CN113466382B

CN113466382B - Content determination method of traditional Chinese medicine preparation for treating cervicodynia

Info

Publication number: CN113466382B
Application number: CN202110817521.6A
Authority: CN
Inventors: 贾庆文; 林永强; 贾美春; 刘洪超; 刘杰; 崔伟亮; 江玉娟; 陈俊亮
Original assignee: SHANDONG MINGREN FURUIDA PHARMACEUTICAL CO Ltd
Current assignee: SHANDONG MINGREN FURUIDA PHARMACEUTICAL CO Ltd
Priority date: 2021-07-19
Filing date: 2021-07-19
Publication date: 2023-04-14
Anticipated expiration: 2041-07-19
Also published as: CN113466382A

Abstract

The invention discloses a content determination method of a traditional Chinese medicine preparation for treating cervicodynia. The invention takes a traditional Chinese medicine cervicodynia preparation sample to be added with methanol for heating reflux extraction, the solvent is recovered till the sample is dry, the residue is dissolved by adding water, and chloroform is used for extracting and removing impurities; extracting the extractive solution with water saturated n-butanol under shaking, washing with ammonia solution, evaporating to dry, and dissolving the residue with methanol to obtain sample solution; then measuring notoginsenoside R in the test solution by high performance liquid chromatography and ultraviolet detector ₁ Ginsenoside R, ginsenoside R _g1 Ginsenoside R, and ginsenoside R _b1 The content of (a). The method improves the treatment method of sample solution to control notoginsenoside R in Notoginseng radix as main ingredient in JINGTONG granule ₁ Ginsenoside Rg ₁ Ginsenoside Rb ₁ The content of the panax notoginseng saponins in the product is detected more truly by comparing the Chinese pharmacopoeia detection method, so that the quality of the preparation is more accurately controlled, the extraction recovery rate of the sample is improved, and the content of the panax notoginseng saponins in the product is more truly detected.

Description

Content determination method of traditional Chinese medicine preparation for treating cervicodynia

Technical Field

The invention relates to a content determination method of a traditional Chinese medicine preparation for treating cervicodynia, and belongs to the technical field of medicine detection.

Background

The preparation for treating the cervical pain (comprising a cervical pain tablet and a cervical pain granule) is a traditional Chinese medicine compound preparation for treating nerve-root type cervical spondylosis, and is prepared from the following medicinal materials in parts by weight: 250g of pseudo-ginseng, 750 g of szechuan lovage rhizome, 500 g of corydalis tuber, 100 g of notopterygium root, 750 g of white paeony root, 1000g of clematis root and 750 g of kudzuvine root. The preparation for treating cervical pain is clinically favored by patients due to good effect of treating the cervical spondylosis caused by the nerve root and obvious curative effect through clinical verification. The cervical pain tablet is not collected in any pharmacopoeia, and the cervical pain granules are collected in 2020 edition of Chinese pharmacopoeia, have the effects of promoting blood circulation, removing blood stasis, promoting qi circulation and relieving pain, are used for nerve root type cervical spondylosis, belong to blood stasis, qi stagnation and venation obstruction symptoms, and have the symptoms of neck, shoulder and upper limb pain, and stiff or scurrying and pain.

The quality standard of the existing neck pain granule carries the determination of the content of the panax notoginseng saponins in the panax notoginseng, and the ginsenoside Rg is respectively determined ₁ Ginsenoside Rb and ginsenoside Rb ₁ And notoginsenoside R ₁ And calculating the total amount of each bag to be not less than 50mg. In the process of measuring the content of the panax notoginseng saponins, the preparation of a sample solution is complicated, the cost is high, and the problem of obviously low recovery rate (the recovery rate is 81-87%) occurs, so that the accuracy of content measurement is directly influenced. Therefore, the quality detection of the preparation product for treating the neck pain is not accurate, and the quality standard needs to be improved.

Disclosure of Invention

Aiming at the problem that the recovery rate of a traditional Chinese medicine preparation for treating cervicodynia in pharmacopoeia is obviously low, the invention provides a content determination method of the traditional Chinese medicine preparation for treating cervicodynia.

The technical scheme of the invention is as follows: a content determination method of a traditional Chinese medicine preparation for treating cervicodynia is characterized in that,

(1) Preparation of test solution

Extraction: adding methanol into the test sample of the Chinese medicinal preparation for treating cervicodynia, reflux-extracting under heating, recovering solvent to dryness, dissolving the residue in water, extracting with chloroform under shaking to remove impurities, and discarding chloroform solution;

and (3) extraction: extracting the extractive solution with water saturated n-butanol under shaking, mixing n-butanol extractive solutions, washing with ammonia test solution, mixing ammonia washing solutions, extracting with water saturated n-butanol under shaking, and mixing the extractive solution with the above n-butanol solution;

volume fixing: evaporating to dryness, dissolving the residue with methanol, transferring to a measuring flask, adding methanol to scale, shaking, filtering, and collecting the filtrate as sample solution;

