CN113899841B

CN113899841B - Method for detecting pinoresinol diglucoside in ginkgo leaf extraction intermediate or preparation thereof

Info

Publication number: CN113899841B
Application number: CN202111323678.XA
Authority: CN
Inventors: 方同华; 李天翥; 岳大彪; 周广红; 林珊; 佟美鸿; 国艳玲
Original assignee: Harbin Zhenbao Pharmaceutical Co ltd; HEILONGJIANG ZBD PHARMACEUTICAL CO Ltd
Current assignee: Harbin Zhenbao Pharmaceutical Co ltd; HEILONGJIANG ZBD PHARMACEUTICAL CO Ltd
Priority date: 2021-11-08
Filing date: 2021-11-08
Publication date: 2023-05-12
Anticipated expiration: 2041-11-08
Also published as: CN113899841A

Abstract

The invention provides a detection method of pinoresinol diglucoside in ginkgo leaf extraction intermediates or preparations thereof, which is based on reversed-phase high-performance liquid chromatography and comprises establishment of chromatographic conditions and preparation of a sample solution; wherein the chromatographic conditions are: chromatographic column: TSK gel ODS-100V, 4.6mm.times.150mm, 3 μm, mobile phase: methanol is used as a mobile phase A, water is used as a mobile phase B, gradient elution is carried out, and the column temperature is as follows: 40-46 ℃, flow rate: 0.65-0.75 mL/min, detection wavelength: 275-282 nm. In the preparation of the test sample, a sample to be tested firstly passes through an activated neutral alumina solid-phase extraction column.

Description

Method for detecting pinoresinol diglucoside in ginkgo leaf extraction intermediate or preparation thereof

Technical Field

The invention belongs to the fields of analytical chemistry and pharmaceutical quality control, and in particular relates to a method for qualitatively and/or quantitatively detecting pinoresinol diglucoside in ginkgo leaf extraction intermediates or preparations thereof.

Background

The Ginkgo leaf extract intermediate is solid or liquid extract which is prepared by extracting and separating dry leaves of Ginkgo (Ginkgo biloba L.) belonging to the Ginkgoaceae and does not contain auxiliary materials, and the solid extract intermediate is called as Ginkgo leaf extract in the edition 2020 of Chinese pharmacopoeia. Ginkgo leaf preparations which have been marketed include tablets, injections and the like. The Shuxuening injection is a sterilized water injection prepared from an intermediate extracted from ginkgo leaf. The literature reports that ginkgo leaf intermediates and the main active ingredients of the ginkgo leaf intermediates and the ginkgo leaf preparations are flavonoids and ginkgo lactones. In recent years, through intensive researches on ginkgo leaf extraction intermediates and chemical components of preparations thereof, the inventor finds that the ginkgo leaf extraction intermediates also contain a plurality of lignans, such as pinoresinol diglucoside, (7S, 8R) -dihydro-dehydro-guaifenesin 4-O-beta-D-glucoside, acanthopanaxoside E and the like. The lignans component has the functions of resisting oxidation, protecting myocardial cells, resisting myocardial toxicity, dilating blood vessels, resisting platelet aggregation, reducing blood fat and the like, and is related to the functional indications of the ginkgo leaf extracted intermediate and the preparation thereof. Therefore, the lignans should also be used as the active ingredients of the ginkgo leaf extraction intermediate and the preparation thereof, and the quality control is necessary, thereby being beneficial to quality control tracing, ensuring the safety of medication, better guiding production and providing scientific basis for comprehensively establishing the quality control standard of the ginkgo leaf extraction intermediate and the preparation thereof.

The ginkgo leaf extract intermediate and the preparation thereof have a plurality of lignans contained in the ginkgo leaf extract intermediate, but the content is low, the polarities of some of the lignans are very similar, and the interference of flavonoid components on lignans detection ensures that a representative component, namely pinoresinol diglucoside, in the ginkgo leaf extract intermediate has certain difficulty in achieving a good separation state on high performance liquid chromatography (HPLC-UV).

In the prior art, the report of the detection method of lignans components is concentrated on shizandra berry and a traditional Chinese medicine preparation containing shizandra berry, for example, a Chinese patent application of publication No. CN111380964A (7 months and 7 days in publication No. 2020) for establishing a fingerprint of shizandra berry medicinal material and erigeron breviscapus capsule, and a Chinese patent application of publication No. CN108226316A (29 months and 2018) for simultaneously measuring 7 lignans content in shizandra berry. The prior art has a few reports on the detection method of pinoresinol diglucoside in ginkgo leaf extraction intermediates or preparation thereof.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a method for detecting pinoresinol diglucoside in ginkgo leaf extraction intermediates or preparations thereof. The method is based on high performance liquid chromatography, and has a degree of separation of pinoresinol diglucoside of about 1.5, and can accurately and quantitatively determine the content of pinoresinol diglucoside in folium Ginkgo extract intermediate or its preparation.

