CN113030340A

CN113030340A - Method for measuring content of characteristic components of radix bupleuri in radix bupleuri and preparation thereof by liquid phase coupling high-resolution mass spectrometry

Info

Publication number: CN113030340A
Application number: CN202110387399.3A
Authority: CN
Inventors: 范妙璇; 郭洪祝; 傅欣彤; 陈有根; 闫海霞; 李岳; 张宪; 杜小伟; 陈晶
Original assignee: Beijing Institute for Drug Control
Current assignee: Beijing Institute for Drug Control
Priority date: 2021-04-09
Filing date: 2021-04-09
Publication date: 2021-06-25
Anticipated expiration: 2041-04-09
Also published as: CN113030340B

Abstract

The invention relates to a method for measuring the content of bupleurum characteristic components in bupleurum and a preparation thereof by liquid phase coupling high resolution mass spectrometry, in particular to a method for measuring the content of saikosaponin in bupleurum medicinal material decoction pieces and bupleurum extracts, such as aqueous extracts thereof or bupleurum yellow preparations. The CHAIHUANG preparation is selected from CHAIHUANG granule, CHAIHUANG capsule, CHAIHUANG tablet, CHAIHUANG oral liquid, and CHAIHUANG soft capsule; preferably, the Chaihuang preparation is selected from Chaihuang granule, Chaihuang capsule, and Chaihuang tablet. The saikosaponin comprises one or more of the following 13 saponins: saikosaponin A, saikosaponin B1, saikosaponin B2, saikosaponin C, saikosaponin D, saikosaponin F, saikosaponin G, saikosaponin H, saikosaponin I, nipagoside K, saikosaponin S, Prosapogenin D, and Prosapogenin F. The method can simultaneously determine the characteristic components of 13 bupleurum in a short time and quantify the components.

Description

Method for measuring content of characteristic components of radix bupleuri in radix bupleuri and preparation thereof by liquid phase coupling high-resolution mass spectrometry

Technical Field

The invention belongs to the technical field of medicines, relates to a method for measuring the quality of a traditional Chinese medicine and a preparation thereof, and particularly relates to a method for measuring the content of a characteristic component of radix bupleuri in a radix bupleuri medicinal material, decoction pieces and an extract thereof, such as an aqueous extract or a radix bupleuri preparation, by using a liquid phase coupling high-resolution mass spectrometry technology. The method can simultaneously measure the characteristic components of 13 bupleurum in 20 minutes and quantify the components.

Background

Bupleurum chinense (Bupleurii RADIX), a dried root of Bupleurum scorzonerifolium Willd, or Bupleurum scorzonerifolium DC, a plant of Umbelliferae, is collected in the first part of the Chinese pharmacopoeia 2020. According to the different characteristics, it is called "Bei chai Hu" and "nan chai Hu" respectively. Collected in spring and autumn, removed stems and leaves and silt, and dried.

The chaihuang preparation is prepared with bupleurum root and skullcap root as two kinds of Chinese medicinal materials, and may be granule, tablet, capsule, soft capsule, etc. Has the effects of clearing heat and relieving exterior syndrome, and is clinically used for treating wind-heat type common cold with symptoms of fever, general malaise, headache, blurred vision and sore throat. In addition, the chaihuang preparation also has the efficacy of clearing heat and diminishing inflammation, and is clinically used for upper respiratory tract infection, cold and fever.

Each 1000 pieces of Chaihuang tablet collected in the first part of China pharmacopoeia 2020 edition is prepared by extracting 1000g of radix bupleuri and 1000g of radix Scutellariae and adding a proper amount of auxiliary materials. Each 1000ml of Chaihuang oral liquid collected in the first part of the China pharmacopoeia 2020 edition is prepared by extracting 500g of radix bupleuri and 500g of radix scutellariae and adding a proper amount of auxiliary materials. Each 1000g of the Chaihuang granules collected by the national food and drug administration national drug Standard WS3-B-2009-95-2008 is prepared from 1250g of radix bupleuri and 90g of radix scutellariae extract by adding a proper amount of auxiliary materials. The Chaihuang capsule collected by the national food and drug administration national drug Standard YBZ02362006 is prepared from radix bupleuri and radix scutellariae, and each capsule contains more than 45mg of baicalin. These preparations all control their quality by baicalin content, and lack monitoring of bupleurum in the preparation.

The bupleurum component is mainly saikoponin, and is composed of a series of characteristic saponins, such as saikoponin A (Saikosaponin A), saikoponin B1(Saikosaponin B1), saikoponin B2(Saikosaponin B2), Saikosaponin C (Saikosaponin C), saikoponin D (Saikosaponin D), saikoponin F (Saikosaponin F), saikoponin G (Saikosaponin G), saikoponin H (Saikosaponin H), saikoponin I (Saikosaponin I), nipagin K (Nepasaikosaponin K), saikoponin S (Saikosaponin S), Prosapogenin D, Prosapogenin F, etc. 13 types, and the chemical structures and properties of these saponins are similar to each other than 13 types, and the chemical structures and physical and chemical properties of these saponins are not measured by conventional liquid chromatography using an ultraviolet detector while separating them.

Although there are reports in the literature on simultaneous determination of a few of the saponins, there is no method for simultaneous determination of 13 saikosaponin types.