(2) Measuring notoginsenoside R in sample solution by high performance liquid chromatography and ultraviolet detector ₁ Ginsenoside Rg ₁ Ginsenoside Rb and ginsenoside Rb ₁ The content of (a);

the chromatographic conditions were: octadecylsilane chemically bonded silica is used as a filling agent; taking acetonitrile as a mobile phase A and water as a mobile phase B, performing gradient elution, wherein the detection wavelength is 203nm, the flow rate is 1.0ml/min, and the gradient elution procedure is as follows:

。

further, the preparation of the test solution is as follows: taking a sample, grinding, taking 1-1.5g, precisely weighing, precisely adding 50ml of methanol, weighing, heating and refluxing for 1 hour, cooling, weighing again, complementing the weight loss with methanol, shaking up, filtering, precisely taking 25ml of a subsequent filtrate, recovering the solvent to dryness, adding 20ml of water into the residue for dissolving, shaking up and extracting for 2 times with chloroform, 20ml each time, discarding the chloroform solution, further shaking up and extracting for 4 times with water-saturated n-butyl alcohol, 20ml each time, combining the n-butyl alcohol solutions, washing for 2 times with an ammonia test solution, 20ml each time, separately taking the n-butyl alcohol solution, combining the ammonia washing solution and extracting for 2 times with water-saturated n-butyl alcohol, 20ml each time, combining the extracting solution and the n-butyl alcohol solution, evaporating to dryness, adding methanol into the residue and dissolving and transferring to a 10ml measuring flask, adding methanol to a scale, filtering, shaking up, and taking a subsequent filtrate to obtain the product.

The preparation for relieving cervicodynia comprises a tablet for relieving cervicodynia and a granule for relieving cervicodynia.

Further, the chromatographic column may be: thermo BDS Hypersil C18; shim-pack GISS C18 Shijin; agilent extended C18, 5 μm in specification, 4.6X 250mm.

The invention has the beneficial effects that: the traditional Chinese medicine compound preparation has various medicinal flavors and complex components, and the method for measuring the content controls the notoginsenoside R in the panax notoginseng as the main medicine in the granular preparation for treating the cervicodynia by innovatively improving the treatment method of the test solution ₁ Ginsenoside Rg ₁ Ginsenoside Rb ₁ The content of the panax notoginseng saponins in the product is more truly detected, thereby more accurately controlling the quality of the preparation, improving the extraction recovery rate (the recovery rate is 96-102%) of the test sample compared with the detection method of Chinese pharmacopoeia.

Drawings

FIG. 1 is an HPLC chromatogram of different mobile phases of example 1;

FIG. 2 is a chromatogram of a sample from a different column according to example 1;

FIG. 3 is a washing condition examination of impurity removal before extraction in example 1;

FIG. 4 shows notoginsenoside R of example 1 ₁ A standard curve;

FIG. 5 shows ginsenoside R of example 1 _g1 A standard curve;

FIG. 6 shows ginsenoside R of example 1 _b1 A standard curve;

FIG. 7 is a proprietary experimental HPLC chromatogram of example 1;

FIG. 8 is a proprietary experimental HPLC chromatogram of example 2.

Detailed Description

The above-described scheme is further illustrated below with reference to specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. The conditions used in the examples may be further adjusted according to the conditions of the particular manufacturer, and the conditions not specified are generally the conditions in routine experiments. Wherein the preparation of the ammonia test solution comprises the following steps: adding water into 400ml of concentrated ammonia solution to 1000ml to obtain the product.

Example 1: content determination of neck pain granules

1. Determination of content

1. Instrument, reagent and test sample

The instrument comprises: shimadzu LC-20A high performance liquid chromatograph; mettlerToledo XSE205 electronic balance;

control and source:

notoginseng radix saponin R ₁ The Chinese food and drug testing research institute provides, batch number: 110745-201619, content is 95.0%;

ginsenoside Rg ₁ The Chinese food and drug testing institute provides, batch number: 110703-201832, the content is 92.4%;

ginsenoside Rb ₁ The Chinese food and drug testing institute provides, batch number: 110704 to 201827, the content is 91.2 percent.

Reagent: acetonitrile was chromatographically pure, water was purified water prepared from Millipore, and all other reagents were analytically pure.

A sample to be tested: cervicodynia granules (Shandong Mingren Furuida pharmaceutical Co., ltd.) batch number: 19101019, 20101006, 20101007, 20101009, 20101010, 20101011, 20101012, 20101013, 20101014, 20101015, 20101006.

2. Chromatographic conditions

Measuring by high performance liquid chromatography (high performance liquid chromatography 0512 of the four parts of China pharmacopoeia 2020 edition).

Octadecylsilane chemically bonded silica is used as a filling agent, the column temperature is 35 ℃, and the sample injection amount is as follows: 10 mu l of the mixture; acetonitrile is used as a mobile phase A, water is used as a mobile phase B, gradient elution is carried out according to the following table, the detection wavelength is 203nm, and the flow rate is 1.0ml/min.