In order to achieve the technical effects, the invention adopts the following technical scheme:

a detection method of pinoresinol diglucoside in ginkgo leaf extraction intermediate or preparation thereof is based on reversed-phase high performance liquid chromatography, and comprises establishment of chromatographic conditions and preparation of sample solution; wherein the chromatographic conditions are:

chromatographic column: TSK gel ODS-100V, 4.6mm.times.150mm, 3 μm,

mobile phase: methanol is taken as a mobile phase A, water is taken as a mobile phase B, gradient elution is carried out according to the specification in the table 1,

TABLE 1 gradient elution procedure

Column temperature: 40-46 DEG C

Flow rate: 0.65-0.75 mL/min,

detection wavelength: 275-282 nm;

the preparation of the sample solution comprises the following operations:

I. taking a neutral alumina solid phase extraction column (with the specification of 100-200 meshes and 1g/6 ml), washing with 10ml of methanol and water respectively at the flow rate of 1-2 ml/min in sequence, and repeating the steps for two times to obtain an activated neutral alumina solid phase extraction column;

II, taking a sample to be detected, preparing a solution containing 1.0mg of total flavonol glycosides per 1ml by using water, precisely measuring 5ml, placing the solution on the activated neutral alumina solid phase extraction column, filtering at a flow rate of 1ml/min, and collecting effluent; and eluting with 30% methanol 50ml at a flow rate of 1ml/min, collecting eluate, mixing the eluate and eluate, evaporating in water bath, dissolving the residue with 30% methanol by ultrasonic method, transferring into a 2ml measuring flask, diluting to scale, shaking, filtering, and collecting filtrate.

It will be appreciated by those skilled in the art that for solid samples to be tested, such as ginkgo leaf extract or solid formulations (e.g. tablets, granules, etc.) prepared from ginkgo leaf extract, a solution of 1.0mg per 1ml of total flavonol glycosides is prepared with water as described in step II above, followed by subsequent treatments. For a liquid sample to be tested, the concentration of the total flavonol glycosides is higher than 1.0mg/ml, and when preparing a sample solution, the solution containing 1.0mg of total flavonol glycosides per 1ml is firstly prepared according to the method in the step II; if the concentration of the total flavonol glycoside is less than or equal to 1.0mg/ml, such as Shuxuening injection, directly taking 5ml for treatment.

Preferably, the column temperature is 45 ℃.

Preferably, the flow rate is 0.7mL/min.

Preferably, the detection wavelength is 277-278 nm.

The neutral alumina solid phase extraction column is commercially available, preferably Ai Jieer (Agela) neutral alumina solid phase extraction column.

Preferably, under the chromatographic conditions, the theoretical plate number should be not less than 4000 as calculated as pinoresinol diglucoside.

Preferably, the detection method further comprises the preparation of a reference substance solution, and the specific operation is as follows:

taking a proper amount of pinoresinol diglucoside reference substance, precisely weighing, adding 30% methanol to prepare a solution containing 18 mug pinoresinol diglucoside per 1 ml.

Preferably, the detection method further comprises an assay specifically comprising:

and respectively precisely sucking 20 mu l of each of the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, and recording a chromatogram under the chromatographic condition.

Preferably, the assay comprises a qualitative assay and/or a quantitative assay; wherein, the specific steps of the qualitative determination are as follows:

respectively precisely sucking 20 mu l of each of the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, recording a chromatogram under the chromatographic condition, taking the retention time of the pinoresinol diglucoside absorption peak in the chromatogram of the reference substance solution as a reference, and checking whether the absorption peak with the same retention time appears in the chromatogram of the sample solution;

the quantitative determination comprises the following specific steps:

and respectively precisely sucking 20 mu l of each of the reference substance solution and the test substance solution, injecting into a high performance liquid chromatograph, recording a chromatogram under the chromatographic condition, and calculating the content of pinoresinol diglucoside in the test substance solution by an external standard method.

Preferably, the ginkgo leaf extract intermediate preparation is Shuxuening injection.

In the present specification, unless specified otherwise, the percentage concentration of the solution refers to the percentage concentration by volume, for example, 30% methanol, that is, the solution of 100 parts by volume, 30 parts by volume of methanol and the balance of water.

Drawings

The invention is further described below with reference to the accompanying drawings.

FIG. 1 shows the UV spectrum of pinoresinol diglucoside.

Figure 2 shows the uv spectrum of the sulzin injection.

FIG. 3 shows the ultraviolet spectrum of the flavonoid quercetin.

