively and effectively control the quality of the Xintong particles, is beneficial to stabilizing the quality of products, ensures the safety and effectiveness of clinical medication, and better meets the requirements of medical treatment and market.

Drawings

FIG. 1 is a UPLC chromatogram of a reference substance, wherein the chromatographic peaks sequentially comprise puerarin, daidzin, 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside, naringin, salvianolic acid B, icariin and tanshinone IIA from left to right;

FIG. 2 is a UPLC chromatogram of a test sample;

FIG. 3 is a UPLC chromatogram of a negative control lacking radix Puerariae;

FIG. 4 is a UPLC chromatogram of a negative control lacking Polygonum multiflorum;

FIG. 5 is a UPLC chromatogram of a negative control lacking immature bitter orange;

FIG. 6 is a UPLC chromatogram of negative control lacking Salvia miltiorrhiza;

FIG. 7 is a UPLC chromatogram of the barrenwort negative control.

Detailed Description

The invention is further illustrated by the following specific examples.

Example 1 UPLC assay methodology Studies of 7 ingredients of Xintong granules

1 drugs and reagents

1.1 drugs and reagents: puerarin control (batch No. 110752-; the methanol and the acetonitrile are chromatographically pure, the water is self-made ultrapure water, the acetonitrile and the methanol are chromatographically pure, the rest reagents are analytically pure, and the water is double distilled water.

1.2 reagent

Xintong granules were provided by the pharmaceutical company, Jianpu, Lunan, and the sample lot numbers are shown in Table 1.

TABLE 1 Xintong granule test sample batch number

2, equipment: waters Acquity Arc high performance liquid chromatograph (usa): 2998PDA detector.

3, chromatographic conditions: a chromatographic column:c18 column (4.6X50 mm, 2.7 μm); acetonitrile is used as a mobile phase A, a phosphoric acid aqueous solution with the volume percentage of 0.1 percent is used as a mobile phase B, and the column temperature:30 ℃; sample introduction volume: 2 mu l of the solution;

table 2 mobile phase gradient content and flow rate variation:

time (min)	Flow rate (ml/min)	Mobile phase A (%)	Mobile phase B (%)
				0-27	0.5→0.3	5％→20％	95％→80％
27-40	0.3	20％→35％	80％→65％
				40-45	0.3→0.5	35％→60％	65％→40％
45-55	0.5	60％	40％
				55-56	0.5	60％→5％	40％→95％

TABLE 3 wavelength switching procedure

Time (min)	Detection wavelength (nm)
		0	250
15	320
		23	283
36	270
		56	270

4 preparation of the solution

4.1 preparation of test solutions

Taking a proper amount of Xintong granules, grinding, taking about 1g, precisely weighing, placing in a 50ml volumetric flask, adding a proper amount of 80% methanol 50ml, carrying out ultrasonic treatment for 30min, cooling, fixing the volume to a scale with 80% methanol, shaking up, filtering to obtain a sample solution;

4.2 preparation of control solutions

Accurately weighing appropriate amount of puerarin, daidzin, 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside, naringin, salvianolic acid B, icariin and tanshinone IIA reference substances, and adding methanol to obtain mixed solution containing 172.16 μ g, 41.64 μ g, 7.38 μ g, 11.84 μ g, 72.88 μ g, 9.28 μ g and 3.12 μ g per 1 mL.

4.3 preparation of negative control solution

Taking negative control containing no radix Puerariae, negative control containing no Polygoni Multiflori radix, negative control containing no fructus Aurantii Immaturus, negative control containing no Saviae Miltiorrhizae radix, and negative control containing no herba Epimedii, and performing the same method according to the preparation of the test solution to obtain negative control solution, wherein the chromatogram shows that there is no interference at the positions of puerarin, daidzin, 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside, naringin, salvianolic acid B, icariin, and tanshinone IIA, as shown in fig. 1-7.

5 Linear relationship investigation

Accurately weighing appropriate amount of reference substance, adding 80% methanol to obtain the following concentrations, measuring under the above chromatographic conditions, respectively injecting 2 μ L sample, and measuring peak area. The results are shown in tables 4, 5, 6, 7, 8, 9 and 10.