3. Preparation of control solutions

Precisely weighing notoginsenoside R ₁ Control 13.51mg was placed in a 25ml measuring flask, dissolved and diluted to the mark with methanol and shaken well. Accurately weighing ginsenoside Rg ₁ Reference substance 14.30mg and ginsenoside Rb ₁ Placing 14.16mg of control substance in a 25ml measuring flask, and precisely transferring the above notoginsenoside R ₁ Adding appropriate amount of methanol into 5ml of control sample, dissolving, diluting to scale, and shaking. (C) _R1 0.1027mg/ml、C _Rg1 0.5285mg/ml、C _Rb1 0.5166mg/ml)。

4. Preparation of test solution

Taking the product, grinding, taking about 1.5g, accurately weighing, accurately adding 50ml of methanol, weighing, heating and refluxing for 1 hour, cooling, weighing again, complementing the lost weight with methanol, shaking up, filtering, accurately taking 25ml of subsequent filtrate, recovering solvent to dryness, adding 20ml of water to dissolve residues, shaking up and extracting 2 times with chloroform, 20ml each time, discarding chloroform solution, further shaking up and extracting 4 times with water-saturated n-butyl alcohol, 20ml each time, combining n-butyl alcohol extracts, washing 2 times with ammonia test solution, 20ml each time, separating n-butyl alcohol solutions, combining ammonia washing solutions, shaking up and extracting 2 times with water-saturated n-butyl alcohol, 20ml each time, combining the extracting solution with the n-butyl alcohol solution, evaporating to dryness, dissolving residues with methanol, transferring to a 10ml measuring flask, adding methanol to scale, shaking up, taking the subsequent filtrate, and obtaining the product.

5. Measurement of

Determining notoginsenoside R in test solution by high performance liquid chromatography and ultraviolet detector ₁ Ginsenoside Rg ₁ Ginsenoside Rb and ginsenoside Rb ₁ The content of (a).

2. Methods and discussion

1. Comparison of Mobile phases

Referring to the first part of the 'Chinese pharmacopoeia' 2020 edition and related documents, three gradient elution mobile phases are examined, which are shown in tables 1 to 3:

TABLE 1 mobile phase (1)

TABLE 2 mobile phase (2)

TABLE 3 mobile phase (3)

The sample solution and the control solution were each drawn up by 10. Mu.l, and injected into a liquid chromatograph to perform measurement under three mobile phase conditions. The results are shown in FIG. 1 and Table 4.

TABLE 4 examination of the Mobile phase

Note: notoginseng radix saponin R in mobile phase 1 ₁ Cannot be separated from the hetero-peak.

From the measurement results, it can be seen that the target peak notoginsenoside R is obtained when the mobile phase 1 is used for gradient elution ₁ Cannot be completely separated; gradient elution is carried out by using a mobile phase 2, separation of three target chromatographic peaks and adjacent peaks is good, and the separation degree meets the requirement; notoginseng radix saponin R in mobile phase 3 ₁ Suspected periwinkle, ginsenoside Rb ₁ The separation degree is not good, so that the mobile phase 2 is determined as the mobile phase, and the notoginsenoside R is determined ₁ Ginsenoside Rg ₁ And ginsenoside Rb ₁ Has a retention time of 27.63, 31.59 and 58.66min, respectively.

2. Comparison of chromatographic columns

Three chromatographic columns were investigated: (1) thermo BDS Hypersil C18; (2) shim-pack GISS C18 Shijin; (3) agilent extended C18, the specification is 5 μm,4.6 × 250mm; the test was carried out under the conditions determined in the above-mentioned content measurement. The results are shown in FIG. 2 and Table 5.

TABLE 5 examination of the columns

The results show that the notoginsenoside R ₁ Ginsenoside Rg ₁ Ginsenoside Rb and ginsenoside Rb ₁ The chromatographic peak has good shape, the separation degree is more than 1.5, and the application range of test conditions is wide. Determining the number of theoretical plates according to notoginsenoside R by referring to the above measurement results and pharmacopoeia ₁ The peak calculation should be not less than 3000.

3. Examination of method for preparing test solution

(1) Investigation of sample size

Preparation of a test solution: taking a proper amount of the product under the condition of different loading amounts, grinding, respectively taking about 1g, 1.5g and 2g, precisely weighing, precisely adding 50ml of methanol, weighing, heating and refluxing for 1 hour, cooling, weighing again, supplementing the reduced weight with methanol, shaking up, filtering, precisely taking 25ml of subsequent filtrate, recovering solvent to dryness, adding 20ml of water to the residue to dissolve, shaking up and extracting with chloroform for 2 times, 20ml each time, discarding chloroform solution, shaking up and extracting with water-saturated n-butanol for 4 times, 20ml each time, combining n-butanol extractive solutions, washing with ammonia test solution for 2 times, 20ml each time, collecting n-butanol solutions, combining ammonia washing solutions, shaking up and extracting with water-saturated n-butanol for 2 times, 20ml each time, combining the extractive solution with the above n-butanol solution, evaporating to dryness, dissolving the residue with methanol, transferring to a 10ml measuring flask, adding methanol to the scale, shaking up, filtering, and taking the subsequent filtrate.

Precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, measuring, and calculating content. The results are shown in Table 6.

TABLE 6 sample size examination results

Referring to the above results, the amount of solvent used, the liquid phase signal response and the uniformity of sampling were comprehensively considered, and the amount of sampling was determined to be 1.5g.

(2) Examination of extraction solvent

Preparation of test solution, taking appropriate amount of the product under different loading amount, grinding, taking 3 parts, each part is about 1.5g, precisely weighing, adding 50ml of methanol, diluted ethanol and 70% ethanol respectively, weighing, heating and refluxing for 1 hour, cooling, weighing again, supplementing the lost weight with corresponding solvent, shaking, filtering, precisely weighing the filtrate 25ml, recovering solvent to dry, adding 20ml of water to the residue for dissolving, shaking and extracting with chloroform for 2 times, each 20ml, discarding chloroform solution, shaking and extracting with water saturated n-butanol for 4 times, each 20ml, mixing n-butanol extractive solutions, washing with ammonia test solution for 2 times, each 20ml, separating n-butanol solution, mixing ammonia washing solutions, shaking and extracting with water saturated n-butanol for 2 times, each 20ml, mixing the extractive solution with the above n-butanol solution, evaporating, dissolving the residue with methanol, transferring to a 10ml measuring flask, adding methanol to scale, shaking, filtering, and continuously taking the filtrate.

Precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, measuring, and calculating content. The results are shown in Table 7.

TABLE 7 examination of extraction solvent

The extraction solvent was determined to use methanol, considering that methanol was used without secondary configuration and the results of the measurement were slightly higher in the solvent under investigation.

(3) Investigation of extraction time

Preparing a test solution, taking the product, grinding, taking 3 parts, each part being about 1.5g, precisely weighing, precisely adding 50ml of methanol, weighing, respectively heating and refluxing for 0.5 hour, 1 hour and 1.5 hours, cooling, weighing again, supplementing the reduced weight with methanol, shaking up, filtering, precisely weighing 25ml of subsequent filtrate, recovering the solvent to dryness, adding 20ml of water into the residue for dissolving, shaking up and extracting for 2 times with chloroform, each time 20ml, discarding the chloroform solution, further shaking up and extracting for 4 times with water-saturated n-butanol, each time 20ml, combining the n-butanol solutions, washing for 2 times with ammonia test solution, each time 20ml, respectively taking the n-butanol solution, combining the ammonia washing solutions, shaking up and extracting for 2 times with water-saturated n-butanol, each time 20ml, combining the extracting solution with the n-butanol solution, evaporating to dryness, adding methanol into the residue for dissolving, transferring to a 10ml measuring flask, adding methanol to scale, shaking up, filtering, and taking the subsequent filtrate.

Precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, measuring, and calculating content. The results are shown in Table 8.

TABLE 8 extraction of time finding results

The test results show that 1 hour can basically ensure the complete extraction of the target component.

(4) Examination of washing conditions for impurity removal before extraction

The preparation of the test solution takes the product, grinds it, takes 2 parts, each part is about 1.5g, accurately weighs, accurately adds methanol 50ml, weighs, heat reflux for 1 hour, cool it, weigh again, make up the weight lost with methanol, shake it evenly, filter, accurately weigh it takes 25ml filtrate, reclaim solvent to dry, the residue adds water 20ml to dissolve, process as stated in table 9, discard the washing liquid, shake and extract 4 times with water saturated n-butanol, 20ml each time, combine n-butanol extract, wash 2 times with ammonia test solution, 20ml each time, divide n-butanol solution, combine ammonia washing liquid, shake and extract 2 times with water saturated n-butanol, 20ml each time, the extract is combined with above-mentioned n-butanol solution, evaporate to dryness, the residue adds methanol and dissolves and transfers to 10ml measuring flask, add methanol to the scale, shake it evenly, take the continued filtrate, get the final product.

Precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring. The results are shown in FIG. 3.

TABLE 9 examination of washing conditions before extraction

Method 1 without pre-washing, and the result (figure 3) shows that the target peak ginsenoside Rg ₁ The front portion had interference and sample 2 was substantially removed after chloroform treatment, so it was determined that chloroform was used to shake wash 2 times before n-butanol extraction.

