CN113640413B - A detection method of American ginseng components in ginsengguipi pills - Google Patents

Abstract

The invention discloses a method for detecting Chinese and western ginseng components in a ginseng spleen-invigorating pill, which comprises a high performance liquid chromatography-evaporative light scattering detector inspection method and a high performance liquid chromatography-tandem mass spectrometry inspection method. The method for detecting the components of the American ginseng and the Chinese western medicine in the ginseng spleen-invigorating pill can effectively improve the accuracy and the stability of sample detection through a reasonable detection method, parameter design and complete sample pretreatment measures, is convenient to popularize and use, can be used for detecting by two detection methods at the same time, and has strong applicability.

Description

A detection method of American ginseng components in ginsengguipi pills

technical field

The invention belongs to the technical field of traditional Chinese medicine detection, and more specifically relates to a method for detecting American ginseng components in ginseng guipi pills.

Background technique

Ginseng Guipi Pill is derived from the addition and subtraction of Guipi Pill in "Jisheng Fang" in the Song Dynasty. The prescription is composed of ten herbs such as ginseng, astragalus, angelica, longan meat, and jujube kernel. It has the effects of nourishing qi and blood, invigorating the spleen and nourishing the heart. Ginseng is sweet in taste, slightly bitter, warm in nature and flat. Return spleen, lung meridian, heart meridian. Invigorate qi, solidify, promote body fluid, soothe the nerves, improve intelligence; the main chemical components are saponins, flavonoids, volatile oils, etc. According to the Chinese Pharmacopoeia, both ginseng and American ginseng contain ginsenosides Rg1, Re and Rb1, ginseng contains a characteristic component of ginsenoside Rf, and American ginseng contains a characteristic component of pseudo-ginsenoside F ₁₁ . In order to strictly control the quality of this product, it is necessary to control the quality of ginseng in the prescription.

The existing standards lack a specific detection method for ginseng adulterated ingredients (American ginseng) in Renshen Guipi Pills. The detection methods for ginseng adulteration in other preparations are not reasonable enough and have narrow applicability. The pre-treatment of samples is not perfect, which affects the final detection. Accuracy and stability are not easy to promote and use.

In summary, how to design a detection method for American ginseng components in Ginseng Guipi Pills, which can effectively improve the accuracy and stability of sample detection and facilitate popularization and use, is an urgent problem to be solved at present.

Contents of the invention

The purpose of the present invention is to solve the above technical problems, and provide a detection method of American ginseng components in Ginseng Guipi Pills. The honey in the sample of Guipi Pills, on the other hand, improves the effect of impurity removal through the modification of the filter membrane in the sample filter, which can effectively improve the accuracy and stability of sample detection, which is convenient for popularization and use, and can be used for high-efficiency liquid at the same time It can be determined by phase chromatography-evaporative light scattering detector (HPLC-ELSD) inspection method and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) inspection method, with strong applicability.

The present invention realizes above-mentioned object through following technical scheme, a kind of detection method of American ginseng component in ginseng Guipi pills, comprises the following steps:

(1) Preparation of the test solution:

a. Remove honey from Ginseng Guipi Pills: Take 2 Ginseng Guipi Pills, cut them into pieces, add them to 50 ml of water, stir at 40-45°C for 10-15min, then filter, collect solid slag and filtrate, filtrate After the monosaccharide molecules are removed by semi-permeable membrane dialysis, the obtained product is mixed with the solid residue to obtain the material to be tested;

b. Take the above-mentioned material to be tested, add 100ml of water-saturated n-butanol, heat and reflux for 1 hour, filter, wash the filtrate twice with ammonia test solution, 50ml each time, discard the ammonia washing solution, take n-butanol solution, and use Wash with 50 ml of water saturated with n-butanol, evaporate the n-butanol layer to dryness, dissolve the residue in 2 ml of methanol, filter with a sample filter, and take the filtrate as the test solution;

(2) Preparation of reference solution:

Take Pseudoginsenoside F ₁₁ reference substance, weigh it accurately, add methanol to make reference substance solution;

(3) Preparation of negative control solution:

Take the missing ginseng medicinal material, prepare a negative control sample according to the prescription process, weigh 18 g, and then make a negative control solution lacking ginseng with the preparation method of the test solution;

(4) Determination method:

Use high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) inspection method or high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) inspection method to determine, set up the detection conditions, draw the test solution, control 10-20 μL each of sample solution and negative control solution, and inject and measure;

(5) Result judgment:

In the chromatogram of the test product, no chromatographic peak with the same retention time as the chromatographic peak of the reference substance should not appear.

Further, step (4) is detected by high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD), and the chromatographic conditions are as follows: chromatographic column: Capcell pak C18 (4.6 mm×250 mm, 5 μm), Mobile phase: acetonitrile-water (volume ratio 30:70); evaporative light scattering detector: gas flow rate: 1.6 L/min, flow rate: 1ml/min, column temperature: 30°C;

At this time, the preparation method of the reference substance solution in step (2) is: accurately weigh 10.05 mg of the pseudo-ginsenoside F ₁₁ reference substance, put it in a 50 ml measuring bottle, add methanol to dissolve and dilute to the mark, and shake well to obtain the reference substance solution .