For example, Wenshuang literature (Wenshuang et al, HPLC method for determining saikosaponin a, b in bupleurum root granule)₁C, d and baicalin content, journal of drug analysis, 01 st 2017, page 148-152) describe the determination of saikosaponin a, b in bupleurum particles₁C, d and baicalin content by HPLC. The method adopts SB-C₁₈Chromatography column (250mm × 4.6mm, 5.0 μm) with acetonitrile (A) -0.01% phosphoric acid water solution (B) as mobile phase, gradient elution (0-30min, 30% A → 60% A; 30-35min, 60% A), flow rate of 1mL/min, and detection wavelength of 210 nm. As a result: ultrasonic extracting with 5% concentrated ammonia methanol solution for 30min to obtain saikosaponin a and saikosaponin b₁C, d and baicaleinThe quantity concentration is respectively in the range of 3.6-36.0, 6.0-60.0, 8.4-84.0, 9.2-92.0 and 10.0-100.0 mug/mL, the linear relation is good (r is more than or equal to 0.9992); the method has good precision, stability and repeatability, and RSD is less than 2.0%; saikosaponin a, b₁The average recovery rates of c, d and baicalin are 97.5%, 95.3%, 99.7%, 98.4% and 95.1%, and the RSD is less than 2.0%; saikosaponin a, b in 3 batches of samples₁D and baicalin content ranges of 1.560-1.573, 7.501-7.587, 9.204-9.356 and 11.635-11.817mg/g respectively, and saikosaponin c is not detected. The authors of the literature believe that the method can be used to verify that saikosaponin a, b in bupleurum root particles₁And c, d and baicalin content measurement can provide scientific basis for overall quality control of the Chaihuang granules.

Yan Jie literature (Jie, et al, HPLC determination of content of saikoside a, c, d in overground and underground parts of 4 Sichuan radix bupleuri, J. Chinese Experimental formulary, 13 th 2014, pages 73-76) describes a method for determining content of saikoside a, c, d in different varieties and different medicinal parts of Sichuan radix bupleuri. The method adopts Agilent TC-C18 chromatographic column (4.6mm multiplied by 250mm, 5 mu m), mobile phase acetonitrile-water, gradient elution, flow rate of 1mL/min, detection wavelength of 210nm and column temperature of 30 ℃. As a result, the saikosaponin a, c and d have good linear relations with peak areas in the ranges of 0.753-22.590, 0.461-13.830 and 0.726-21.780 mu g respectively, and the average sample adding recovery rates are 100.83%, 99.96% and 101.12% respectively. The authors of the literature believe that the difference in saikosaponin content is greater for different species and different medicinal parts.

The present inventors tried to adopt the above literature methods and found that they could not simultaneously measure the above 13 saikosaponins. Therefore, the skilled in the art expects to have new methods for determining the content of saikosaponin in bupleurum medicinal materials, decoction pieces and extracts thereof, such as aqueous extracts and bupleurum root preparations, especially for simultaneously determining the content of the 13 saikosaponin.

Disclosure of Invention

The invention aims to provide a method for measuring the content of saikosaponin in bupleurum medicinal materials, decoction pieces and extracts thereof, such as aqueous extracts and bupleurum root preparations, and particularly provides a method for simultaneously measuring the content of 13 saikosaponin in the bupleurum medicinal materials, the decoction pieces and the extracts thereof, such as aqueous extracts and bupleurum root preparations. The present inventors have surprisingly found that the chromatography-mass spectrometry detection of 13 saponins can be accomplished within 20 minutes by the method of the present invention. The present invention has been completed based on such findings.

Therefore, the invention provides a method for measuring the content of saikosaponin in bupleurum medicinal materials, decoction pieces and extracts thereof, such as aqueous extracts or bupleurum root preparations, which adopts a high performance liquid chromatography high resolution mass spectrometry method to simultaneously measure the saikosaponin in the bupleurum root preparations and calculate the content of the saikosaponin.

The method according to the first aspect of the present invention, wherein said chaihuang preparation is selected from the group consisting of chaihuang granules, chaihuang capsules, chaihuang tablets, chaihuang oral liquid, chaihuang soft capsules. Preferably, the Chaihuang preparation is selected from Chaihuang granule, Chaihuang capsule, and Chaihuang tablet.

The method according to the first aspect of the present invention, wherein the saikosaponin comprises one or more combinations selected from the following 13 saponins: saikosaponin A (Saikosaponin A), Saikosaponin B1(Saikosaponin B1), Saikosaponin B2(Saikosaponin B2), Saikosaponin C (Saikosaponin C), Saikosaponin D (Saikosaponin D), Saikosaponin F (Saikosaponin F), Saikosaponin G (Saikosaponin G), Saikosaponin H (Saikosaponin H), Saikosaponin I (Saikosaponin I), nipagin K (Nepasaikosaponin K), saikoponin S (Saikosaponin S), Prosapogenin D, Prosapogenin F.

The liquid phase coupled high resolution mass spectrometry can be selected from: waters QTof high-resolution mass spectrometry, Agilent iFunnel Q-TOF high-resolution mass spectrometry, the mo Fisher quadrupole-electrostatic field orbitrap high-resolution mass spectrometry (UPLC-Q active-MS), the mo Fisher ion well-orbitrip high-resolution mass spectrometry, SCIEX QTOF high-resolution mass spectrometry, AB Qtof high-resolution time-of-flight mass spectrometry, Shimadzu Q-TOF high-resolution mass spectrometry, and the like.

The method according to the first aspect of the present invention, wherein the chromatographic conditions of the liquid chromatography-mass spectrometry (such as ultra high performance liquid chromatography tandem electrostatic field orbitrap mass spectrometry) are as follows: the column was phenomenex C18 (2.1X 150mm, 1.7 μm), and 0.1% formic acid solution was used as mobile phase A and methanol was used as mobile phase B, and the elution was carried out in a gradient at a flow rate of 0.25 mL/min. The elution gradient was as follows: 0-1min, 2% B; 1.0-2.5min, 2% -60% B; 2.5-6.5min, 60% -90% B; 6.5-7.5min, 90% -100% B; 7.5-9.5min, 100% B; 9.5-9.6min, 100% -2% B; 9.6-15.0min, 12% B. The column temperature was 25 ℃ and the amount of sample was 5. mu.L.