(5) Examination of the number of extractions

Preparation of test solution, taking appropriate amount of the product under different loading amount, grinding, taking 3 parts, each part is about 1.5g, precisely weighing, precisely adding 50ml of methanol, weighing, heating and refluxing for 1 hour, cooling, weighing again, supplementing the weight loss with methanol, shaking up, filtering, precisely weighing 25ml of filtrate, recovering solvent to dry, dissolving the residue with 20ml of water, shaking up and extracting with chloroform for 2 times, each time 20ml, discarding chloroform solution, shaking up and extracting with water saturated n-butanol for 3 times, 4 times, 5 times, each time 20ml, mixing n-butanol solutions, washing with ammonia test solution for 2 times, each time 20ml, separating n-butanol solution, mixing ammonia washing solution, shaking up and extracting with water saturated n-butanol for 2 times, each time 20ml, mixing the extractive solution with the above n-butanol solution, evaporating to dryness, dissolving the residue with methanol, transferring into a 10ml measuring flask, adding methanol to scale, shaking up, filtering, and taking the filtrate to obtain the final product.

Precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring. The results are shown in Table 10.

TABLE 10 examination of the number of extractions

As can be seen from the measurement results, the results of n-butanol extraction 4 times are equivalent to those of n-butanol extraction 5 times, and are slightly higher than those of n-butanol extraction 3 times, so that n-butanol extraction 4 times is determined.

(6) Examination of washing conditions after extraction

Preparation of test solution the appropriate amount of the product under the condition of different loading amounts is taken, ground, 3 parts of the product, each part of which is about 1.5g, precisely weighed, precisely added with 50ml of methanol, weighed, heated and refluxed for 1 hour, cooled, weighed again, the loss-reduced weight is complemented with methanol, shaken evenly, filtered, precisely taken for 25ml of continuous filtrate, the solvent is recovered till dry, the residue is added with 20ml of water for dissolving, chloroform is used for shaking and extracting for 2 times, each time 20ml is used for discarding chloroform liquid, water saturated n-butyl alcohol is used for shaking and extracting for 4 times, each time 20ml is used for washing, the n-butyl alcohol liquid is combined, ammonia test solution is used for washing for 2 times, each time 20ml is used for separating and extracting the n-butyl alcohol liquid, ammonia washing liquid is combined, water saturated n-butyl alcohol is used for shaking and extracting for 1 time, 2 times and 3 times, each time 20ml is used for shaking, extracting the extracting solution is combined with the n-butyl alcohol liquid, dried by distillation, the residue is added with methanol for dissolving and transferred to a 10ml measuring bottle, methanol is added to the scale, filtered evenly, and the filtrate is obtained.

Precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, measuring, and calculating content. The results are shown in Table 11.

TABLE 11 examination of backwash alkali number

According to the measurement result, the alkali liquor can be backwashed by n-butyl alcohol for 2 times to basically ensure that the lost target component can be recovered, so that the ammonia test solution is determined to be extracted by using water saturated n-butyl alcohol for 2 times.

3. Methodology validation

1. Linear relation

Precisely sucking 1. Mu.l, 3. Mu.l, 5. Mu.l, 10. Mu.l, 15. Mu.l, 20. Mu.l and 30. Mu.l of the control solutions, respectively, injecting into a liquid chromatograph, and measuring peak area integral values of the three control solutions, respectively. And respectively taking the sampling amount of the three photographs as a horizontal coordinate and the peak area integral value as a vertical coordinate, and performing linear regression to obtain a regression equation. The results are shown in tables 12 to 14.

TABLE 12 notoginsenoside R ₁ Linear relationship examination results

Notoginseng radix saponin R ₁ The regression equation of (1) is y =360654x +3791.3, and the correlation coefficient r =1.0000.

TABLE 13 ginsenoside Rg ₁ Linear relationship examination result

Ginsenoside Rg ₁ Regression equation y =424514x +16338, and correlation coefficient r =1.0000.

TABLE 14 ginsenoside Rb ₁ Linear relationship examination result

Ginsenoside Rb ₁ The regression equation y =306750x +15678, and the correlation coefficient r =1.0000.

The test result shows that the notoginsenoside R ₁ The sample injection amount of the reference substance is 0.1027-3.0810 mug, and the sample injection amount and the peak area integral value form a good linear relation; ginsenoside Rg ₁ The sample injection amount of the reference substance is in a good linear relation with the peak area integral value between 0.5285 and 15.855 mug; ginsenoside Rb ₁ The sample amount of the control sample is 0.5166-15.498 mug, and the sample amount and the peak area integral value have good linear relation, as shown in FIGS. 4-6.

2. Precision test

Precisely sucking 10 μ l of the control solution, injecting into liquid chromatograph, continuously sampling for 5 times, and measuring peak area integral value. The results are shown in Table 15.

TABLE 15 results of precision test

The test result shows that the precision of the instrument is good.

3. Sample stability test

Taking the product, and preparing into test solution according to the preparation method of test solution provided in the content determination. And respectively injecting samples once at 0, 2, 4, 8, 12 and 24 hours, wherein 10 mu l of samples are injected each time, and measuring peak area integral values of the three target components to observe the stability of the components to be detected of the test solution in the detection process. The results are shown in Table 16.

TABLE 16 stability test results

The test result shows that the test solution has good stability within 24 hours and can meet the measurement requirement.