Further, step (4) is detected by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), and the chromatographic conditions are:

Column Waters ACQUITY UPLC® BEH-C ₁₈ (2.1mm×100 mm, 1.7 μm); acetonitrile as mobile phase A, water as mobile phase B; flow rate 0.4 ml/min; column temperature 40 ℃; injection volume 3μL;

Gradient elution: 0-12 min, 8%-80% A; 12-14 min, 80%-90% A; 14-14.2 min, 90%-8% A; 14.2-20 min, 8% A; acetonitrile - The volume ratio of water is 30:70;

The mass spectrometry conditions are as follows: time-of-flight mass spectrometry is used as the detector, electrospray ionization ion source is used, negative ion mode is used for detection, Leucine-enkephalin is used as the calibration solution, the scanning range is m/z 50-1200, the sampling interval is 0.5 s, and the capillary voltage is 2.5 kV, the cone voltage is 40V, the ion source temperature is 100°C, the desolvation temperature is 450°C, the desolvation gas flow rate is 800 L•h ^-1 , the cone gas flow rate is 40 L•h ^-1 , and the collision gas is argon; The low collision energy is 6V, and the high collision energy is 20-40V;

At this time, the preparation method of the reference substance solution in step (2) is: take pseudo-ginsenoside F ₁₁ reference substance, weigh it accurately, add methanol to make a solution containing 50 µg per 1 ml, shake well, and obtain the reference substance solution.

Further, the preparation method of the semipermeable membrane in step (1) is:

A. After mixing the alkaline basalt nano powder with 0.1-0.3 times the amount of silicone oil, disperse it in a disperser at 1000-2000r/min for 1-2 hours to obtain nano powder, mix the nano powder with 40-60 times water Mix to make a dispersion;

B. Take a quantitative filter paper, use a glass rod to immerse it in 75% cold sulfuric acid, take out the filter paper after soaking for 70-80s, spray saturated carbonic acid solution on the surface of the filter paper, the spraying amount is 6-10%, put the filter paper in place after spraying Rinse in the dispersion in step A for 1-3 times, then soak in dilute ammonia water for 3-5 minutes, take it out and dry it in the air to obtain a semipermeable membrane.

Further, the filter membrane of the sample filter in step (1) is a modified regenerated cellulose filter membrane, and the pore size of the modified regenerated cellulose filter membrane is 0.2-0.5 μm.

Further, the preparation method of the modified regenerated cellulose filter membrane is:

S1. Preparation of controlled-release pore-forming agent: granulate the pore-forming agent for later use, dissolve poly-N2 isopropylacrylamide in deionized water at 24-26°C to make a material solution, and prepare the material solution at 31 Spray onto the surface of the granulated pore-forming agent at -33°C and 1-1.5MPa, then raise the temperature to 40-50°C and keep it warm for 20-30 minutes to obtain a controlled-release pore-forming agent;

S2. Preparation of film-making solution: dissolving cellulose in a solvent, then adding polylactic acid, controlled-release pore-forming agent and additives, mixing evenly, defoaming and filtering, to obtain a film-making solution;

S3. Membrane production: at a temperature of 80-84°C, cast the film-making solution prepared in step S2 on a glass plate to form a flat film, and then place the flat film at 40-50°C in a ventilated environment for 3-5s. Then stop the ventilation, lower the temperature of the flat film to below 30°C, let it stand for 30-50 minutes, then let it stand for 5-10 seconds in a ventilated environment, then put the flat film into the coagulation bath, solidify for 10-15 minutes to form a film, and use deionized The residual solvent in the membrane is washed with water, and then dried at room temperature to obtain a modified regenerated cellulose filter membrane.

Further, in step S1, the mass ratio of the material liquid to the pore-forming agent is (0.08-0.12): 1, and the mass concentration of polyN2 isopropylacrylamide in the material liquid is 4-8%; in step S2, the cellulose , polylactic acid, controlled-release pore-forming agent and additives have a mass ratio of 1: (0.3-0.6): (0.15-0.3): (0.02-0.05).

Further, in step S1, the pore-forming agent includes pore-forming agent A and pore-forming agent B, the mass ratio of which is 1: (0.3-0.6), and the pore-forming agent A and pore-forming agent B are granulated separately, The pore-forming agent A is polyethylene glycol, and the pore-forming agent B is sodium chloride and sodium alginate in a mass ratio of 1:(1.5-3).

Further, the solvent in step S2 is dimethyl sulfoxide/tetraethylammonium chloride with a ratio of (2-4):1, and the additive is fatty acid glyceride or polysorbate; the ventilation environment in step S3 is: Nitrogen or argon, the ventilation rate is 0.05-0.1m ³ /h.

The invention also discloses the application of the detection method in controlling the quality of ginseng in ginseng Guipi pills.

The beneficial effects of the present invention are:

(1) The present invention can effectively improve the accuracy and stability of sample detection through reasonable detection methods and parameter design, as well as perfect sample pretreatment measures, which is convenient for popularization and use, and can be used for high performance liquid chromatography-evaporative light at the same time Scattering detector (HPLC-ELSD) detection method, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) detection method, strong applicability;

(2) Since the target sample of the present invention, Ginseng Guipi Pills, contains a large amount of honey, the present invention first removes the honey in the Ginseng Guipi Pills with a semi-permeable membrane during sample pretreatment, which can reduce the amount of honey in the subsequent detection. the adverse effects caused;

(3) The surface of the semi-permeable membrane prepared by the present invention contains nano-powder mixed with alkaline basalt nano-powder and silicone oil, which can improve the smoothness of the surface of the semi-permeable membrane, so that in the process of dialysis to remove monosaccharide molecules, reduce Sample adsorption and loss, thus improving accuracy;