The method according to the first aspect of the present invention, wherein the mass spectrometry conditions of the liquid chromatography-mass spectrometry (such as ultra performance liquid chromatography tandem electrostatic field orbitrap mass spectrometry) are as follows: mass spectrum conditions: the positive and negative ion online detection mode is characterized in that ESI source, ion source temperature is 300 ℃, and capillary temperature is 350 ℃; the voltage of the taper hole is 3.0 kV; sheath gas flow 50 psi; auxiliary gas flow 10 psi; the S-Lens RF is 30 percent, and the scanning range m/z is 110-800; resolution 75000.

The method according to the first aspect of the present invention, wherein the method for quantifying molecular weight and chemical formula of said 13 saponins in the determination of liquid chromatography-mass spectrometry (such as ultra high performance liquid chromatography tandem electrostatic field orbitrap mass spectrometry) is as listed below.

The method according to the first aspect of the present invention, wherein the preparation of the mixed reference solution for reference and the preparation of the standard curve are calculated as follows: and precisely weighing a proper amount of reference substances, wherein the sample weights are respectively as follows: 17.56mg of saikosaponin A, 115.46mg of saikosaponin B, 211.98mg of saikosaponin B, 12.84mg of saikosaponin C, 11.56mg of saikosaponin D, 13.86mg of saikosaponin F, 12.44mg of saikosaponin G, 13.55mg of saikosaponin H, 12.83mg of saikosaponin I, 4.91mg of nipagoside K, 13.88mg of saikosaponin S, 11.03mg of Prosapogenin D and 11.03mg of Prosapogenin F are prepared by dissolving with 80% methanol to obtain saikosaponin G497.6 μ G/mL, saikosaponin H542.0 μ G/mL, 1.2 μ G/mL of saikosaponin I, 25 μ G/mL of saikosaponin K, 25 μ G/mL of saikosaponin F, 555.2 μ G/mL of saikosaponin S, 3625 μ G/mL of saikosaponin B, 3632 μ G/mL of saikosaponin B, 2479.2 μ G/mL of saikosaponin D, 11.03mg of saikosaponin F, 11.6 μ G/mL of saikosaponin F, 78 μ G/mL of saikosaponin S, A control mother liquor of Prosapogenin D441.2 μ g/mL and Prosapogenin F441.2 μ g/mL; precisely sucking 750 μ L of each of the 13 reference mother solutions to 10mL volumetric flasks, metering to volume with 80% methanol to scale, and making into 13 saikosaponin mixed reference solutions containing saikosaponin A52.68 μ G/mL, saikosaponin B146.38 μ G/mL, saikosaponin B235.94 μ G/mL, saikosaponin C38.52 μ G/mL, saikosaponin D34.68 μ G/mL, saikosaponin F41.58 μ G/mL, saikosaponin G37.32 μ G/mL, saikosaponin H40.65 μ G/mL, saikosaponin I38.49 μ G/mL, nipagin K36.825 μ G/mL, saikosaponin S41.64 μ G/mL, Prosapogenin D33.09 μ G/mL, and Prosapogenin F33.09 μ G/mL; diluting 13 saikosaponin mixed reference substance solutions by 5 times, 10 times, 25 times, 50 times, 75 times, 100 times, 250 times, 500 times, 750 times and 1000 times respectively, injecting, and calculating a standard curve, wherein the standard curve is used for quantitative calculation of various saponins in the Chaihuang preparation.

The method according to the first aspect of the present invention, wherein the test solution of bupleurum root material to be used in the determination is prepared as follows: taking a 0.5g sample of radix bupleuri, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of methanol solution containing 5% concentrated ammonia test solution, sealing, weighing, carrying out ultrasonic treatment (power 500W, frequency 40kHz) for 30 minutes, taking out, cooling, weighing again, supplementing the weight loss by using the methanol solution containing 5% concentrated ammonia test solution, shaking uniformly, and filtering by using a 0.22 mu m microporous membrane to obtain the Chinese medicinal composition.

The method according to the first aspect of the present invention, wherein the test solution for decocting Bupleurum chinense DC to be used in the measurement is prepared by the following method: decocting 10g of bupleuri radix in water twice for 4 hr each time, filtering decoction, mixing filtrates, and concentrating to obtain extract; taking a sample extract which is equivalent to 0.5g of radix bupleuri, precisely weighing, placing the sample extract in a conical flask with a plug, precisely adding 25ml of methanol solution containing 5% concentrated ammonia test solution, sealing the plug, weighing, carrying out ultrasonic treatment (power 500W, frequency 35kHz) for 30 minutes, taking out, cooling, weighing again, supplementing the weight loss by using the methanol solution containing 5% concentrated ammonia test solution, shaking uniformly, and filtering by using a 0.22 mu m microporous membrane to obtain the Chinese medicinal composition.

The method according to the first aspect of the present invention, wherein the test solution of a chaihuang preparation to be used in the assay is prepared as follows: taking a sample of the Chaihuang preparation which is equivalent to 0.5g of bupleurum containing medicinal material, precisely weighing, placing the sample in a conical flask with a plug, precisely adding 25ml of methanol solution containing 5% concentrated ammonia test solution, sealing the plug, weighing, carrying out ultrasonic treatment (power 500W, frequency 40kHz) for 30 minutes, taking out, cooling, weighing again, complementing the loss weight by using the methanol solution containing 5% concentrated ammonia test solution, shaking up, and filtering by using a 0.22 mu m microporous membrane to obtain the bupleurum containing medicinal material; alternatively, the method according to the first aspect of the present invention, wherein the preparation of the test solution of a faaiba preparation to be used in the assay is prepared by a faaiba preparation test solution modification preparation method comprising the following operations: taking a sample of the chaihuang preparation which is equivalent to 0.5g of bupleurum containing medicinal material, precisely weighing, placing the sample into a conical flask with a plug, adding 25ml of diethyl ether and 0.5ml of 10 percent sodium hydroxide, sealing the plug, carrying out ultrasonic treatment (power 500W and frequency 40kHz) for 10 minutes, filtering, washing residues with 20ml of diethyl ether, volatilizing the diethyl ether from the residues, precisely adding 25ml of methanol solution containing 5 percent concentrated ammonia test solution, sealing the plug, weighing, carrying out ultrasonic treatment (power 500W and frequency 40kHz) for 30 minutes, taking out, cooling, weighing again, complementing the weight loss by methanol solution containing 5 percent concentrated ammonia test solution, shaking up, and filtering by a 0.22 mu m microporous membrane to obtain the chaihuang preparation.