4. Method repeatability inspection

Preparation of a test solution: taking the product, preparing the product into a test solution according to the preparation method of the test solution provided in the content measurement, and preparing 6 parts in parallel.

Precisely sucking 10 μ l of control solution and 10 μ l of each test solution, injecting into liquid chromatograph, measuring, and calculating content, the results are shown in Table 17.

TABLE 17 results of repeatability tests

The test result shows that the repeatability of the content determination method is good.

5. Sample application recovery test

Preparation of a control stock solution: precisely weighing notoginsenoside R ₁ Reference substance 18.06mg, ginsenoside Rg ₁ Control 72.15mg, ginsenoside Rb ₁ 69.75mg of the control was placed in a 500ml measuring flask, dissolved and diluted to the mark by adding methanol, and shaken up to serve as a control stock solution.

Preparation of a test solution: taking the product with known content (batch number: 20101009, sanchinoside Rg) ₁ The content of ginsenoside Rg is 1.985mg/g ₁ The content of 8.029mg/g and ginsenoside Rb ₁ 7.331 mg/g), grinding, collecting about 0.75g, precisely weighing, precisely adding 40ml and 10ml of methanol as stock solution of above control, weighing, heating under reflux for 1 hr, cooling, weighing again, supplementing lost weight with methanol, shaking, filtering, precisely weighing 25ml of filtrate, recovering solvent to dry, dissolving residue with 20ml of water, shaking with chloroform for 2 times, each 20ml, discarding chloroform solution, shaking with water-saturated n-butanol for 4 times, each 20ml, mixing n-butanol solutions, washing with ammonia test solution for 2 times, each 20ml, respectively collecting n-butanol solution, mixing ammonia washing solutions, shaking with water-saturated n-butanol for 2 times, each 20ml, mixing extractive solutions with the above n-butanol solutions, evaporating to dryness, dissolving residue with methanol, transferring to 10ml measuring flask, adding methanol, addingAnd (4) uniformly shaking the methanol to scale, filtering and taking the subsequent filtrate to obtain the product.

Precisely sucking 10 μ l of the reference solution and 10 μ l of each of the test solutions, injecting into liquid chromatograph, measuring, and calculating content. The results are shown in tables 18 to 20. Test results show that the content determination method for the three target components has good recovery rate.

TABLE 18 notoginsenoside R ₁ Sample recovery rate investigation result

TABLE 19 ginsenoside Rg ₁ Sample recovery rate investigation result

TABLE 20 ginsenoside Rb ₁ Investigation result of sample recovery rate

6. Specificity test

Preparation of negative control solution: removing other medicinal materials of Notoginseng radix, preparing into Notoginseng radix-deficient negative control sample according to the prescription proportion, and preparing into negative control solution according to the preparation method of the test solution.

Respectively and precisely sucking 10 mul of each of the reference substance solution, the test article solution under the repeatability test item and the negative reference solution, and injecting the solutions into a liquid chromatograph for measurement. Results in the chromatogram of the test sample having a corresponding chromatographic peak at the same position as the retention time of the chromatogram of the control, and the chromatogram of the negative control having no corresponding chromatographic peak. The other medicinal ingredients in the prescription have no interference to the measurement result. See fig. 7.

4. Sample assay

10 batches of samples of the cervicodynia granules were tested under the conditions determined by the above assay method, and the results are shown in Table 21.

TABLE 21 measurement results of neck pain granule content

The formula amount of the pseudo-ginseng is 250g, and all the pseudo-ginseng is raw powder which is directly fed. The lower limit of the theoretical content of the product is 43 mg/bag calculated by converting the total content limit of three saponins (water content is 14.0 percent and the total content of a dried product is 5.0 percent) and the prepared total amount is 1000g (4 g/bag) specified by the prescription dosage pharmacopoeia, and 10 measured samples are all higher than the limit (67.75 mg/bag-75.64 mg/bag, average 70.33 mg/bag).

In conclusion, the traditional Chinese medicine compound preparation has various medicinal flavors and complex components, and the method for measuring the content controls the notoginsenoside R in the panax notoginseng as the main medicine in the granular preparation for controlling the cervicodynia by innovatively improving the treatment method of the test solution ₁ Ginsenoside Rg ₁ Ginsenoside Rb ₁ The content of the panax notoginseng saponins in the product is detected more truly by comparing the Chinese pharmacopoeia detection method, so that the quality of the preparation is more accurately controlled, the extraction recovery rate of the sample is improved, and the content of the panax notoginseng saponins in the product is more truly detected. The method compares the sample treatment methods of different mobile phase conditions, different chromatographic columns, different sampling amounts, different extraction solvents, different extraction time, different impurity removal methods, different extraction times, different washing conditions and the like, simultaneously researches and discovers that the acetonitrile is used as the mobile phase A, the water is used as the mobile phase B, the optimal gradient elution program improves the separation degree of the pharmacopeia method, and the ultraviolet detector is used for measuring the notoginsenoside R in the panax notoginseng ₁ Ginsenoside Rg ₁ Ginsenoside Rb ₁ A method for detecting the content of (a).