(4) In the present invention, when preparing the semipermeable membrane, the alkaline basalt nanopowder is introduced into the surface of the semipermeable membrane. The alkali content in the alkaline basalt is relatively high, and the surface of the filter paper reacted in concentrated sulfuric acid still has a lot of concentrated Sulfuric acid, at this time, introducing alkaline basalt nano-powder on the surface of the filter paper can strengthen the close combination of the nano-powder and the surface of the filter paper. In addition, the alkaline basalt nano-powder has good fluidity and lubricity, which can enhance the smoothness of the surface of the semi-permeable membrane;

(5) In the preparation of the semipermeable membrane, the present invention disperses silicone oil and alkaline basalt nanopowder at high speed to make nanopowder. On the one hand, after the silicone oil fills the pores of the alkaline basalt nanopowder, a super slippery surface can be formed; on the other hand, Silicone oil is a non-polar substance, and after it is introduced into the surface of the semi-permeable membrane, it can further reduce the adsorption and loss of samples;

(6) In the step B of preparing the semipermeable membrane in the present invention, after the surface of the filter paper is hydrolyzed, a saturated carbonic acid solution is sprayed on its surface, in order to decompose the process of releasing carbon dioxide during subsequent rinsing in the dispersion liquid, so as to promote the process of removing carbon dioxide on the surface of the filter paper. Material exchange, promote the combination of nano powder in the dispersion and the surface of the filter paper;

(7) Since the impurity content of the sample has a great influence on the detection process, the filter membrane of the sample filter of the present invention adopts a modified regenerated cellulose filter membrane, and the main preparation materials are cellulose and polylactic acid, which are environmentally friendly and degradable materials. Suitable for one-time use of sample filters in laboratories;

(8) The modified regenerated cellulose filter membrane prepared by the present invention, through the preparation and addition of a controlled-release pore-forming agent, not only can control the pore-forming time, but also make the pore-forming agent more evenly distributed, which can effectively increase the pore size of the membrane. High efficiency, uniform membrane pore size, high filtration efficiency, and effective removal of impurities;

(9) When preparing the modified regenerated cellulose filter membrane in the present invention, the pore-forming agent used is composed of pore-forming agent A and pore-forming agent B, and pore-forming agent A is polyethylene glycol, which can enhance membrane flux and form The pore agent B is sodium chloride and sodium alginate. When the outer coating material polyN2 isopropylacrylamide gel melts below 30°C, the water in the gel, sodium chloride and sodium alginate are released. Sodium alginate acts as a pore-forming agent in aqueous sodium chloride solution and forms a large number of pores in the membrane. The combination of pore-forming agent A and pore-forming agent B can significantly improve the membrane performance of the modified regenerated cellulose filter membrane .

Description of drawings

Fig. 1 is pseudo-ginsenoside F ₁₁ reference substance chromatogram;

Fig. 2 is negative control sample chromatogram;

Figure 3 is the chromatogram of the test product (A company sample, batch number: 17013851);

Figure 4 is the chromatogram of the test product (B enterprise sample, batch number: 20170205);

Figure 5 is a full-scan base peak diagram of pseudo-ginsenoside F ₁₁ ;

Fig. 6 is the primary mass spectrogram of pseudoginsenoside F ₁₁ ;

Fig. 7 is the secondary mass spectrogram of pseudoginsenoside F ₁₁ ;

Figure 8 is a negative control sample full-scan base peak diagram;

Figure 9 is the full-scan base peak diagram of the test product (sample from company B, batch number: 20170205);

Figure 10 is the primary mass spectrogram of the test product (sample from company B, batch number: 20170205);

Figure 11 is the secondary mass spectrogram of the test product (sample from company B, batch number: 20170205).

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Instruments and Reagents

1. Instrument

Agilent 1260 high-performance liquid chromatography; Agilent Technologies ELSD detector; AcquityUPLC®/Xevo® G2 QTof liquid mass spectrometer, MassLynx® V4.1 mass spectrometry workstation (Waters, USA); Sartorius BT 25S electronic balance.

2. Reagents and drugs

Ginseng Guipi pills: 10 batches of samples (big honey pills) from companies A and B.

Negative control sample: Take raw and auxiliary materials lacking ginseng medicinal materials, and make negative samples according to the preparation process of this product.

Pseudoginsenoside F ₁₁ : Source: China National Institutes for Food and Drug Control Batch number: 110841-201607.

Acetonitrile was chromatographically pure, and experimental water was deionized water.

Example 1

This embodiment provides a method for detecting the components of American ginseng in Ginseng Guipi Pills, comprising the following steps:

(1) Preparation of the test solution:

a. Remove honey from Ginseng Guipi Pills: Take 2 Ginseng Guipi Pills, cut them into pieces, add them to 50 ml of water, stir at 40°C for 10 minutes, then filter, collect the solid residue and filtrate respectively, and use a semipermeable membrane for the filtrate After dialysis to remove monosaccharide molecules, the obtained product is mixed with solid slag to obtain the material to be tested;

b. Take the above-mentioned material to be tested, add 100ml of water-saturated n-butanol, heat and reflux for 1 hour, filter, wash the filtrate twice with ammonia test solution, 50ml each time, discard the ammonia washing solution, take n-butanol solution, and use Wash with 50 ml of water saturated with n-butanol, evaporate the n-butanol layer to dryness, dissolve the residue in 2 ml of methanol, filter with a sample filter, and take the filtrate as the test solution;

(2) Preparation of reference solution:

Accurately weigh 10.05 mg of pseudo-ginsenoside F ₁₁ reference substance, put it in a 50 ml measuring bottle, add methanol to dissolve and dilute to the mark, shake well, and obtain the reference substance solution;

(3) Preparation of negative control solution:

Take the missing ginseng medicinal material, prepare a negative control sample according to the prescription process, weigh 18 g, and then make a negative control solution lacking ginseng with the preparation method of the test solution;

(4) Determination method:

Use high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) inspection method for determination, set up the detection conditions, draw 10 μL each of the test solution, reference solution, and negative control solution, and inject samples for determination;

(5) Result judgment:

In the chromatogram of the test product, no chromatographic peak with the same retention time as the chromatographic peak of the reference substance should not appear.