The method according to the first aspect of the present invention is characterized in that when a liquid chromatography-mass spectrometry (such as ultra high performance liquid chromatography-tandem electrostatic field orbitrap high resolution mass spectrometry) is used for sample determination, 5 μ l of each sample solution is precisely absorbed, the sample solution is respectively injected into a high performance liquid chromatography-mass spectrometry instrument for determination, and the content of each saponin in the test solution is calculated by using a standard curve, so as to obtain the product.

The method of the invention obtains satisfactory technical effect, and especially can simultaneously determine the content of 13 saponins from bupleurum in the chaihuang preparation within a short time of 20 min.

Drawings

FIGS. 1-7 are ion diagrams of LC-MS analysis of 13 saikosaponin, in each of which SSS represents saikosaponin S, CF represents Prosapogenin F, SSCD represents Prosapogenin D, and other abbreviations have similar meanings.

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. The following examples further illustrate the invention without limiting it.

Test example 1: research on UPLC-Q active-MS content determination of characteristic component of radix bupleuri in Chaihuang preparation

The characteristic components of the bupleurum medicinal materials, decoction pieces and aqueous extracts thereof and the bupleurum root preparation are simultaneously measured by adopting an ultra-high performance liquid chromatography tandem electrostatic field orbit trap high resolution mass spectrometry (UPLC-Q active-MS) technology, and the chromatographic conditions and the mass spectrometry conditions are detailed below. According to UPLC-Q active-MS content determination methodology research aiming at saikosaponin characteristic components of the bupleurum root-yellow preparation, a content determination method is established. And samples of chaihuang preparations from multiple manufacturers were collected from all over the country for analysis.

1. Experimental medicinal materials

Samples of commercially available Bupleurum falcatum were collected in 70 batches, which included: 45 batches of bupleurum chinense medicinal materials (8 batches of the bupleurum chinense medicinal materials are adulterated), 11 batches of Tibetan bupleurum chinense medicinal materials, 4 batches of bupleurum chinense medicinal materials, 7 batches of bupleurum chinense medicinal materials, 1 batch of bupleurum scorzonerifolium medicinal materials and 2 batches of solidago decurrens medicinal materials, wherein part of the medicinal materials and the sources and batch numbers (or self-numbering) are detailed in the following table 1.

TABLE 1

2. Saponin reference substance

The detailed information of the reference substances of 13 saikosaponin detected by the invention is shown in the following table 2.

3. Preparation of mixed control solutions and preparation of standard curves:

and precisely weighing a proper amount of reference substances, wherein the sample weights are respectively as follows: 17.56mg of saikosaponin A, 115.46mg of saikosaponin B, 211.98mg of saikosaponin B, 12.84mg of saikosaponin C, 11.56mg of saikosaponin D, 13.86mg of saikosaponin F, 12.44mg of saikosaponin G, 13.55mg of saikosaponin H, 12.83mg of saikosaponin I, 4.91mg of nipagoside K, 13.88mg of saikosaponin S, 11.03mg of Prosapogenin D and 11.03mg of Prosapogenin F are prepared by dissolving with 80% methanol to obtain saikosaponin G497.6 μ G/mL, saikosaponin H542.0 μ G/mL, 1.2 μ G/mL of saikosaponin I, 25 μ G/mL of saikosaponin K, 25 μ G/mL of saikosaponin F, 555.2 μ G/mL of saikosaponin S, 3625 μ G/mL of saikosaponin B, 3632 μ G/mL of saikosaponin B, 2479.2 μ G/mL of saikosaponin D, 11.03mg of saikosaponin F, 11.6 μ G/mL of saikosaponin F, 78 μ G/mL of saikosaponin S, Prosapogenin D441.2 μ g/mL, Prosapogenin F441.2 μ g/mL. Precisely sucking 750 μ L of each of the 13 reference mother solutions to 10mL volumetric flasks, metering to volume with 80% methanol to scale, and making into 13 saikosaponin mixed reference solutions containing saikosaponin A52.68 μ G/mL, saikosaponin B146.38 μ G/mL, saikosaponin B235.94 μ G/mL, saikosaponin C38.52 μ G/mL, saikosaponin D34.68 μ G/mL, saikosaponin F41.58 μ G/mL, saikosaponin G37.32 μ G/mL, saikosaponin H40.65 μ G/mL, saikosaponin I38.49 μ G/mL, nipagin K36.825 μ G/mL, saikosaponin S41.64 μ G/mL, Prosapogenin D33.09 μ G/mL, and Prosapogenin F33.09 μ G/mL. Diluting 13 saikosaponin mixed reference substance solutions by 5 times, 10 times, 25 times, 50 times, 75 times, 100 times, 250 times, 500 times, 750 times and 1000 times respectively, injecting, and calculating a standard curve, wherein the standard curve is used for quantitative calculation of various saponins in the Chaihuang preparation.

4. Measurement conditions

An ultra-high performance liquid chromatography tandem electrostatic field orbit trap high resolution mass spectrometer (UPLC-Q active-MS, which can be called as a chromatography-mass spectrometer, LC-MS, UPLC-MS and the like in the invention) is adopted.