TABLE 4 Puerarin Linear relationship examination results

TABLE 5 Soyabean glycoside Linear relationship examination results

TABLE 62, 3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside Linear relationship examination results

TABLE 7 naringin Linear relationship examination results

TABLE 8 examination of the Linear relationship of salvianolic acid B

TABLE 9 Linear relationship examination of icariin

TABLE 10 tanshinone IIA Linear relationship examination results

And (3) puerarin determination, taking the measured peak area as an ordinate and the concentration (mu g/mL) of a reference substance as an abscissa, drawing a standard curve, and obtaining a regression equation Y which is 23156.0534X-114200.6219 and r which is 0.99990, namely the concentration of the puerarin skin glycoside of the pomelo is good linearly within the range of 43.04-430.40 ug/mL.

The daidzin measurement is carried out by taking the measured peak area as an ordinate and the concentration of a reference substance (mu g/mL) as an abscissa, and drawing a standard curve, wherein the regression equation Y is 20444.8573X-13371.4329, and r is 0.99995, namely the daidzin concentration is good in linearity within the range of 6.94-69.40 mu g/mL.

In the measurement of 2,3,5,4 '-tetrahydroxystilbene-2-O-beta-D-glucoside, a standard curve is drawn by taking the measured peak area as an ordinate and the concentration (mu g/mL) of a reference substance as an abscissa, and the regression equation Y is 49621.7480X-38470.6000 and r is 0.99986, namely, the concentration of 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside is in a range of 2.46-24.60 mu g/mL, and has good linearity.

And (3) measuring the naringin, taking the measured peak area as an ordinate and the concentration (mu g/mL) of a reference substance as an abscissa, drawing a standard curve, and obtaining a regression equation Y which is 12748.3939X-6586.7757 and r which is 0.99979, namely the naringin concentration is good in linearity within the range of 2.96-29.6 mu g/mL.

When the salvianolic acid B is measured, a standard curve is drawn by taking the measured peak area as an ordinate and taking the concentration (mu g/mL) of a reference substance as an abscissa, and the regression equation Y is 8413.9412X-29594.4274, and r is 0.99992, namely the neohesperidin concentration is good in linearity within the range of 18.22-182.20 mu g/mL.

And (3) measuring icariin, taking the measured peak area as an ordinate and the concentration (mu g/mL) of a reference substance as an abscissa, drawing a standard curve, and obtaining a regression equation Y of 16237.8413X-7986.2575 and r of 0.99992, namely the concentration of the aloin is good linearly in the range of 2.32-23.20 mu g/mL.

And (3) measuring tanshinone IIA, taking the measured peak area as an ordinate and the concentration (mu g/mL) of a reference substance as an abscissa, drawing a standard curve, and obtaining a regression equation Y which is 25382.8899X-3052.5616 and r which is 0.99998, namely the concentration of the aloin is good in linearity within the range of 1.04-10.40 mu g/mL.

6 precision test

And precisely sucking the reference substance solution, and repeatedly injecting the sample for 6 times, wherein each time is 2 mu L, and the peak area RSD is less than 2 percent, which indicates that the precision of the instrument is good. The results are shown in Table 11.

TABLE 11 results of precision test

7 stability test

The same sample solution is taken and respectively measured for 0, 2, 4, 6, 8, 12, 16, 20 and 24 hours after preparation, and the result shows that the sample is stable within 24 hours. The results are shown in Table 12.

Table 12 stability test results

8 repeatability test

6 parts of the same lot (07015001) of sample are respectively taken, and the content is respectively measured and calculated according to the sample preparation method operation under the item of 'sample measurement'. The results are shown in tables 13, 14, 15, 16, 17, 18 and 19.

TABLE 13 Puerarin repeatability test results

TABLE 14 Glycine repeatability test results

TABLE 152, 3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside repeatability test results

TABLE 16 Naringin repeatability test results

TABLE 17 salvianolic acid B renaturation test results

TABLE 18 icariin reproducibility test results

TABLE 19 tanshinone IIA repeatability test results

9 recovery test

6 parts of samples with known content are precisely weighed, 5mL of puerarin reference substance solution with 0.8608mg/mL, 8mL of daidzin reference substance solution with 0.1388mg/mL, 4mL of 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside reference substance solution with 0.0492mg/mL, 6mL of naringin reference substance solution with 0.0592mg/mL, 4.5 mL of salvianolic acid B with 0.3644mg/mL, 5mL of icariin with 0.0464mg/mL and 4mL of tanshinone IIA reference substance solution with 0.0208mg/mL are precisely added respectively, and the determination is carried out according to the preparation method under the item ' preparation of test solution '. The results are shown in tables 20, 21, 22, 23, 24, 25 and 26.

TABLE 20 Puerarin recovery test results

TABLE 21 Glycine recovery test results

TABLE 222, 3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside recovery test results

TABLE 23 naringin recovery test results

TABLE 24 recovery test results for salvianolic acid B

TABLE 25 icariin recovery test results

TABLE 26 tanshinone IIA recovery test results

The results of measuring the contents of 7 components in 1024 batches of Xintong granules are shown in Table 27

TABLE 2724 contents of 7 ingredients in bulk Xintong granules

Example 2 UPLC assay of 7 ingredients of Xintong granules

A. Preparation of a test solution: taking a proper amount of Xintong granules, grinding into fine powder, precisely weighing 1g, placing in a 50ml volumetric flask, adding 50ml of 70% methanol, carrying out ultrasonic treatment for 30min, cooling, fixing the volume to a scale with 70% methanol, shaking up, and filtering to obtain a test solution;

B. preparation of control solutions: accurately weighing appropriate amount of puerarin, daidzin, 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside, naringin, salvianolic acid B, icariin and tanshinone IIA reference substances, placing in a volumetric flask, adding 70% methanol for dissolving and diluting to scale, and shaking;

C. UPLC chromatographic conditions: performing gradient elution by using acetonitrile A-0.1% phosphoric acid solution B as a mobile phase; the detection wavelength is 250nm-320 nm; the sample volume is 2 mu L; the mobile phase gradient content, flow rate variation and wavelength switching procedure are shown in the following table:

time (min)	Detection wavelength (nm)
		0	250
15	320
		23	283
36	270
		56	270

D. The determination method comprises the following steps: and D, precisely sucking 2 mu L of the reference solution and the test solution respectively, injecting into a liquid chromatograph, and measuring according to the chromatographic conditions in the step C to obtain the test solution.

Example 3 UPLC assay of 7 ingredients of Xintong granules

A. Preparation of a test solution: taking a proper amount of Xintong granules, grinding into fine powder, precisely weighing 1g, placing in a 50ml volumetric flask, adding 50ml of 90% methanol, carrying out ultrasonic treatment for 30min, cooling, fixing the volume to a scale with 90% methanol, shaking up, and filtering to obtain a test solution;

B. preparation of control solutions: precisely weighing appropriate amount of puerarin, daidzin, 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside, naringin, salvianolic acid B, icariin and tanshinone IIA reference substances, placing in a volumetric flask, adding 90% methanol for dissolving and diluting to scale, and shaking;

C. UPLC chromatographic conditions: performing gradient elution by using acetonitrile A-0.1% phosphoric acid solution B as a mobile phase; the detection wavelength is 250nm-320 nm; the sample volume is 2 mu L; the mobile phase gradient content, flow rate variation and wavelength switching procedure are shown in the following table:

time (min)	Flow rate (ml/min)	Mobile phase A (%)	Mobile phase B (%)
				0-27	0.5→0.3	5％→20％	95％→80％
27-40	0.3	20％→35％	80％→65％
				40-45	0.3→0.5	35％→60％	65％→40％
45-55	0.5	60％	40％
				55-56	0.5	60％→5％	40％→95％

D. The determination method comprises the following steps: and D, precisely sucking 2 mu L of the reference solution and the test solution respectively, injecting into a liquid chromatograph, and measuring according to the chromatographic conditions in the step C to obtain the test solution.