In step (4), the chromatographic conditions are: chromatographic column: Capcell pak C18 (4.6 mm×250 mm, 5 μm), mobile phase: acetonitrile-water (volume ratio 30:70); evaporative light scattering detector: gas flow rate: 1.6 L/min, flow rate: 1ml/min, column temperature: 30°C.

Example 2

This embodiment provides a method for detecting the components of American ginseng in Ginseng Guipi Pills, comprising the following steps:

(1) Preparation of the test solution:

a. Remove honey from Ginseng Guipi Pills: Take 2 Ginseng Guipi Pills, cut them into pieces, add them to 50 ml of water, stir at 45°C for 15 minutes, then filter, collect the solid residue and filtrate respectively, and use a semi-permeable membrane for the filtrate After dialysis to remove monosaccharide molecules, the obtained product is mixed with solid slag to obtain the material to be tested;

b. Take the above-mentioned material to be tested, add 100ml of water-saturated n-butanol, heat and reflux for 1 hour, filter, wash the filtrate twice with ammonia test solution, 50ml each time, discard the ammonia washing solution, take n-butanol solution, and use Wash with 50 ml of water saturated with n-butanol, evaporate the n-butanol layer to dryness, dissolve the residue in 2 ml of methanol, filter with a sample filter, and take the filtrate as the test solution;

(2) Preparation of reference solution:

Take Pseudoginsenoside F ₁₁ reference substance, weigh it accurately, add methanol to make a solution containing 50 µg per 1 ml, shake well, and obtain the reference substance solution;

(3) Preparation of negative control solution:

Take the missing ginseng medicinal material, prepare a negative control sample according to the prescription process, weigh 18 g, and then make a negative control solution lacking ginseng with the preparation method of the test solution;

(4) Determination method:

Use high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) inspection method for determination, set up the detection conditions, draw 20 μL each of the test solution, reference solution, and negative control solution, and inject samples for determination;

(5) Result judgment:

In the chromatogram of the test product, no chromatographic peak with the same retention time as the chromatographic peak of the reference substance should not appear.

In step (4), the chromatographic conditions are:

Column Waters ACQUITY UPLC® BEH-C ₁₈ (2.1mm×100 mm, 1.7 μm); acetonitrile as mobile phase A, water as mobile phase B; flow rate 0.4 ml/min; column temperature 40 ℃; injection volume 3μL;

Gradient elution: 0-12 min, 8%-80% A; 12-14 min, 80%-90% A; 14-14.2 min, 90%-8% A; 14.2-20 min, 8% A; acetonitrile - The volume ratio of water is 30:70;

The mass spectrometry conditions are as follows: time-of-flight mass spectrometry is used as the detector, electrospray ionization ion source is used, negative ion mode is used for detection, Leucine-enkephalin is used as the calibration solution, the scanning range is m/z 50-1200, the sampling interval is 0.5 s, and the capillary voltage is 2.5 kV, the cone voltage is 40V, the ion source temperature is 100°C, the desolvation temperature is 450°C, the desolvation gas flow rate is 800 L•h ^-1 , the cone gas flow rate is 40 L•h ^-1 , and the collision gas is argon; The low collision energy is 6V, and the high collision energy is 20-40V.

Example 3

On the basis of Example 1, this example also provides a method for detecting the components of American ginseng in Ginseng Guipi Pills. The method for preparing the semipermeable membrane in step (1) is as follows:

A. After mixing the alkaline basalt nano powder with 0.1 times the amount of silicone oil, place it in a disperser at 1000r/min to disperse for 1 hour to obtain a nano powder, mix the nano powder with 40 times water to make a dispersion;

B. Take a quantitative filter paper, use a glass rod to immerse it in 75% cold sulfuric acid, take out the filter paper after soaking for 70s, spray saturated carbonic acid solution on the surface of the filter paper, the spraying amount is 6%, and put the filter paper into the step A after spraying Rinse once in the dispersion liquid, then soak in dilute ammonia water for 3 minutes, take it out and dry it in the air to obtain a semi-permeable membrane.

All the other are identical with embodiment 1.

Example 4

On the basis of Example 2, this example also provides a method for detecting the components of American ginseng in Ginseng Guipi Pills. The method for preparing the semipermeable membrane in step (1) is as follows:

A. After mixing the alkaline basalt nano-powder with 0.3 times the amount of silicone oil, place it in a 2000r/min disperser for 2 hours to obtain a nano-powder, mix the nano-powder with 60 times the amount of water to make a dispersion;

B. Take a quantitative filter paper, use a glass rod to immerse it in 75% cold sulfuric acid, take out the filter paper after soaking for 80s, spray saturated carbonic acid solution on the surface of the filter paper, the spraying amount is 10%, put the filter paper into the step A after spraying Rinse in the dispersion for 3 times, then soak in dilute ammonia water for 5 minutes, take it out and dry it in the air to obtain a semipermeable membrane.

All the other are identical with embodiment 2.

Example 5

On the basis of Example 1, this example also provides a method for detecting American ginseng components in Ginseng Guipi Pills. The filter membrane of the sample filter in step (1) is a modified regenerated cellulose filter membrane, and the modified The pore size of the regenerated cellulose filter membrane is 0.2 μm.