Fig. 4 shows HPLC chromatograms of a sulcinine injection obtained on a neutral alumina solid phase extraction column at different loading amounts when examining the test sample solution preparation method in example 1, wherein:

a: the loading amount of the neutral alumina solid phase extraction column is 0ml (the injection of the Shuxuening injection is directly injected);

b: the loading amount of the neutral alumina solid phase extraction column is 2ml; wherein B-1 is a chromatogram at 277nm and B-2 is a chromatogram at 354nm;

c: the loading amount of the neutral alumina solid phase extraction column is 5ml; wherein, C-1 is a chromatogram at 277nm, and C-2 is a chromatogram at 354 nm.

Fig. 5 shows HPLC chromatograms of the sample solution of example 2 eluted under different gradient elution procedures, wherein:

a: gradient elution procedure 1;

b: gradient elution procedure 2;

c: gradient elution procedure 3;

d: gradient elution procedure 4;

e: gradient elution procedure 5;

f: gradient elution procedure 6.

FIG. 6 shows an HPLC chromatogram of the sample solution of example 2 eluted with a gradient elution procedure 5 on different chromatographs of the same type.

FIG. 7 shows the HPLC profile of the sample solution obtained from the different brands and specifications of the chromatographic column of example 3, wherein:

a: column TSK gel ODS-100V (4.6X105 mm,3 μm);

b: column Agilent XDB-C18 column (4.6X150 mm,5 μm);

c: chromatographic column Thermoacclaim 120C 18 chromatographic column (4.5X250 mm,5 μm).

FIG. 8 shows the HPLC profile of the sample solution treated with different brands of neutral alumina solid phase extraction columns of example 4, wherein:

a: ai Jieer neutral alumina solid phase extraction column;

b: a microphone Lin Zhongxing alumina solid phase extraction column;

c: shanghai Kangang neutral alumina solid phase extraction column.

FIG. 9 shows HPLC patterns of a control solution and a test solution in a specificity study test in example 5; wherein:

a: chromatograms of the control solutions at 277nm;

b: chromatograms of the test solution at 277nm;

c: chromatograms of the test solutions at 354 nm.

FIG. 10 shows a standard curve established in a linear test of example 5, in which the horizontal axis (x-axis) is the content of pinoresinol diglucoside (. Mu.g/ml) and the vertical axis (y-axis) is the peak area of the peak of the pinoresinol diglucoside chromatogram.

FIG. 11 shows an HPLC chromatogram of the three-batch Shuxuening injection determined in example 6; wherein:

a: chromatograms of test samples prepared by Shuxuening injection (ZSA 20190904);

b: chromatograms of test samples prepared by Shuxuening injection (ZSA 20190914);

c: chromatograms of test samples prepared from Shuxuening injection (ZSA 20190920).

FIG. 12 shows HPLC chromatograms of three batches of ginkgo leaf extract intermediates measured in example 7; wherein:

a: chromatogram of the test sample prepared from ginkgo leaf extraction intermediate (G13201201);

b: chromatogram of the test sample prepared from ginkgo leaf extraction intermediate (G13201202);

c: chromatograms of test samples prepared from ginkgo leaf extract intermediates (G13201203).

Detailed Description

The invention provides a detection method of pinoresinol diglucoside in ginkgo leaf extraction intermediates or preparations thereof, which is based on reversed-phase high-performance liquid chromatography and comprises the following steps:

chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica (column TSK gel ODS-100V 4.6mm.times.150mm, 3 μm) was used as a filler; using methanol as mobile phase A and water as mobile phase B, and performing gradient elution according to the specifications in Table 1; the flow rate is 0.65-0.75 ml (preferably 0.7 ml) per minute, and the detection wavelength is 275-282 nm (preferably 277 nm); column temperature 40-46 deg.c, preferably 45 deg.c. The theoretical plate number should be not less than 4000 as calculated on pinoresinol diglucoside.

Preparation of a control solution: taking a proper amount of pinoresinol diglucoside reference substance, precisely weighing, and adding 30% methanol to prepare a solution containing 18 mug per 1 ml.

Preparation of test solution: taking a sample to be detected, preparing a solution containing 1.0mg of total flavonol glycosides per 1ml by using water, precisely measuring 5ml, placing the solution on an activated neutral alumina solid phase extraction column (100-200 meshes, 1g/6ml; activation treatment, namely washing with 10ml of methanol and water at a flow rate of 1-2 ml/min in sequence and repeating twice), filtering at a flow rate of 1ml/min, and collecting effluent; and eluting with 30% methanol 50ml at a flow rate of 1ml/min, mixing the above effluent and eluate, evaporating in water bath, dissolving the residue with 30% methanol under ultrasound, transferring into a 2ml measuring flask, diluting to scale, shaking, filtering, and collecting filtrate.

Assay: respectively precisely sucking 20 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.

The inventor takes Shuxuening injection (Heilongjiang treasure island pharmaceutical industry Co., ltd.) as a sample, carefully surveys chromatographic columns, neutral alumina solid phase extraction columns, mobile phase elution programs and the like, and establishes the detection method through repeated experiments and fumbling.

The invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are for illustration of the invention only and are not intended to limit the scope of the invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials and the like used in the examples described below are commercially available products unless otherwise specified. Wherein, the purchase conditions of partial reagents and control products are as follows:

shuxuening injection: heilongjiang treasure pharmaceutical Co., ltd., lot number: ZSA20190904 (total flavonol glycoside 0.89mg per 1ml injection), ZSA20190914 (total flavonol glycoside 0.90mg per 1ml injection), ZSA20190920 (total flavonol glycoside 0.87mg per 1ml injection);

pinoresinol diglucoside control: chinese food and drug assay institute, lot number: 111537-201706.

Example 1Preparation of test solutions

The ultraviolet absorption spectrum of pinoresinol diglucoside is shown in figure 1. The Shuxuening injection is an injection preparation of an intermediate extracted from ginkgo leaf, and the ultraviolet absorption spectrum of the Shuxuening injection is shown in figure 2. The ultraviolet absorption spectrum of flavonoid quercetin is shown in figure 3. Fig. 1 shows that the pinoresinol diglucoside has the maximum absorption at 277-280 nm, fig. 2 shows that the Shuxuening injection has the absorption at 277-280 nm, and fig. 3 shows that the flavonoid component quercetin in the Shuxuening injection also has the absorption at 250nm and 360nm, so that when the ultraviolet detector is adopted to detect the content of the pinoresinol diglucoside, the flavonoid component in the Shuxuening injection has interference on the pinoresinol diglucoside and needs to be removed. For this purpose, the sulzin injection is first passed through an activated neutral alumina solid phase extraction column to remove interfering components. This example examined the loading of neutral alumina solid phase extraction columns.

Instrument: waters e2695 high performance liquid chromatograph;

chromatographic column: TSK gel ODS-100V (4.6X105 mm,3 μm);

column temperature: 45 ℃;

sample injection amount: 20 μl;

flow rate: 0.5-0.7 ml/min;

detection wavelength: 277nm,354nm;

neutral alumina solid phase extraction column: bona Ai Jieer technologies Co., ltd., specification: 100-200 meshes, 1g/6ml.

Sample solution preparation: washing the neutral alumina solid phase extraction column with 10ml of methanol and water at a flow rate of 1-2 ml/min, and repeating twice to obtain the activated neutral alumina solid phase extraction column. Precisely sucking a certain volume of Shuxuening injection, loading, filtering at a flow rate of 1ml/min, and collecting effluent; and eluting with 30% methanol 50ml at a flow rate of 1ml/min, mixing the above effluent and eluate, evaporating in water bath, dissolving the residue with 30% methanol under ultrasound, transferring into a 2ml measuring flask, diluting to scale, shaking, filtering, and collecting filtrate.

1. Direct sample injection

Precisely sucking 2ml of Shuxuening injection, placing into a 5ml measuring flask, adding water for dilution, and fixing the volume to the scale. 10 μl of the sample solution was precisely aspirated, and injected into a high performance liquid chromatograph, the mobile phase and gradient elution procedure are shown in Table 2, and the chromatogram is shown as A in FIG. 4.

TABLE 2 gradient elution procedure

Time (min)	Water (%)	Methanol (%)
			0	80	20
30	70	30
			40	20	80
41	80	20
			60	80	20

2.2 ml injection sample

Precisely sucking 2ml of Shuxuening injection, and preparing a test solution according to the operation of preparing the test solution. 20 μl of the sample solution was precisely aspirated, and injected into a high performance liquid chromatograph, the mobile phase and gradient elution procedure are shown in Table 3, and the chromatograms are shown in FIG. 4 as B-1 (277 nm) and B-2 (354 nm).

TABLE 3 gradient elution procedure

Time (min)	Methanol-water (21:79,%)	Methanol (%)
			0	100	0
45	100	0
			45.5	0	100
55	0	100
			55.5	100	0
90	100	0

3. 5ml injection sample

Precisely sucking 5ml of Shuxuening injection, and preparing a test solution according to the operation of preparing the test solution. 20 μl of the sample solution was precisely aspirated, and injected into a high performance liquid chromatograph, the mobile phase and gradient elution procedure are shown in Table 1, and the chromatograms are shown in C-1 (277 nm) and C-2 (354 nm) in FIG. 4.

4. 8ml injection sample

The eluent is yellow when the loading volume is 8ml, which indicates that the neutral alumina solid phase extraction column is overloaded, and the subsequent operation is stopped.

Conclusion: as can be seen from a comparison of A and B-1 in FIG. 4, the components interfering with the measurement of pinoresinol diglucoside in the Shuxuening injection can be removed by using a neutral alumina solid phase extraction column. As can be seen from a comparison of B-1 and C-1, and B-2 and C-2 in FIG. 4: the concentration of the extraction column is slightly lower when the loading volume of the extraction column is 2ml, and the response value of a chromatographic peak is small and is easily influenced by baseline fluctuation. Therefore, the loading volume of the sulxuening injection of the neutral alumina solid phase extraction column is preferably 5ml.