Chromatographic conditions are as follows: the column was phenomenex C18 (2.1X 150mm, 1.7 μm), and 0.1% formic acid aqueous solution was used as mobile phase A and methanol was used as mobile phase B, and the elution was carried out by gradient elution at a flow rate of 0.25 mL/min. The elution gradient was as follows: 0-1min, 2% B; 1.0-2.5min, 2% -60% B; 2.5-6.5min, 60% -90% B; 6.5-7.5min, 90% -100% B; 7.5-9.5min, 100% B; 9.5-9.6min, 100% -2% B; 9.6-15.0min, 12% B. The column temperature was 25 ℃ and the amount of sample was 5. mu.L.

Mass spectrum conditions: the positive and negative ion online detection mode is characterized in that ESI source, ion source temperature is 300 ℃, and capillary temperature is 350 ℃; the voltage of the taper hole is 3.0 kV; sheath gas flow 50 psi; auxiliary gas flow 10 psi; the S-Lens RF is 30 percent, and the scanning range m/z is 110-800; resolution 75000.

The molecular weight and chemical formula of each control are shown in table 2 below.

TABLE 2

No	Name of Chinese	English name	Method for quantifying molecular weight	Chemical formula (II)	Reference supplier
						1	Saikosaponin A	Saikosaponin A	825.46419(+ HCOOH)	C42H68O13	Chinese food and drug assay Research institute
2	Saikosaponin B1	Saikosaponin B1	825.46419(+ HCOOH)	C42H68O13	Lemeitian medicine \| dester Biological organisms
						3	Saikosaponin B2	Saikosaponin B2	825.46419(+ HCOOH)	C42H68O13	Lemeitian medicine \| dester Biological organisms
4	Saikosaponin C	Saikosaponin C	971.5221(+ HCOOH)	C48H78O17	Lemeitian medicine \| dester Biological organisms
						5	Saikosaponin D	Saikosaponin D	825.46419(+ HCOOH)	C42H68O13	Chinese food and drug assay Research institute
6	Saikosaponin G	Saikosaponin G	825.46419(+ HCOOH)	C42H68O13	Lemeitian medicine \| dester Biological organisms
						7	Saikosaponin H	Saikosaponin H	971.5221(+ HCOOH)	C48H78O17	Lemeitian medicine \| dester Biological organisms
8	Root of Chinese Thorowax Saponin K	Nepasaikosap onin K	989.53267(+ HCOOH)	C48H80O18	NATURE STANDARD
						9	Saikosaponin S	Saikosaponin S	987.51702(+ HCOOH)	C48H78O18	The technology of Beijing Saibao includes Limited company
10	Saikosaponin I	Saikosaponin I	971.5221(+ HCOOH)	C48H78O17	Lemeitian medicine \| dester Biological organisms
						11	Prosapoge nin D	Prosaikogeni n D	663.41137(+ HCOOH)	C36H58O8	Lemeitian medicine \| dester Biological organisms
12	Saikosaponin F	Saikosaponin F	973.53775(+ HCOOH)	C48H80O17	Lemeitian medicine \| dester Biological organisms
						13	Prosapoge nin F	Prosaikogeni n F	617.40589	C36H58O8	The technology of Beijing Saibao includes Limited company

5. Preparation and determination of samples

Preparing a bupleurum medicinal material test solution: taking a 0.5g sample of radix bupleuri, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of methanol solution containing 5% concentrated ammonia test solution, sealing, weighing, carrying out ultrasonic treatment (power 500W, frequency 40kHz) for 30 minutes, taking out, cooling, weighing again, supplementing the weight loss by using the methanol solution containing 5% concentrated ammonia test solution, shaking uniformly, and filtering by using a 0.22 mu m microporous membrane to obtain the Chinese medicinal composition.

Preparation of a radix bupleuri water extract (namely decoction) test solution: decocting 10g of bupleuri radix in water twice for 4 hr each time, filtering the decoctions, mixing the filtrates, and concentrating to obtain extract. Taking a sample extract which is equivalent to 0.5g of radix bupleuri, precisely weighing, placing the sample extract in a conical flask with a plug, precisely adding 25ml of methanol solution containing 5% concentrated ammonia test solution, sealing the plug, weighing, carrying out ultrasonic treatment (power 500W, frequency 35kHz) for 30 minutes, taking out, cooling, weighing again, supplementing the weight loss by using the methanol solution containing 5% concentrated ammonia test solution, shaking uniformly, and filtering by using a 0.22 mu m microporous membrane to obtain the Chinese medicinal composition.

Preparing a chaihuang preparation test solution: weighing Bupleurum root extract sample 0.5g, placing in conical flask with plug, adding methanol solution 25ml containing 5% concentrated ammonia solution, sealing, weighing, ultrasonic treating (power 500W, frequency 40kHz) for 30min, taking out, cooling, weighing, supplementing the weight loss with methanol solution containing 5% concentrated ammonia solution, shaking, and filtering with 0.22 μm microporous membrane.

The determination method comprises the following steps: precisely sucking 5 μ l of each sample solution, respectively injecting into high performance liquid chromatography-mass spectrometer, measuring, and calculating content of each saponin in the test solution with standard curve.

6. Methodology specificity review

Under the above conditions of chromatography-mass spectrometry, 13 saikosaponin ion diagrams are shown in FIGS. 1-7. As can be seen from the figure, 13 saikosaponin were well separated. In the figure, SSS means saikosaponin S, CF means Prosapogenin F, SSCD means Prosapogenin D, and other abbreviations have similar meanings. Furthermore, all 13 saponins could be tested within 20min under the chromatography-mass spectrometry conditions described above in the present invention.