The preparation method of the modified regenerated cellulose filter membrane is:

S1. Preparation of controlled-release pore-forming agent: granulate the pore-forming agent for later use, dissolve poly-N2 isopropylacrylamide in deionized water at 24°C to prepare a material solution, and prepare the obtained material solution at 31°C, Spray onto the surface of the granulated pore-forming agent under the condition of 1 MPa, then raise the temperature to 40°C and keep it warm for 20 minutes to obtain the controlled-release pore-forming agent;

S2. Preparation of film-making solution: dissolving cellulose in a solvent, then adding polylactic acid, controlled-release pore-forming agent and additives, mixing evenly, defoaming and filtering, to obtain a film-making solution;

S3. Membrane production: at a temperature of 80°C, cast the film-making solution prepared in step S2 on a glass plate to form a flat film, then place the flat film at 40°C in a ventilated environment for 3s, then stop the ventilation, and place the flat film Cool the film below 30°C, let it stand for 30 minutes, then let it stand for 5 seconds in a ventilated environment, then put the flat film into the coagulation bath, solidify for 10 minutes to form a film, wash off the residual solvent in the film with deionized water, and then dry it at room temperature After drying, the modified regenerated cellulose filter membrane is obtained.

In step S1, the mass ratio of the material solution to the pore-forming agent is 0.08:1, and the mass concentration of poly-N2 isopropylacrylamide in the material solution is 4%; in step S2, cellulose, polylactic acid, controlled-release pore-forming The mass ratio of agent and additive is 1:0.3:0.15:0.02.

In step S1, the pore-forming agent includes pore-forming agent A and pore-forming agent B, the mass ratio of which is 1:0.3, and the pore-forming agent A and pore-forming agent B are granulated separately, and the pore-forming agent A is Polyethylene glycol, the pore-forming agent B is sodium chloride and sodium alginate, the mass ratio of which is 1:1.5.

The solvent in step S2 is dimethyl sulfoxide/tetraethylammonium chloride with a ratio of 2:1, and the additive is fatty acid glyceride; the ventilation environment in step S3 is: nitrogen, and the ventilation rate is 0.05m ³ /h .

All the other are identical with embodiment 1.

Example 6

On the basis of Example 2, this example also provides a method for detecting American ginseng components in Ginseng Guipi Pills. The filter membrane of the sample filter in step (1) is a modified regenerated cellulose filter membrane, and the modified The pore size of the regenerated cellulose filter membrane is 0.5 μm.

The preparation method of the modified regenerated cellulose filter membrane is:

S1. Preparation of controlled-release pore-forming agent: granulate the pore-forming agent for later use, dissolve poly-N2 isopropylacrylamide in deionized water at 26°C to make a material solution, and prepare the obtained material solution at 33°C, Spray onto the surface of the granulated pore-forming agent under the condition of 1.5MPa, then raise the temperature to 50°C and keep it warm for 30 minutes to obtain the controlled-release pore-forming agent;

S2. Preparation of film-making solution: dissolving cellulose in a solvent, then adding polylactic acid, controlled-release pore-forming agent and additives, mixing evenly, defoaming and filtering, to obtain a film-making solution;

S3. Membrane production: at a temperature of 84°C, cast the film-making solution prepared in step S2 on a glass plate to form a flat film, then place the flat film at 50°C in an aeration environment for 5s, then stop the ventilation, and place the flat film Cool the film to below 30°C, let it stand for 50 minutes, then let it stand for 10 seconds in a ventilated environment, then put the flat film into the coagulation bath, solidify for 15 minutes to form a film, wash off the residual solvent in the film with deionized water, and then dry it at room temperature After drying, the modified regenerated cellulose filter membrane is obtained.

In step S1, the mass ratio of the material solution to the pore-forming agent is 0.12:1, and the mass concentration of poly-N2 isopropylacrylamide in the material solution is 8%; in step S2, cellulose, polylactic acid, controlled-release pore-forming The mass ratio of agent and additive is 1:0.6:0.3:0.05.

In step S1, the pore-forming agent includes pore-forming agent A and pore-forming agent B, the mass ratio of which is 1:0.6, and the pore-forming agent A and pore-forming agent B are granulated separately, and the pore-forming agent A is Polyethylene glycol, the pore-forming agent B is sodium chloride and sodium alginate, the mass ratio of which is 1:3.

The solvent in step S2 is dimethyl sulfoxide/tetraethylammonium chloride with a ratio of 4:1, and the additive is polysorbate; the ventilation environment in step S3 is: argon, and the ventilation rate is 0.1m ³ /h .

All the other are identical with embodiment 2.

Example 7

This embodiment provides a method for detecting the components of American ginseng in Ginseng Guipi Pills, comprising the following steps:

(1) Preparation of the test solution:

a. Remove honey from Ginseng Guipi Pills: Take 2 Ginseng Guipi Pills, cut them into pieces, add them to 50 ml of water, stir at 42°C for 12 minutes, then filter, collect the solid slag and filtrate respectively, and use a semi-permeable membrane for the filtrate After dialysis to remove monosaccharide molecules, the obtained product is mixed with solid slag to obtain the material to be tested;

b. Take the above-mentioned material to be tested, add 100ml of water-saturated n-butanol, heat and reflux for 1 hour, filter, wash the filtrate twice with ammonia test solution, 50ml each time, discard the ammonia washing solution, take n-butanol solution, and use Wash with 50 ml of water saturated with n-butanol, get the n-butanol layer and evaporate to dryness, dissolve the residue with 2 ml of methanol, filter with a sample filter, and take the subsequent filtrate as the test solution;