Example 2Investigation of mobile phase and gradient elution procedure

Instrument: waters e2695 high performance liquid chromatograph;

chromatographic column: TSK gel ODS-100V (4.6X105 mm,3 μm);

column temperature: 45 ℃;

sample injection amount: 20 μl;

flow rate: 0.5-0.7 ml/min;

detection wavelength: 277nm;

Sample solution preparation: sequentially washing the neutral alumina solid phase extraction column with 10ml of methanol and water at a flow rate of 1-2 ml/min, and repeating the steps twice to obtain the activated neutral alumina solid phase extraction column. Precisely sucking 5ml of SHUXUENING injection, sampling, filtering at a flow rate of 1ml/min, and collecting effluent; and eluting with 30% methanol 50ml at a flow rate of 1ml/min, mixing the above effluent and eluate, evaporating in water bath, dissolving the residue with 30% methanol under ultrasound, transferring into a 2ml measuring flask, diluting to scale, shaking, filtering, and collecting filtrate.

1. Gradient elution procedure 1 is shown in table 4; the chromatogram is shown in FIG. 5A.

TABLE 4 gradient elution procedure 1

Time (min)	Methanol (%)	Water (%)
			0	22	78
40	22	78
			41	80	20
55	80	20
			55.5	22	78
80	22	78

2. Gradient elution procedure 2 is shown in table 5; the chromatogram is shown in FIG. 5B.

TABLE 5 gradient elution procedure 2

3. Gradient elution procedure 3 is shown in table 6; the chromatogram is shown in FIG. 5C.

TABLE 6 gradient elution procedure 3

Time (min)	(methanol: water: formic acid=22:78:0.2,%)	Methanol (%)
			0	100	0
45	100	0
			45.5	0	100
55	0	100
			55.5	100	0
90	100	0

4. Gradient elution procedure 4 is shown in table 7; the chromatogram is shown in FIG. 5D.

TABLE 7 gradient elution procedure 4

Time (min)	Methanol (%)	Water (%)
			0	20	80
45	25	75
			45.5	100	0
59.5	100	0
			60	20	80
80	20	80

5. Gradient elution procedure 5 is shown in table 8; the chromatogram is shown in FIG. 5E.

TABLE 8 gradient elution procedure 5

Time (min)	Methanol (%)	Water (%)
			0	20	80
45	23	77
			45.5	100	0
59.5	100	0
			60	20	80
80	20	80

6. Gradient elution procedure 6 is shown in table 9; the chromatogram is shown in F of FIG. 5.

TABLE 9 gradient elution procedure 6

Time (min)	Methanol (%)	Water (%)
			0	20	80
45	22	78
			45.5	100	0
59.5	100	0
			60	20	80
80	20	80

Conclusion: as can be seen by comparing the chromatograms obtained under the different elution procedures in FIG. 5, the gradient elution procedure 5 has the best effect of separating pinoresinol diglucoside, and the degree of separation from the front and rear adjacent chromatographic peaks reaches 1.5. Thus, preliminary determination of the gradient elution procedure 5 is a preferred gradient elution procedure.

On three high performance liquid chromatographs of the same type (Waters e2695 high performance liquid chromatograph, waters e2695 (4) high performance liquid chromatograph and Waters e2695 (5) high performance liquid chromatograph), elution was performed according to the gradient elution program 5, and a chromatogram was recorded, see fig. 6. The degree of separation of pinoresinol diglucoside in the chromatograms obtained on the three instruments is shown in Table 10.

TABLE 10 influence of different chromatographs of the same model on the degree of separation of pinoresinol diglucoside

Degree of separation	Waters e2695	Waters e2695(4)	Waters e2695(5)
				Degree of separation from the front	1.59	1.49	1.48
Degree of separation from the rear peak	1.64	1.52	1.53

The data in table 10 show that gradient elution procedure 5 achieved baseline separation (degree of separation 1.5) of pinoresinol diglucoside on all 3 instruments examined. The specificity of the gradient elution program is good.

Through the study and investigation of the present example, it was confirmed that the mobile phase consisted of methanol (phase a) and water (phase B), and gradient elution was performed, and the elution procedure was preferably the above-described gradient elution procedure 5, i.e., the elution procedure shown in tables 1 and 8.

Example 3Selection of chromatograph and chromatographic column

1. Selection of chromatographic columns

Instrument: waters e2695 high performance liquid chromatograph;

column temperature: 45 ℃;

sample injection amount: 20 μl;

mobile phase: methanol-water, gradient elution was performed as shown in table 1;

flow rate: 0.5-0.7 ml/min;

detection wavelength: 277nm;

1. Chromatographic column 1

Model: TSK gel ODS-100V (4.6X105 mm,3 μm).