7. Linear relationship and quantitative limit

Selecting a Chaihuang preparation (No. 41 Chaihuang granules), adding 13 saikosaponin mixed reference substance solutions (1.333333333 times of the saikosaponin mixed reference substance solution), 2 times of the saikosaponin mixed reference substance solution (c), 4 times of the saikosaponin mixed reference substance solution (c), 10 times of the saikosaponin mixed reference substance solution (c), 13.33333333 times of the saikosaponin mixed reference substance solution (c), 20 times of the saikosaponin mixed reference substance solution (c), 40 times of the saikosaponin mixed reference substance solution (r), 100 times of the saikosaponin mixed reference substance solution (c) and 200 times of the saikosaponin mixed reference substance solution (c), making a matrix standard curve, and performing linear regression on the sample injection concentration (X, ng/L) by taking the peak area (Y) of a chromatographic peak of a compound to be detected as a vertical coordinate. The linear range, regression equation and correlation coefficient, the results are shown in table 4 below. The data of the lowest addition concentration point in the linear equation are used for calculating the detection limit of 13 saponins according to the signal-to-noise ratio of 3 times, and the quantitative limit of 13 saponins is calculated according to the signal-to-noise ratio of 10 times, and the result is shown in table 4. These results indicate that the chaihuang preparation still has excellent linear relationship after being added with the reference substance, and the detection limit and the quantification limit can meet the general requirements of drug analysis.

8. Methodological recovery and precision testing

Taking 0.125g, 0.25g, 0.5g and 9 parts in total of the bupleurum root preparation (bupleurum root particles with the number 40), respectively adding 50, 100 and 200 mu L of a mixed reference substance solution of the saikoside according to the low level, the middle level and the high level, wherein each added reference level is 3 parts in parallel, preparing a sample solution according to the method, measuring, and calculating the recovery rate and the relative standard deviation of each saikoside, which are shown in the table 3.

TABLE 3

The results in the above table on the recovery and precision of the process indicate that the process has a large fluctuation in the recovery of individual saponins and a relatively large standard deviation of the recovery of different saponins, which is not ideal from the viewpoint of pharmaceutical analysis. In order to overcome the above problems, the operation of the present invention for the item "preparation of a fasuvin preparation test solution" in "preparation and measurement of samples" described above in test example 1 is changed to the following manner (which may be referred to as "fasuvin preparation test solution modified preparation method" in the present invention): taking a sample of the chaihuang preparation which is equivalent to 0.5g of bupleurum containing medicinal material, precisely weighing, placing the sample into a conical flask with a plug, adding 25ml of diethyl ether and 0.5ml of 10 percent sodium hydroxide, sealing the plug, carrying out ultrasonic treatment (power 500W and frequency 40kHz) for 10 minutes, filtering, washing residues with 20ml of diethyl ether, volatilizing the diethyl ether from the residues, precisely adding 25ml of methanol solution containing 5 percent concentrated ammonia test solution, sealing the plug, weighing, carrying out ultrasonic treatment (power 500W and frequency 40kHz) for 30 minutes, taking out, cooling, weighing again, complementing the weight loss by methanol solution containing 5 percent concentrated ammonia test solution, shaking up, and filtering by a 0.22 mu m microporous membrane to obtain the chaihuang preparation. The present inventors have surprisingly found that excellent process recovery and precision of all 13 saponins can be obtained when the above mentioned chaihuang preparation test solution is modified preparation method when preparing the test solution. In a supplementary test, supplementary test a, the above modified preparation method of the chaihuang preparation test solution was used for methodological recovery and precision tests: taking 0.125g, 0.25g, 0.5g and 9 parts in total of a fagopyrum preparation (fagopyrum cymosum granules with the number of 40), respectively adding saikosaponin mixed reference substance solutions (50, 100 and 200 mu L) according to the low, middle and high levels, wherein each added reference level is parallel to 3 parts, preparing a test sample solution according to the fagopyrum cymosum preparation test sample solution modification preparation method, measuring, and calculating the recovery rate and relative standard deviation of each saikosaponin; as a result, the precision (RSD) of the 13 saponins is in the range of 0.84% -2.37%, for example, the RSD of the saikosaponin G is 2.37%, the RSD of the Prosapogenin D is 1.51%, and the recovery rate of the 13 saponins is in the range of 89% -95%, for example, the recovery rates of the saikosaponin A measured three times are respectively 92.4%, 90.1% and 91.7%; this result indicates that the improved preparation method using the chaihuang preparation test solution can significantly improve the methodological recovery rate and precision. In a supplementary test, supplementary test B, referring to supplementary test a, but when the ether-sodium hydroxide in the modified preparation method of chaihuang preparation sample solution is changed into ether, i.e. no sodium hydroxide is added, the same methodology recovery rate and precision tests are carried out, and as a result, the precision of 13 saponins fluctuates within the range of 1.36-11.62%, for example, the precision of Prosapogenin F is 8.68%, indicating that sodium hydroxide is indispensable in the modified preparation method. In a supplementary test, supplementary test C, referring to supplementary test A, but when the ether in the ether-sodium hydroxide in the modified preparation method of the chaihuang preparation sample solution is changed into acetone with the same amount, the same methodology recovery rate and precision tests are carried out, and as a result, the precision of 13 saponins fluctuates within the range of 0.92-13.24%, for example, the precision of saikosaponin I is 5.61%, which indicates that the ether cannot be easily replaced in the modified preparation method. Therefore, in one embodiment of the present invention, when preparing the test solution of the faggot preparation, the above modified preparation method of the test solution of the faggot preparation is adopted. In a supplementary trial, supplementary trial D, the recovery and relative standard deviation of each saikosaponin was determined/calculated with reference to supplementary trial a but the tested bupleurum root preparation was bupleurum root capsule No. 47; as a result, the precision (RSD) of 13 saponins was in the range of 0.97% to 2.21%, for example, RSD of saikosaponin G was 1.85%, and the recovery rates of 13 saponins were in the range of 91% to 96%, for example, the recovery rates of saikosaponin A measured three times were 93.7%, 91.5%, and 94.4%, respectively. In a supplementary trial, supplementary trial E, the recovery and relative standard deviation of each saikosaponin was determined/calculated with reference to supplementary trial a but the tested bupleurum preparation was a number 50 bupleurum tablet; as a result, the precision (RSD) of 13 saponins was in the range of 1.14% -2.63%, for example, RSD of saikosaponin G was 2.14%, and the recovery rate of 13 saponins was in the range of 88% -94%, for example, the recovery rates of saikosaponin A measured three times were 92.3%, 89.2%, and 90.7%, respectively. Therefore, the method is suitable for measuring the content of 13 saponins in various bupleurum root preparations. In other supplementary experiments, it has been found that, in the above "preparation of the bupleurum medicinal material test sample solution" and "preparation of the bupleurum decoction test sample solution" in "5, preparation and determination of the sample", when the bupleurum medicinal material and the bupleurum decoction test sample are tested by using the two, the recovery rate can reach more than 97%, and the precision RSD is below 2%, which indicates that the methodological recovery rate and precision are excellent for the bupleurum medicinal material and the bupleurum decoction test sample.