(2) Preparation of reference solution:

Accurately weigh 10.05 mg of pseudo-ginsenoside F ₁₁ reference substance, put it in a 50 ml measuring bottle, add methanol to dissolve and dilute to the mark, shake well, and obtain the reference substance solution;

(3) Preparation of negative control solution:

Take the missing ginseng medicinal material, prepare a negative control sample according to the prescription process, weigh 18 g, and then make a negative control solution lacking ginseng with the preparation method of the test solution;

(4) Determination method:

Use the high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) inspection method to determine, set up the detection conditions, absorb 15 μL each of the test solution, reference solution, and negative control solution, and inject samples for determination;

(5) Result judgment:

In the chromatogram of the test product, no chromatographic peak with the same retention time as the chromatographic peak of the reference substance should not appear.

In step (4), the chromatographic conditions are the same as in Example 1.

The preparation method of the semipermeable membrane in step (1) is:

A. After mixing the alkaline basalt nano powder with 0.2 times the amount of silicone oil, place it in a disperser at 1500r/min and disperse for 1.5 hours to obtain a nano powder, mix the nano powder with 50 times of water to make a dispersion;

B. Take a quantitative filter paper, use a glass rod to immerse it in 75% cold sulfuric acid, take out the filter paper after soaking for 75s, spray saturated carbonic acid solution on the surface of the filter paper, the spraying amount is 8%, and put the filter paper into the step A after spraying Rinse twice in the dispersion liquid, then soak in dilute ammonia water for 4 minutes, take it out and dry it in the air to obtain a semi-permeable membrane.

The filter membrane of the sample filter in step (1) is a modified regenerated cellulose filter membrane, and the pore size of the modified regenerated cellulose filter membrane is 0.35 μm.

The preparation method of the modified regenerated cellulose filter membrane is:

S1. Preparation of controlled-release pore-forming agent: granulate the pore-forming agent for later use, dissolve poly-N2 isopropylacrylamide in deionized water at 25°C to prepare a material solution, and prepare the obtained material solution at 32°C, Spray onto the surface of the granulated pore-forming agent under the condition of 1.25MPa, then raise the temperature to 45°C and keep it warm for 25 minutes to obtain the controlled-release pore-forming agent;

S2. Preparation of film-making solution: dissolving cellulose in a solvent, then adding polylactic acid, controlled-release pore-forming agent and additives, mixing evenly, defoaming and filtering, to obtain a film-making solution;

S3. Membrane production: at a temperature of 82°C, cast the film-making solution prepared in step S2 on a glass plate to form a flat film, then place the flat film at 45°C in an aeration environment for 4s, then stop the ventilation, and place the flat film Cool the film to below 30°C, let it stand for 40 minutes, then let it stand for 8 seconds in a ventilated environment, then put the flat film into the coagulation bath, solidify for 12 minutes to form a film, wash off the residual solvent in the film with deionized water, and then dry it at room temperature After drying, the modified regenerated cellulose filter membrane is obtained.

In step S1, the mass ratio of the material solution to the pore-forming agent is 0.1:1, and the mass concentration of poly-N2 isopropylacrylamide in the material solution is 6%; in step S2, cellulose, polylactic acid, controlled-release pore-forming The mass ratio of agent and additive is 1:0.45:0.22:0.03.

In step S1, the pore-forming agent includes pore-forming agent A and pore-forming agent B, the mass ratio of which is 1:0.45, and the pore-forming agent A and pore-forming agent B are granulated separately, and the pore-forming agent A is Polyethylene glycol, the pore-forming agent B is sodium chloride and sodium alginate, the mass ratio of which is 1:2.2.

The solvent in step S2 is dimethyl sulfoxide/tetraethylammonium chloride with a ratio of 3:1, and the additive is polysorbate; the ventilation environment in step S3 is nitrogen, and the ventilation rate is 0.08m ³ /h.

Example 8

This embodiment provides a method for detecting the components of American ginseng in Ginseng Guipi Pills, comprising the following steps:

(1) Preparation of the test solution:

a. Remove honey from Ginseng Guipi Pills: Take 2 Ginseng Guipi Pills, cut them into pieces, add them to 50 ml of water, stir at 42°C for 12 minutes, then filter, collect the solid slag and filtrate respectively, and use a semi-permeable membrane for the filtrate After dialysis to remove monosaccharide molecules, the obtained product is mixed with solid slag to obtain the material to be tested;

b. Take the above-mentioned material to be tested, add 100ml of water-saturated n-butanol, heat and reflux for 1 hour, filter, wash the filtrate twice with ammonia test solution, 50ml each time, discard the ammonia washing solution, take n-butanol solution, and use Wash with 50 ml of water saturated with n-butanol, evaporate the n-butanol layer to dryness, dissolve the residue in 2 ml of methanol, filter with a sample filter, and take the filtrate as the test solution;

(2) Preparation of reference solution:

Take Pseudoginsenoside F ₁₁ reference substance, weigh it accurately, add methanol to make a solution containing 50 µg per 1 ml, shake well, and obtain the reference substance solution;

(3) Preparation of negative control solution:

Take the missing ginseng medicinal material, prepare a negative control sample according to the prescription process, weigh 18 g, and then make a negative control solution lacking ginseng with the preparation method of the test solution;

(4) Determination method:

Use high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) inspection method for determination, set up the detection conditions, draw 15 μL each of the test solution, reference solution, and negative control solution, and inject samples for determination;

(5) Result judgment:

In the chromatogram of the test product, no chromatographic peak with the same retention time as the chromatographic peak of the reference substance should not appear.