Example 2: content determination of tablets for relieving neck pain

1. Determination of content

1. Instrument, reagent and test sample

The instrument comprises the following steps: shimadzu LC-20A high performance liquid chromatograph; a mettler toledo XSE205 electronic balance;

control and source: notoginseng radix saponin R ₁ The Chinese food and drug testing research institute provides, batch number: 110745-201619, in an amount of 95.0%;

ginsenoside Rg ₁ The Chinese food and drug testing research institute provides, batch number: 110703-201832, in an amount of 92.4%;

ginsenoside Rb ₁ The Chinese food and drug testing research institute provides, batch number: 110704 to 201827, the content is 91.2 percent.

A test sample: cervicodynia tablet (Shandong Mingren Furuida pharmaceutical Co., ltd.) batch number: 20201001, 20201002, 20201003, 20201004, 20201005, 20201006, 20201007, 19201026, 19201029, 19201033.

2. Chromatographic conditions

Octadecylsilane chemically bonded silica is used as a filling agent; column temperature 35 ℃, sample injection amount: 10 mu l of the mixture; acetonitrile is used as a mobile phase A, water is used as a mobile phase B, gradient elution is carried out according to the following table, the detection wavelength is 203nm, and the flow rate is 1.0ml/min.

3. Preparation of control:

precisely weighing notoginsenoside R ₁ Putting 13.51mg of a reference substance into a 25ml measuring flask, adding methanol to dissolve and dilute the reference substance to a scale, and shaking up; accurately weighing ginsenoside Rg ₁ Reference substance 14.30mg and ginsenoside Rb ₁ Placing 14.16mg of control substance in a 25ml measuring flask, and precisely transferring the above notoginsenoside R ₁ Adding appropriate amount of methanol into 5ml of control sample, dissolving, diluting to scale, and shaking. (C) _R1 0.1027mg/ml、C _Rg1 0.5285mg/ml、C _Rb1 0.5166mg/ml)。

4. Preparing a test solution:

taking the product, grinding, taking about 1g, precisely weighing, precisely adding 50ml of methanol, weighing, heating and refluxing for 1 hour, cooling, weighing again, complementing the lost weight with methanol, shaking up, filtering, precisely taking 25ml of a subsequent filtrate, recovering the solvent to dryness, adding 20ml of water into the residue for dissolving, shaking up and extracting for 2 times with chloroform, 20ml each time, discarding the chloroform solution, further shaking up and extracting for 4 times with water saturated n-butyl alcohol, 20ml each time, combining the n-butyl alcohol extract, washing for 2 times with an ammonia test solution, 20ml each time, separately taking the n-butyl alcohol solution, combining the ammonia washing solutions, shaking up and extracting for 2 times with water saturated n-butyl alcohol, 20ml each time, combining the extracting solution with the n-butyl alcohol solution, evaporating to dryness, adding methanol to dissolve the residue and transferring to a 10ml measuring flask, adding methanol to scale, shaking up and filtering, and taking a subsequent filtrate to obtain the product.

5. Measurement of

Measuring the contents of notoginsenoside R1, ginsenoside Rg1 and ginsenoside Rb1 in the sample solution by high performance liquid chromatography and ultraviolet detector.

2. Methods and discussion

The test procedure is as described in the results and discussion of example 1, with the best results of the test results being shown in the test conditions for the assay described above.

3. Methodology validation

1. Sample stability test

This product was collected and prepared into a test solution according to the method for preparing a test solution as provided in example 2 above. And respectively injecting samples once at 0, 2, 4, 8, 12 and 24 hours, wherein 10 mu l of samples are injected each time, and measuring peak area integral values of the three target components to observe the stability of the components to be detected of the test solution in the detection process. The results are shown in Table 22.

TABLE 22 stability test results

2. Method repeatability inspection

Preparation of a test solution: the product was collected and prepared into a sample solution by the method for preparing a sample solution as described in example 2, and 6 parts of the sample solution were prepared in parallel.

Injecting 10 μ l of precision control solution and 10 μ l of each sample solution into liquid chromatograph, measuring, and calculating content. The results are shown in Table 23.

TABLE 23 repeatability test results

The test result shows that the repeatability of the content measuring method is good.

3. Sample application recovery test

Preparation of a control stock solution: precisely weighing notoginsenoside R ₁ Reference substance 18.06mg, ginsenoside Rg ₁ Control 72.15mg, ginsenoside Rb ₁ 69.75mg of the control was placed in a 500ml measuring flask, dissolved and diluted to the mark with methanol, and shaken up to serve as a control stock solution.