9. Detection of Chaihuang preparation sample

52 samples of the chaihuang preparation series obtained by the invention, including 46 batches of granules, 3 batches of capsules and 3 batches of tablets, are selected and measured according to the operation method (including the operation of the chaihuang preparation test solution modification preparation method), and the measurement results of the content of the characteristic components in a part of typical chaihuang preparations are shown in the table 4 (unit: mu g/g).

TABLE 4

As can be seen from the results in the table, the LC-MS method can simultaneously determine the contents of 13 saponins in the Chaihuang granules, Chaihuang capsules and Chaihuang tablets.

10. Measurement result of radix bupleuri

The method is adopted to measure the contents of 13 saponins in the bupleurum medicinal material. The results show that 13 saikosaponin components have the highest saikosaponin a and highest saikosaponin d, the content of bupleurum chinense and bupleurum falcatum is higher than 100 mu g/g except for individual samples, the content of a and d of bupleurum chinense and bupleurum scorzonerifolium are obviously lower than that of bupleurum chinense, the content of saponins such as solidago virgaurea, starwort root and the like in other non-bupleurum genera are extremely low, and a small amount of bupleurum chinense or bupleurum chinense is. The Nepasaikosaponin K content is higher than that of other quality bupleurum (except part of adulterated bupleurum) by about 25-650 times, so the index can be used as an index component for distinguishing the bupleurum. The content of Prosapogenin D in the bupleurum chinense is found to be about 4 to 200 times higher than that of other bupleurum chinense, so that the index can be used as an index component for distinguishing the bupleurum chinense as a reference.

Comparing the changes before and after decocting bupleuri radix, the results show that after decocting, saikosaponin I, G, S, H, B1 and B2 are greatly increased, saikosaponin A, D is greatly reduced, and saikosaponin F, hypo-glycoside F and C are also reduced. Nepasaikosaponin K and hypoglycoside D also tended to decrease, but the magnitude was not large. Therefore, the results are more stable as a characteristic index.

In the present invention, for convenience of description, the term "before decocting" may be understood as "decoction pieces of medicinal materials", and the term "after decocting" may be understood as "extract"

The results of some bupleurum root herbs are shown in table 5a and table 5 b.

TABLE 5a

Continue to watch 5a (B1 ~ S)

Continuation table 5a (G-F)

TABLE 5b

Continue to watch 5B (B1 ~ S)

Continue to watch 5b (G-F)

The above-described embodiments are merely preferred embodiments for fully illustrating the present application, and the scope of the present application is not limited thereto. The equivalent substitution or change made by the person skilled in the art on the basis of the present application is within the protection scope of the present application. The protection scope of this application is subject to the claims.

Claims

1. Determine the method for saikosaponin content in Bupleurum medicinal materials, decoction pieces and extracts thereof such as its water extract or or Bupleurum chinensis preparation, the method adopts liquid phase coupled with high-resolution mass spectrometry (such as ultra-high performance liquid chromatography in series electrostatic Field Orbitrap High Resolution Mass Spectrometry) (UPLC-Q Exactive-MS) simultaneously measured saikosaponins in the Bupleurum Radix, decoction pieces and their extracts such as their water extracts or Bupleurum chinensis preparations and calculated their content.

2. method according to claim 1, wherein said Chaihuang preparation is selected from Chaihuang granule, Chaihuang capsule, Chaihuang tablet, Chaihuang oral liquid, Chaihuang soft capsule; Chaihuang capsules, Chaihuang tablets.

3. The method according to claim 1, wherein the saikosaponin comprises one or more combinations selected from the following 13 saponins: saikosaponin A (Saikosaponin A), saikosaponin B1 (Saikosaponin B1), saikosaponin B1 Saikosaponin B2 (Saikosaponin B2), saikosaponin C (Saikosaponin C), saikosaponin D (Saikosaponin D), saikosaponin F (Saikosaponin F), saikosaponin G (Saikosaponin G), saikosaponin H (Saikosaponin) H), Saikosaponin I (Saikosaponin I), Nepasaikosaponin K, Saikosaponin S (Saikosaponin S), Prosapogenin D, Prosapogenin F.

4. The method according to claim 1, wherein the chromatographic conditions of the ultra-high performance liquid chromatography tandem electrostatic field orbitrap high-resolution mass spectrometry method are as follows: Mobile phase A, methanol as mobile phase B, gradient elution, flow rate 0.25mL/min; elution gradient as follows: 0-1min, 2%B; 1.0-2.5min, 2%-60%B; 2.5- 6.5min, 60%-90%B; 6.5-7.5min, 90%-100%B; 7.5-9.5min, 100%B; 9.5-9.6min, 100%-2%B; 9.6-15.0min, 12% B; the column temperature is 25°C, and the injection volume is 5 μL.

5. The method according to claim 1, wherein the mass spectrometry conditions of the ultra-high performance liquid chromatography tandem electrostatic field orbitrap high-resolution mass spectrometry method are as follows: mass spectrometry conditions: positive and negative ion online detection modes, ESI source, ion source temperature 300 ℃, Capillary temperature 350°C; cone voltage 3.0kV; sheath gas flow 50psi; auxiliary gas flow 10psi; S-Lens RF 30%, scanning range m/z 110-800; resolution 75000.