In step (4), the chromatographic conditions and mass spectrometry conditions are the same as in Example 2.

The preparation method of the semipermeable membrane in step (1) is the same as that in Example 7.

The filter membrane of the sample filter in step (1) is a modified regenerated cellulose filter membrane, the pore size of the modified regenerated cellulose filter membrane is 0.35 μm, and its preparation method is the same as that of Example 7.

Application of the detection method of the present invention in controlling the quality of ginseng in ginseng Guipi pills

Select 10 batches of samples of Ginseng Guipi Pills (Big Honey Pills) from 2 production companies (Company A, Company B) after deduplication of batch numbers, and test according to the detection methods in Examples 7 and 8 respectively to determine whether pseudo-ginseng is detected Saponin F ₁₁ , the results are shown in the table below:

Table 1

From the results in Table 1, it can be seen that in the 10 batches of samples of Ginseng Guipi pills from A company, none of the pseudo-ginsenoside F ₁₁ was detected, and the product was qualified; in the 10 batches of samples of B company’s Ginseng Guipi pills, all the pseudo-ginsenoside F was detected _11. It is suspected that American ginseng scraps are used instead of ginseng for feeding.

In order to judge the detection results in more detail and intuitively, the present invention also provides related atlases, see Figures 1-11.

As shown in Figure 1, the chromatogram of the pseudo-ginsenoside F ₁₁ reference substance showed its characteristic chromatographic peaks; as shown in Figure 2, no pseudo-ginsenoside F ₁₁ was detected in the chromatogram of the negative control sample.

As shown in Figure 5, the full-scan basal peak figure of pseudoginsenoside F ₁₁ reference substance shows its characteristic base peak; Saponin F11.

The present invention adopts embodiment 7 (HPLC-ELSD examination method), detects to A enterprise batch number 17013851 sample, and its chromatogram is shown in Fig. 3, does not appear in Fig. 3 the chromatographic peak consistent with Fig. 1 reference substance chromatographic peak retention time, confirms Pseudo-ginsenoside F ₁₁ was not detected in the batch number 17013851 sample of enterprise A.

The present invention adopts embodiment 8 (HPLC-MS/MS inspection method) to detect the batch number 20170205 sample of B enterprise, and its chromatogram is shown in Figure 4, and a chromatographic peak consistent with the retention time of the chromatographic peak of the reference substance in Figure 1 appears in Figure 4, It was confirmed that pseudo-ginsenoside F ₁₁ was detected in the sample of batch number 20170205 of enterprise B; then confirmed by mass spectrometry, see Figure 9-11 for details, and it was confirmed that pseudo-ginsenoside F ₁₁ was detected.

The beneficial effect of the present invention is that: the present invention provides a detection method for American ginseng components in Ginseng Guipi Pills, through reasonable detection methods and parameter design, and perfect sample pretreatment measures, the accuracy and accuracy of sample detection can be effectively improved. Stability, easy to popularize and use, and can be used for high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) inspection method, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) inspection method determination, applicability powerful.

Finally, it should be noted that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it is still The technical solutions recorded in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (3)

1. A detection method for Chinese and western ginseng components in a ginseng spleen-invigorating pill is characterized by comprising the following steps: the method comprises the following steps:

(1) Preparation of a test solution:

a. removing honey in the ginseng spleen-invigorating pills: taking 2 pills of ginseng spleen-invigorating pills, cutting into pieces, adding 50ml water, stirring for 10-15min at 40-45 ℃, then filtering, respectively collecting solid residues and filtrate, dialyzing the filtrate with a semipermeable membrane to remove monosaccharide molecules, and mixing the obtained product with the solid residues to obtain a material to be detected;

b. taking the material to be detected, adding 100ml of water saturated n-butyl alcohol, heating and refluxing for 1 hour, filtering, washing the filtrate with ammonia test solution for 2 times, 50ml each time, discarding the ammonia washing solution, taking the n-butyl alcohol solution, washing with 50ml of water saturated with n-butyl alcohol, taking the n-butyl alcohol layer to dry, dissolving the residue with methanol 2 ml, filtering with a sample filter, and taking the subsequent filtrate as a sample solution;

(2) Preparation of control solutions:

collecting pseudoginsenoside F ₁₁ Accurately weighing reference substance, and adding methanol to obtain reference substance solution;

(3) Preparation of negative control solution:

taking a ginseng-lacking medicinal material, preparing a negative control sample according to a prescription process, weighing 18 g, and preparing a ginseng-lacking negative control solution by the same method as the preparation method of the test sample solution;

(4) The determination method comprises the following steps:

detecting by high performance liquid chromatography-evaporative light scattering detector detection method or high performance liquid chromatography-tandem mass spectrometry detection method, setting detection conditions, respectively sucking 10-20 μ L of sample solution, reference solution, and negative reference solution, and performing sample injection detection;

(5) And (5) judging a result:

in the chromatogram of the test sample, a chromatographic peak with the same retention time as that of the chromatographic peak of the reference sample cannot appear;

the preparation method of the semipermeable membrane in the step (1) comprises the following steps:

A. mixing the alkaline basalt nano powder with 0.1-0.3 times of silicone oil, placing the mixture in a dispersion machine of 1000-2000r/min for dispersing for 1-2h to obtain nano powder, and mixing the nano powder with 40-60 times of water to prepare dispersion liquid;