The chromatogram is shown as A in FIG. 7.

2. Chromatographic column 2

Model: agilent XDB-C18 column (4.6X105 mm,5 μm).

The chromatogram is shown in FIG. 7B.

3. Chromatographic column 3

Model: thermo acclaim 120C 18 chromatographic column (4.5X250 mm,5 μm).

The chromatogram is shown as C in FIG. 7.

The peak purity of pinoresinol diglucoside in the chromatograms obtained on the three chromatographic columns is shown in Table 11.

TABLE 11 influence of different chromatographic columns on peak purity of pinoresinol diglucoside

Generally, the purity of the chromatographic peak is greater than 990 or the purity angle is smaller than the purity threshold value through software calculation, so that the purity of the peak meets the requirement, and the separation degree of the chromatographic peak is good. Figures 7 and 11 show that the separation effect of columns of different brands and specifications is significantly different. The chromatographic peaks of pinoresinol diglucoside are respectively wrapped with the chromatographic peaks in front of and behind the chromatographic peaks by adopting Agilent XDB-C18 (4.6X105 mm,5 μm) and Thermo acllim 120C 18 (4.5X105 mm,5 μm), and neither the peak purity nor the separation degree can meet the requirements. When a TSK gel ODS-100V (4.6X105 mm,3 μm) column is used, the peak shape of pinoresinol diglucoside is the best, and the peak purity meets the requirements, so that the column is preferably used.

Example 4Investigation of different brands of neutral alumina solid phase extraction columns

Instrument: waters e2695 high performance liquid chromatograph;

chromatographic column: TSK gel ODS-100V (4.6X105 mm,3 μm);

column temperature: 45 ℃;

sample injection amount: 20 μl;

flow rate: 0.5-0.7 ml/min;

detection wavelength: 277nm.

1. Ai Jieer neutral alumina solid phase extraction column: bona Ai Jieer technologies Co., ltd., specification: 100-200 meshes, 1g/6ml.

The chromatogram is shown as A in FIG. 8.

2. The microphone Lin Zhongxing alumina solid phase extraction column has the specification of 100-200 meshes and 1g/6ml.

The chromatogram is shown in FIG. 8B.

3. Shanghai Kangang neutral alumina solid phase extraction column with specification of 100-200 meshes and 1g/6ml.

The chromatogram is shown as C in FIG. 8.

The content measurement results of the test sample solutions treated by the three neutral alumina solid phase extraction columns are shown in Table 12.

TABLE 12 determination of the content of pinoresinol diglucoside after treatment with different neutral alumina solid-phase extraction columns

Content μg/ml	Ai Jieer	Microphone forest	Shanghai Xiyang
				Content determination
1	5.296	4.963	4.474
				Content determination 2	5.296	4.685	5.041

Conclusion: fig. 8 and table 12 show that different brands of neutral alumina solid phase extraction columns have a significant effect on the determination of pinoresinol diglucoside content in the Shuxuening injection. Preferably Ai Jieer neutral alumina solid phase extraction column.

Example 5Methodology investigation of the detection method established by the invention

Through the researches and investigations described in the above examples 1-4, a detection method of pinoresinol diglucoside in ginkgo leaf extraction intermediates or preparations thereof is established, wherein the detection method is based on reversed-phase high performance liquid chromatography and comprises the following steps:

chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica (column TSK gel ODS-100V 4.6mm.times.150mm, 3 μm) was used as a filler; using methanol as mobile phase A and water as mobile phase B, and performing gradient elution according to the specifications in Table 1; the flow rate is 0.7ml per minute, and the detection wavelength is 277nm; column temperature 45 ℃. The theoretical plate number should be not less than 4000 as calculated on pinoresinol diglucoside.

Preparation of test solution: taking a sample to be detected, preparing a solution containing 1.0mg of total flavonol glycosides per 1ml with water, precisely measuring 5ml, placing the solution on an activated Ai Jieer neutral alumina solid phase extraction column (100-200 meshes, 1g/6ml; activation treatment, washing with 10ml of methanol and water at a flow rate of 1-2 ml/min in sequence and repeating twice), filtering at a flow rate of 1ml/min, and collecting effluent; and eluting with 30% methanol 50ml at a flow rate of 1ml/min, mixing the above effluent and eluate, evaporating in water bath, dissolving the residue with 30% methanol under ultrasound, transferring into a 2ml measuring flask, diluting to scale, shaking, filtering, and collecting filtrate.

Assay: respectively precisely sucking 20 μl of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring, and recording chromatogram.