Preparation of a test solution: taking the product (batch number: 20101006, sanchinoside Rg) according to the weight difference ₁ The content is 2.524mg/g, and ginsenoside Rg ₁ The content of 10.325mg/g and ginsenoside Rb ₁ 9.424 mg/g), grinding, taking about 0.5g, precisely weighing, precisely adding 40ml of methanol and 10ml of methanol in stock solution of the above reference substances, weighing, heating and refluxing for 1 hour, cooling, weighing again, supplementing the weight loss with methanol, shaking up, filtering, precisely weighing 25ml of subsequent filtrate, recovering solvent to dryness, adding 20ml of water to residue for dissolving, shaking up with chloroform for 2 times, 20ml each time, discarding chloroform solution, shaking up with water-saturated n-butanol for 4 times, 20ml each time, combining n-butanol extractive solutions, washing with ammonia test solution for 2 times, 20ml each time, collecting n-butanol solution, combining ammonia washing solutions, shaking up with water-saturated n-butanol for 2 times, 20ml each time, combining the extractive solution with the above-mentioned alcohol solution, evaporating to dryness, adding methanol to dissolve residue, transferring to a 10ml measuring flask, adding methanol to scale, shaking up, taking the subsequent filtrate, and obtaining the final product.

Precisely sucking 10 μ l of control solution and 10 μ l of each test solution, injecting into liquid chromatograph, measuring, and calculating content. The results are shown in tables 24 to 26.

TABLE 24 notoginsenoside R ₁ Sample recovery rate investigation result

TABLE 25 ginsenoside Rg ₁ Sample recovery rate investigation result

TABLE 26 ginsenoside Rb ₁ Sample recovery rate investigation result

Test results show that the content determination method for the three target components has good recovery rate.

4. Specificity test

Preparation of negative control solution: removing other medicinal materials of Notoginseng radix, preparing into Notoginseng radix-deficient negative control sample according to prescription proportion, and preparing into negative control solution according to the preparation method of the sample solution.

Respectively and precisely sucking 10 mul of each of the reference substance solution, the test substance solution under the repeatability test item and the negative reference solution, and injecting the solution into a liquid chromatograph for measurement. Results in the chromatogram of the test sample having a corresponding chromatographic peak at the same position as the retention time of the chromatogram of the control, and the chromatogram of the negative control having no corresponding chromatographic peak. The other medicinal materials in the prescription have no interference to the measurement result, and the figure is shown in figure 8.

4. Determination of sample solutions

The results of the measurements on 10 batches of cervicodynia tablet samples according to the conditions determined by the above content measurement method are shown in table 27.

Table 27 assay results of cervicodynia tablets

The formula amount of the pseudo-ginseng is 250g, and all the pseudo-ginseng is raw powder which is directly fed. The lower limit of the theoretical content of the product is 10.75 mg/tablet according to the total content limit of three saponins (water content is 14.0 percent, the total amount of a dried product is 5.0 percent) and the total amount of the prepared product is 1000 tablets (0.67 g per tablet) specified by the prescription dosage pharmacopoeia, and 10 tested samples are all higher than the limit (14.17 mg/tablet-15.54 mg/tablet, average 14.93 mg/tablet).

Claims

1. A content determination method of a traditional Chinese medicine preparation for treating neck pain is characterized in that the preparation for treating neck pain is a tablet or a granule for treating neck pain, and the determination method comprises the following steps:

(1) Preparation of test solution

Taking a sample, grinding, taking 1-1.5g, precisely weighing, precisely adding 50ml of methanol, weighing, heating and refluxing for 1 hour, cooling, weighing again, complementing the weight loss with methanol, shaking up, filtering, precisely taking 25ml of a subsequent filtrate, recovering the solvent to dryness, adding 20ml of water into the residue for dissolving, shaking up and extracting for 2 times with chloroform, each time 20ml, discarding the chloroform solution, continuing to shake up and extract for 4 times with water-saturated n-butanol, each time 20ml, combining the n-butanol solutions, washing for 2 times with an ammonia test solution, each time 20ml, respectively taking the n-butanol solution, combining the ammonia washing solution with water-saturated n-butanol for 2 times, each time 20ml, combining the obtained extracting solution with the n-butanol solution, evaporating to dryness, dissolving the residue with methanol, transferring to a 10ml measuring flask, adding methanol to scale, filtering, shaking up, and taking a subsequent filtrate to obtain a sample solution;

(2) Measuring notoginsenoside R in sample solution by high performance liquid chromatography and ultraviolet detector ₁ Ginsenoside Rg ₁ Ginsenoside Rb ₁ The content of (A);

the chromatographic conditions were: octadecylsilane chemically bonded silica is used as a filling agent; taking acetonitrile as a mobile phase A and water as a mobile phase B, and carrying out gradient elution, wherein the gradient elution procedure is as follows:

。

2. the method for measuring the content of the traditional Chinese medicine preparation for treating cervicodynia according to claim 1, wherein a chromatographic column comprises: thermo BDS Hypersil C18; shim-pack GISS C18 or Agilent extended C18 of Shimadzu, the specification is 5 μm,4.6 × 250mm.