B. taking a certain amount of filter paper, immersing the filter paper in 75% cold sulfuric acid by using a glass rod, taking out the filter paper after soaking for 70-80s, spraying a saturated carbonic acid solution on the surface of the filter paper, wherein the spraying amount is 6-10%, putting the filter paper into the dispersion liquid obtained in the step A after spraying is finished, rinsing for 1-3 times, putting the filter paper into dilute ammonia water, soaking for 3-5min, taking out and airing to obtain a semipermeable membrane;

the filtering membrane of the sample filter in the step (1) is a modified regenerated cellulose filtering membrane, and the aperture of the modified regenerated cellulose filtering membrane is 0.2-0.5 mu m;

the preparation method of the modified regenerated cellulose filter membrane comprises the following steps:

s1, preparing a controlled release pore-forming agent: granulating the pore-forming agent for later use, dissolving poly (N-isopropylacrylamide) in deionized water at 24-26 ℃ to prepare a material liquid, spraying the obtained material liquid on the surface of the granulated pore-forming agent at 31-33 ℃ and 1-1.5MPa, then heating to 40-50 ℃ and preserving heat for 20-30min to obtain the controlled-release pore-forming agent;

s2, preparing a membrane preparation liquid: dissolving cellulose in a solvent, then adding polylactic acid, a controlled-release pore-forming agent and an additive, uniformly mixing, defoaming and filtering to obtain a membrane-making solution;

s3, film making: casting the membrane-forming solution prepared in the step S2 on a glass plate at the temperature of 80-84 ℃ to form a flat membrane, standing the flat membrane for 3-5S at the temperature of 40-50 ℃ in an aeration environment, stopping aeration, cooling the flat membrane to below 30 ℃, standing for 30-50min, standing for 5-10S in the aeration environment, putting the flat membrane into a coagulating bath, curing for 10-15min to form a membrane, washing away residual solvent in the membrane by using deionized water, and airing at room temperature to obtain the modified regenerated cellulose filtering membrane;

in the step S1, the mass ratio of the material liquid to the pore-forming agent is (0.08-0.12): 1, the mass concentration of poly (N-isopropyl acrylamide) in the material liquid is 4-8%; in the step S2, the mass ratio of the cellulose to the polylactic acid to the controlled-release pore-forming agent to the additive is 1: (0.3-0.6): (0.15-0.3): (0.02-0.05);

in the step S1, the pore-forming agent comprises a pore-forming agent A and a pore-forming agent B, and the mass ratio of the pore-forming agent A to the pore-forming agent B is 1: (0.3-0.6), separately granulating the pore-forming agent A and the pore-forming agent B, wherein the pore-forming agent A is polyethylene glycol, the pore-forming agent B is sodium chloride and sodium alginate, and the mass ratio of the pore-forming agent A to the pore-forming agent B is 1: (1.5-3);

the solvent in the step S2 is (2-4): 1, dimethyl sulfoxide/tetraethyl ammonium chloride, and the additive is fatty glyceride or polysorbate; the aeration environment in step S3 is: nitrogen or argon with a ventilation of 0.05-0.1m ³ /h。

2. The method for detecting the American ginseng components in the ginseng spleen-invigorating pill according to claim 1, which is characterized in that:

and (4) detecting by adopting a high performance liquid chromatography-evaporative light scattering detector inspection method, wherein the chromatographic conditions are as follows: a chromatographic column: capcell pak C18,4.6 mm. Times.250mm, 5 μm, mobile phase: acetonitrile-water, volume ratio 30; evaporative light scattering detector: gas flow rate: 1.6 L/min, flow rate: 1ml/min, and the column temperature is 30 ℃;

in this case, the preparation method of the reference solution in the step (2) is as follows: accurately weighing pseudoginsenoside F ₁₁ And (3) placing a reference substance 10.05 mg in a 50ml measuring flask, adding methanol to dissolve and dilute the solution to a scale, and shaking the solution uniformly to obtain a reference substance solution.

3. The method for detecting the American ginseng components in the ginseng spleen-invigorating pill according to claim 1, which is characterized in that:

and (4) detecting by adopting a high performance liquid chromatography-tandem mass spectrometry inspection method, wherein the chromatographic conditions are as follows:

chromatographic column Waters ACQUITY UPLC BEH-C ₁₈ 2.1mm × 100mm,1.7 μm; acetonitrile is taken as a mobile phase A, and water is taken as a mobile phase B; the flow rate is 0.4 ml/min; the column temperature is 40 ℃; 3 mu L of sample volume;

gradient elution: 0-12 min, 8-80% A; for 12-14 min, 80-90% of A; 14-14.2 min,90% -8% of A; 14.2-20 min,8% A; the volume ratio of acetonitrile to water is 30;

the mass spectrum conditions are as follows: taking a flight time mass spectrum as a detector, adopting an electrospray ionization ion source and a negative ion mode for detection, taking Leucine-enkephalin as a calibration solution, and scanning the range: m/z is 50-1200, the sampling interval is 0.5s, the capillary voltage is 2.5kV, the taper hole voltage is 40V, the ion source temperature is 100 ℃, the desolvation temperature is 450 ℃, and the desolvation gas flow rate is 800 L.h ^-1 Cone hole gas flow rate of 40 L.h ^-1 The collision gas is argon; the collision low energy is 6V, and the collision high energy is 20-40V;

in this case, the preparation method of the reference solution in the step (2) is as follows: collecting pseudoginsenoside F ₁₁ And (3) precisely weighing a reference substance, adding methanol to prepare a solution containing 50 microgram per 1ml, and shaking up to obtain the reference substance solution.

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