According to the embodiment, the sulzin injection is taken as a sample to be detected, the sample solution to be detected is prepared by directly loading the sample solution to the activated Ai Jieer neutral alumina solid phase extraction column, and methodology investigation is carried out on the detection method from the aspects of specificity, repeatability, linearity, stability, accuracy, durability, reproducibility and the like.

1. Investigation of specificity

The detection method adopts a neutral alumina solid-phase extraction method to remove the components causing interference in the sample to be detected. The sample solution to be tested is prepared by the method by taking the Shuxuening injection (batch number ZSA 20190904) as a sample to be tested, and is sampled under the chromatographic condition for measurement, and a chromatogram is recorded, as shown in figure 9. Relevant parameters of the pinoresinol diglucoside chromatographic peak are shown in table 13.

TABLE 13 parameters of the chromatographic peaks of pinoresinol diglucoside

Chromatographic peak name	Retention time	Degree of separation	Degree of asymmetry	Theoretical plate number	Peak purity
						Pinoresinol diglucoside	36.960	1.5	0.94	21754	994

Table 13 shows that the detection method established by the invention is free from the interference of flavonoid components, the chromatographic peak separation degree is 1.5, the peak purity value is more than 990, and the method has strong specificity and can be suitable for the content measurement of the pinoresinol diglucoside in the Shuxuening injection.

2. Repeatability of

6 parts of Shuxuening injection (batch number ZSA 20190904) is taken, a test solution is prepared according to the method, the content of pinoresinol diglucoside is measured and calculated, and the result is shown in Table 14.

TABLE 14 repeatability test results

The results in table 14 show that RSD for the content of the sulxuening injection (lot ZSA 20190904) measured on 6 parallel samples is less than 3.0%, indicating good reproducibility of the method.

3. Linearity of

6 parts of reference substance solutions with different concentrations are prepared according to the table 15, 20 μl of the reference substance solutions are respectively sucked precisely, and are injected into a high performance liquid chromatograph for measurement, and the results are shown in table 15. And drawing a standard curve by taking the content (mug/ml) of pinoresinol diglucoside as a horizontal axis (x axis) and the peak area of a pinoresinol diglucoside chromatographic peak as a vertical axis (y axis), and establishing a regression equation. The standard curve is shown in fig. 10, and the regression equation is shown below:

y＝13,314.7013x+11,067.6013

the results show that pinoresinol diglucoside has good linear relationship in the range of 6.602 mu g/ml to 165.060 mu g/ml.

Table 15 results of linear relationship investigation

4. Stability of

The sample solution is prepared by taking the Shuxuening injection (batch number ZSA 20190904) according to the preparation method of the sample solution, and is placed at room temperature, measured at 0, 3, 6, 12, 24, 36 and 48 hours respectively, the chromatograms are recorded, the peak areas of the pinoresinol diglucoside chromatographic peaks are obtained through integration, and the results are shown in Table 16.

TABLE 16 stability test results

The results in Table 16 show that the pinoresinol diglucoside content was stable over 48 hours of the test solution.

5. Accuracy of

0.4ml, 0.8ml and 1.2ml of pinoresinol diglucoside reference substance solution (content 17.42 mug/ml) are respectively measured to an activated neutral alumina solid phase extraction column, and each volume is 3 parts in parallel. Then 2.5ml of Shuxuening injection (batch No. ZSA 20190904) with known pinoresinol diglucoside content of 5.296 mug/ml is respectively taken, each neutral alumina solid phase extraction column is respectively added, and the mixture is treated according to a preparation method of a sample solution to obtain the sample solution. Precisely sucking 20 μl of the sample solution, injecting into high performance liquid chromatograph, recording chromatogram, measuring pinoresinol diglucoside content, and calculating recovery rate according to the following formula:

sample recovery = (total measured-amount present in sample)/actual addition x 100%.

The results are shown in Table 17.

TABLE 17 recovery test results

The data in Table 17 show that the recovery rate of 3 concentrations of pinoresinol diglucoside is between 92.2% and 102.9%, and RSD is less than 3.0%, which shows that the method has good accuracy.

6. Durability of

Taking Shuxuening injection with batch number of ZSA20190904, preparing test solution by the preparation method, and respectively examining the content change condition of pinoresinol diglucoside measured on different brands of liquid chromatographs under the conditions of fine adjustment of column temperature, flow rate and organic phase proportion of the test solution, detection wavelength, methanol proportion of solid phase extraction column eluent and elution volume of the solid phase extraction column. The results are shown in Table 18.

Table 18 durability test results

The results in table 18 show that slight adjustments in the parameters or conditions described above have substantially no effect on the assay. The detection method established by the invention has good durability.

7. Reproducibility of

3 batches of Shuxuening injections (batch numbers: ZSA20190904, ZSA20190912, ZSA 20190914) were taken in different analytical laboratories, and were tested for the content of pinoresinol diglucoside by the method established according to the invention, the results being shown in Table 19.