6. The method according to claim 1, wherein the quantitative molecular weight and chemical formula of the 13 saponins in the ultra-high performance liquid chromatography tandem electrostatic field orbitrap high-resolution mass spectrometry method are as described in the description.

7. according to the method for claim 1, wherein the method for the preparation of the mixed reference substance solution used for calculating reference and the preparation of standard curve is as follows: each precision takes by weighing an appropriate amount of reference substance, and each sample size is respectively: saikosaponin A 17.56 mg, saikosaponin B115.46mg, saikosaponin B2 11.98mg, saikosaponin C 12.84mg, saikosaponin D 11.56mg, saikosaponin F13.86mg, saikosaponin G 12.44mg, saikosaponin H 13.55mg , saikosaponin I 12.83mg, saikosaponin K 4.91mg, saikosaponin S 13.88mg, Prosapogenin D 11.03mg, Prosapogenin F 11.03mg, precisely add 80% methanol to dissolve, respectively prepared containing saikosaponin A 702.4μg/mL, saikosaponin B1 618.4μg/mL, saikosaponin B2 479.2μg/mL, saikosaponin C 513.6μg/mL, saikosaponin D 462.4μg/mL, saikosaponin F 554.4μg/mL, saikosaponin G 497.6μg/mL, saikosaponin H 542.0μg/mL, saikosaponin I 513.2μg/mL, saikosaponin K 491.0μg/mL, saikosaponin S 555.2μg/mL, Prosapogenin D 441.2 μg/mL, Prosapogenin F44 1.2μg/mL reference substance mother solution; Precisely pipette 750μL of each of the above 13 reference substance mother solutions into a 10mL volumetric flask, and make up to the mark with 80% methanol to prepare saikosaponin A 52.68μg/mL , saikosaponin B1 46.38μg/mL, saikosaponin B 235.94μg/mL, saikosaponin C 38.52μg/mL, saikosaponin D 34.68μg/mL, saikosaponin F 41.58μg/mL, saikosaponin G 37.32 μg/mL, saikosaponin H 40.65 μg/mL, saikosaponin I 38.49 μg/mL, saikosaponin K 36.825 μg/mL, saikosaponin S 41.64 μg/mL, Prosapogenin D 33.09 μg/mL, Prosapogenin F33.09μg/mL 13 kinds of saikosaponin mixed reference solution; 13 kinds of saikosaponin mixed reference solution were diluted 5 times, 10 times, 25 times, 50 times, 75 times, 100 times, 250 times, 500 times, 750 times and 1000 times, inject the needle, and calculate the standard curve, which is used for the quantitative calculation of various saponins in Chaihuang preparation.

8. according to the method of claim 1, wherein the method for the preparation of the Bupleurum medicinal material need testing solution that needs to use in the measurement is as follows: get the sample of Bupleurum 0.5g, accurately weigh, place in the conical flask with stopper, accurately add 25ml of methanol solution containing 5% concentrated ammonia test solution, sealed, weighed, ultrasonically treated (power 500W, frequency 40kHz) for 30 minutes, taken out, allowed to cool, and weighed again, with 5% concentrated ammonia test solution. Methanol solution to make up for the lost weight, shake well, and filter through a 0.22 μm microporous membrane; and/or

Wherein the preparation method of the test solution of the Bupleurum chinensis water that needs to be used in the determination (that is, decocting) is as follows: respectively get various Bupleurum edulis 10g, add water and decoct twice, each 4 hours, the decoction is filtered, The filtrates were combined and concentrated to make an extract; take a sample extract equivalent to 0.5 g of Bupleurum, accurately weighed, placed in a conical flask with a stopper, and accurately added 25 ml of methanol solution containing 5% concentrated ammonia test solution, and the plug was tightly plugged. , weighed, ultrasonically treated (power 500W, frequency 35kHz) for 30 minutes, taken out, let cool, weighed again, used methanol solution containing 5% concentrated ammonia test solution to make up the lost weight, shake well, use 0.22μm Filtration through a microporous membrane.

9. according to the method of claim 1, wherein the method for the preparation of the Chaihuang preparation need testing solution that needs to use in measuring is as follows: get the Chaihuang preparation sample that is equivalent to containing Bupleurum chinensis medicinal material 0.5g, accurately weighed, put a stopper In the conical flask, accurately add 25ml of methanol solution containing 5% concentrated ammonia test solution, close the stopper, weigh the fixed weight, ultrasonically treat it (power 500W, frequency 40kHz) for 30 minutes, take it out, let it cool, and weigh it again. The methanol solution of 5% concentrated ammonia test solution makes up for the lost weight, shakes up, and filters through a 0.22 μm microporous filter membrane; The preparation of the preparation test solution was prepared by the modified preparation method of the Chaihuang preparation test solution including the following operations: take a sample of Chaihuang preparation containing 0.5 g of Chaihu medicinal material, accurately weigh it, and place a stopper. In the conical flask, add 25ml of ether and 0.5ml of 10% sodium hydroxide, seal it, ultrasonically treat it (power 500W, frequency 40kHz) for 10 minutes, filter, wash the residue with 20ml of ether, evaporate the residue to dry ether, add 5 25ml of methanol solution of % concentrated ammonia test solution, sealed, weighed, ultrasonically treated (power 500W, frequency 40kHz) for 30 minutes, taken out, let cool, weighed again, and used methanol solution containing 5% concentrated ammonia test solution Make up for the lost weight, shake well, and filter through a 0.22 μm microporous membrane.

10. The method according to claim 1, wherein when using ultra-high performance liquid chromatography tandem electrostatic field orbitrap high-resolution mass spectrometry to measure the sample, precision draws 5 μl of each need testing solution, and injects high performance liquid chromatography-mass spectrometry respectively, Determination, use the standard curve to calculate the content of various saponins in the test solution.