TABLE 19 repeatability test results

Analytical laboratory	ZSA20190904	ZSA20190912	ZSA20190914
					1	4.9μg/ml	11.4μg/ml	9.7μg/ml
2	4.7μg/ml	10.0μg/ml	8.5μg/ml
				Deviation (%)	2.1	6.6	6.6

The results in Table 19 show that the content deviation of pinoresinol diglucoside measured by the detection method in different analysis laboratories is less than 11.0%, and accords with the rule of the verification and guidance of the analysis method 9101 in the fourth edition of the Chinese pharmacopoeia 2020, which shows that the method has good reproducibility.

Example 6Determination of pinoresinol diglucoside content in Shuxuening injection

Preparation of test solution: precisely measuring 5ml of Shuxuening injection, placing the injection on an activated Ai Jieer neutral alumina solid phase extraction column (100-200 meshes, 1g/6ml; activation treatment: washing with 10ml of methanol and water at a flow rate of 1-2 ml/min respectively, repeating twice), filtering at a flow rate of 1ml/min, and collecting effluent; and eluting with 30% methanol 50ml at a flow rate of 1ml/min, mixing the above effluent and eluate, evaporating in water bath, dissolving the residue with 30% methanol under ultrasound, transferring into a 2ml measuring flask, diluting to scale, shaking, filtering, and collecting filtrate.

Assay: respectively precisely sucking 20 μl of the reference solution and 20 μl of the sample solution, injecting into a liquid chromatograph, measuring, and calculating the content of pinoresinol diglucoside in the sample solution by external standard method.

The chromatogram is shown in FIG. 11, and the measurement results are shown in Table 20.

Table 20 determination of the content of pinoresinol diglucoside in Shuxuening injection

Detection result	ZSA20190904	ZSA20190914	ZSA20190920
				Content (μg/ml)	4.9	9.7	8.2

Example 7Determination of pinoresinol diglucoside content in ginkgo leaf extraction intermediate

Preparation of ginkgo leaf extraction intermediates: the ginkgo leaf extract is prepared according to the method of the harvest of Chinese pharmacopoeia (2020 edition, P434). Lot number: g13201201, G13201202, G13201203.

Preparation of test solution: taking ginkgo leaf extract intermediate, preparing 1.0mg of solution containing total flavonol glycoside per 1ml with water, precisely measuring 5ml, placing the solution on an activated Ai Jieer neutral alumina solid phase extraction column (100-200 meshes, 1g/6ml; pre-washing with 10ml of methanol and water respectively at a flow rate of 1-2 ml/min, repeating twice), filtering at a flow rate of 1ml/min, and collecting effluent; and eluting with 30% methanol 50ml at a flow rate of 1ml/min, mixing the above effluent and eluate, evaporating in water bath, dissolving the residue with 30% methanol under ultrasound, transferring into a 2ml measuring flask, diluting to scale, shaking, filtering, and collecting filtrate.

The chromatogram is shown in FIG. 12, and the measurement results are shown in Table 21.

TABLE 21 determination of the content of pinoresinol diglucoside in the Ginkgo biloba extract intermediate

Detection result	G13201201	G13201202	G13201203
				Content (μg/ml)	15.0	15.4	15.9

Claims

1. A method for detecting pinoresinol diglucoside in folium Ginkgo extract intermediate or its preparation, wherein the folium Ginkgo extract intermediate preparation is SHUXUENING injection; the detection method is based on reversed-phase high performance liquid chromatography, and comprises establishment of chromatographic conditions and preparation of a sample solution; wherein the chromatographic conditions are:

chromatographic column: TSK gel ODS-100V, 4.6mm.times.150mm, 3 μm,

mobile phase: methanol is taken as a mobile phase A, water is taken as a mobile phase B, gradient elution is carried out according to the specifications in the following table,

column temperature: 45 c,

flow rate: 0.65-0.75 mL/min,

detection wavelength: 275-282 nm;

the preparation of the sample solution comprises the following operations:

I. taking a Ai Jieer neutral alumina solid-phase extraction column with the specification of 100-200 meshes and 1g/6ml, washing with 10ml of methanol and water respectively at the flow rate of 1-2 ml/min, and repeating twice to obtain an activated neutral alumina solid-phase extraction column;

2. The method of claim 1, wherein the flow rate is 0.7mL/min.

3. The detection method according to claim 1, wherein the detection wavelength is 277-278 nm.

4. The method according to claim 1, wherein the theoretical plate number is not less than 4000 in terms of pinoresinol diglucoside under the chromatographic conditions.

5. The method according to claim 1, further comprising the preparation of a control solution, comprising the specific operations of:

6. The method of claim 5, further comprising the steps of:

7. The detection method according to claim 6, wherein the assay comprises a qualitative assay and/or a quantitative assay; wherein, the specific steps of the qualitative determination are as follows:

the quantitative determination comprises the following specific steps: