CN113325095A

CN113325095A - UPLC fingerprint construction method and detection method of garden burnet medicinal material

Info

Publication number: CN113325095A
Application number: CN202110489021.4A
Authority: CN
Inventors: 李国卫; 贾小舟; 何民友; 索彩仙; 杨小龙; 卢晓莹; 凌志洲; 魏梅; 孙冬梅; 陈向东; 程学仁
Original assignee: Guangdong Yifang Pharmaceutical Co Ltd; Shandong Yifang Pharmaceutical Co Ltd; Zhejiang Yifang Pharmaceutical Co Ltd
Current assignee: Guangdong Yifang Pharmaceutical Co Ltd; Shandong Yifang Pharmaceutical Co Ltd; Zhejiang Yifang Pharmaceutical Co Ltd
Priority date: 2021-04-29
Filing date: 2021-04-29
Publication date: 2021-08-31
Anticipated expiration: 2041-04-29
Also published as: CN113325095B

Abstract

The invention relates to a UPLC fingerprint construction method and a detection method of sanguisorba officinalis medicinal materials. The method is simple and reliable, has good reproducibility, can comprehensively reflect the overall quality of the sanguisorba officinalis medicinal material, and further can effectively improve the accuracy and stability of the quality detection of the sanguisorba officinalis medicinal material.

Description

UPLC fingerprint construction method and detection method of garden burnet medicinal material

Technical Field

The invention relates to the technical field of traditional Chinese medicine quality control, in particular to a construction method and a detection method of UPLC fingerprint of a garden burnet medicinal material.

Background

The "Chinese pharmacopoeia" 2020 edition records that Sanguisorba officinalis is dried root of Sanguisorba officinalis L. or Sanguisorba longata L. var. longifolia (Bert.) yuet Li, which is a Rosaceae plant. The latter is known as Mian Di Yu. Sanguisorba is bitter, sour, astringent and slightly cold in taste; it enters liver and large intestine meridians; has effects in cooling blood, stopping bleeding, removing toxic substance, and healing sore, and can be used for treating hematochezia, hemorrhoidal bleeding, bloody dysentery, metrorrhagia, scald due to hot water and fire, carbuncle, swelling, and sore.

Generally, the sanguisorba needs to be picked and dug when sprouting or withering in autumn in spring, fibrous roots of the sanguisorba need to be removed in sequence after picking and digging, and then the sanguisorba is cleaned and dried, or sliced and dried when the sanguisorba is fresh, so that the quality difference of the sanguisorba medicinal materials can be caused by different growing places, different treatment processes and the like of the sanguisorba, and therefore, whether the corresponding sanguisorba medicinal materials meet the medicinal standard or not is difficult to evaluate. Therefore, it is highly desirable to construct an accurate and stable quality evaluation standard for sanguisorba officinalis.

Disclosure of Invention

Therefore, a UPLC fingerprint spectrum construction method and a detection method of the sanguisorba officinalis medicinal material are needed to be provided. The sanguisorba officinalis medicinal material fingerprint constructed by the method can be used for quality evaluation of the sanguisorba officinalis medicinal material, and the accuracy and stability of sanguisorba officinalis medicinal material detection are improved.

A method for constructing a fingerprint of a garden burnet medicinal material comprises the following steps:

preparing gallic acid reference solution, radix Sangusorbae reference medicinal material solution and multiple batches of radix Sangusorbae sample solutions;

detecting the gallic acid reference solution, the radix Sangusorbae reference medicinal material solution and each batch of radix Sangusorbae sample solution by ultra-high liquid chromatography to obtain corresponding chromatograms;

establishing a sanguisorba officinalis medicinal material comparison characteristic map according to the chromatogram of the sanguisorba officinalis reference medicinal material; identifying common peaks in the chromatogram of each sanguisorba officinalis test sample, selecting peaks which are consistent with the retention time of the sanguisorba officinalis reference medicinal material from the common peaks as characteristic peaks, and enabling the characteristic peaks to correspond to the characteristic peaks corresponding to the sanguisorba officinalis reference characteristic spectrum to construct the sanguisorba officinalis medicinal material fingerprint spectrum.

In some embodiments, the sanguisorba officinalis medicinal material control characteristic spectrum is established by the following method:

adopting a traditional Chinese medicine chromatogram fingerprint spectrum similarity evaluation system, taking a peak corresponding to gallic acid as a reference peak, calculating the relative retention time and the relative peak area of each characteristic peak of the sanguisorba officinalis reference medicinal material, generating a reference spectrum according to an average method or a median method, and establishing the sanguisorba officinalis reference characteristic spectrum.

In some embodiments, the mobile phase a of the hplc is methanol, and the phase B is 0.08% to 0.12% phosphoric acid in water.

In some embodiments, the elution procedure of the hplc is: 0-6min, the A phase is 3-6%, and the B phase is 97-94%; 6-8min, the A phase is 6-11%, and the B phase is 94-89%; 8-20min, 11% of phase A and 89% of phase B; 20-25min, 11-42% of phase A and 89-58% of phase B; 25-30min, 42% for phase A and 58% for phase B; 30-35min, the A phase accounts for 42-55%, and the B phase accounts for 58-45%; 35-40min, wherein the A phase is 55-97%, and the B phase is 45-3%; for 40-45min, the A phase accounts for 97%, and the B phase accounts for 3%; 45-46min, the A phase is 97-3%, and the B phase is 3-97%; 46-60min, 3% of phase A and 97% of phase B.

In some of these embodiments, the flow rate is 0.1mL/min to 0.3mL/min and the detection wavelength is 270 nm.

In some of these embodiments, the characteristic peaks comprise at least three of a first peak, a second peak, a third peak, and a fourth peak;

the retention time of the first peak is 4.49-4.67min, the retention time of the second peak is 13.28-13.50min, the retention time of the third peak is 17.53-17.70min, and the retention time of the fourth peak is 30.37-30.71 min.

wherein the first peak is a peak having the following mass spectral information: m/z in positive ion mode is 171.0288 + -3 ppm, or m/z in negative ion mode is 169.0132 + -3 ppm;

the second peak is a peak having the following mass spectral information: m/z in positive ion mode is 315.0710 + -3 ppm, or m/z in negative ion mode is 345.0829 + -3 ppm;

the third peak is a peak with the following mass spectral information: m/z in positive ion mode is 291.0861 + -3 ppm, or m/z in negative ion mode is 289.0720 + -3 ppm;

the fourth peak is a peak with the following mass spectral information: the m/z in the negative ion mode was 300.9991. + -.3 ppm.

In some of these embodiments, the second mass spectrum of the peak having a first peak with m/z of 171.0288 ± 3ppm has fragment ion peaks at least at the following m/z: 153.0183 + -3 ppm, 127.0391 + -3 ppm, 125.0235 + -3 ppm, 109.0288 + -3 ppm, 107.0132 + -3 ppm, 81.0341 + -3 ppm; or

A second mass spectrum of a peak having a first peak m/z of 169.0132 ± 3ppm has at least the following fragment ion peaks at m/z: 125.0232 + -3 ppm; or

A second mass spectrum of a peak having a m/z of 315.0710 ± 3ppm of the second peak having fragment ion peaks at least at the following m/z: 153.0182 + -3 ppm; or

A second mass spectrum of a peak having a m/z of 345.0829 ± 3ppm of the second peak having fragment ion peaks at least at the following m/z: 239.0594 + -3 ppm, 148.0155 + -3 ppm, 223.0280 + -3 ppm, 87.0438 + -3 ppm; or

A secondary mass spectrum of a peak having a third peak m/z of 291.0861 ± 3ppm has at least the following fragment ion peaks at m/z: 139.0390 + -3 ppm, 123.0442 + -3 ppm, 147.0441 + -3 ppm; or

A secondary mass spectrum of a peak having a third peak m/z of 289.0720 ± 3ppm has at least the following fragment ion peaks at m/z: 109.0282 + -3 ppm, 125.0232 + -3 ppm, 123.0440 + -3 ppm, 151.0390 + -3 ppm; or

A secondary mass spectrum of a peak having a fourth peak m/z of 300.9991 ± 3ppm has at least the following fragment ion peaks at m/z: 229.0138. + -. 3 ppm.

In some of these embodiments, the first peak is a peak corresponding to gallic acid, the third peak is a peak corresponding to catechin or epicatechin, and the fourth peak is a peak corresponding to ellagic acid.

In some of these embodiments, the concentration of the gallic acid control solution is between 0.8mg/mL and 1.2 mg/mL.

In some embodiments, the concentration of the sanguisorbae officinalis test solution is 3mg/mL to 6 mg/mL.

A detection method of sanguisorba officinalis medicinal materials comprises the following steps:

preparing a test solution of a garden burnet medicinal material;

detecting the test solution by adopting an ultra-high performance liquid chromatography-mass spectrometry combined technology to obtain a corresponding spectrogram;

the spectrogram is analyzed by the sanguisorba officinalis medicinal material fingerprint constructed by the method.

In some embodiments, the chromatographic conditions of the hplc are: the phase A is methanol, and the phase B is 0.08-0.12% formic acid aqueous solution;

the elution procedure was: 0-6min, the A phase is 3-6%, and the B phase is 97-94%; 6-8min, the A phase is 6-11%, and the B phase is 94-89%; 8-20min, 11% of phase A and 89% of phase B; 20-25min, 11-42% of phase A and 89-58% of phase B; 25-30min, 42% for phase A and 58% for phase B; 30-35min, the A phase accounts for 42-55%, and the B phase accounts for 58-45%; 35-40min, wherein the A phase is 55-97%, and the B phase is 45-3%; for 40-45min, the A phase accounts for 97%, and the B phase accounts for 3%; 45-46min, the A phase is 97-3%, and the B phase is 3-97%; 46-60min, 3% of phase A and 97% of phase B;

the mass spectrum conditions are as follows: HESI ion source, the sheath gas flow rate is 30-40arb, the auxiliary gas flow rate is 8-12arb, the spraying voltage is 3-4KV, the S-lens voltage is 45-55V, the heating temperature is 340-360 ℃, the capillary temperature is 340-360 ℃, the scanning collection is respectively carried out in positive and negative ion modes, the scanning mass range is m/z 100-1200, and the normalized collision energy is 20, 40 and 60eV respectively. .

(1) preparing a test solution and a reference solution of the garden burnet medicinal material;

(2) respectively detecting the test solution and the reference solution by adopting an ultra-high performance liquid chromatography-mass spectrometry combined technology to obtain a test sample spectrum and a reference spectrum of the sanguisorba officinalis medicinal material; and analyzing the quality of the radix Sangusorbae sample according to the sample spectrum and the reference spectrum by using peaks corresponding to at least two of gallic acid, catechin (epicatechin) and ellagic acid as characteristic peaks.

The invention has the following beneficial effects:

the invention adopts UPLC method to detect and analyze the garden burnet samples of each batch, finds out the characteristic peak of each garden burnet sample, constructs the fingerprint, and lays the foundation for the quality control of the garden burnet medicinal material. The method is simple and reliable, has good reproducibility, can comprehensively reflect the overall quality of the sanguisorba officinalis medicinal material, and further can effectively improve the accuracy and stability of the quality detection of the sanguisorba officinalis medicinal material.

Drawings

FIG. 1 is a comparison graph of characteristic spectrum of radix Sangusorbae and reference substance;

FIG. 2 is a characteristic spectrum of a sanguisorba officinalis reference medicinal material;

FIG. 3 is a sanguisorba officinalis medicinal material comparison feature map established based on FIG. 2;

FIG. 4 is a characteristic spectrum of 23 lots of radix Sangusorbae sample medicinal materials;

FIG. 5 is a total ion flow diagram and an ultraviolet absorption chromatogram of a sanguisorba officinalis test sample solution;

FIG. 6 is a first order mass spectrometry scan of peak number 1 (positive ion mode);

FIG. 7 is a first order mass spectrometry scan of peak number 1 (negative ion mode);

FIG. 8 is a secondary mass spectrum (positive ion mode) of the m/z 171.0288 peak;

FIG. 9 is a secondary mass spectrum (negative ion mode) of the m/z 169.0132 peak;

figure 10 secondary ion fragment mass spectrometry mirror image (negative ion mode);

FIG. 11 shows the structure of gallic acid;

FIG. 12 shows the possible fragmentation patterns (positive ion mode) for the m/z 171.0288 peak;

FIG. 13 shows a possible cleavage pattern (negative ion mode) for the m/z 169.0132 peak;

FIG. 14 is a first order mass spectral scan of peak number 2 (positive ion mode);

FIG. 15 is a first order mass spectrometry scan of peak number 2 (negative ion mode);

FIG. 16 is a secondary mass spectrum (positive ion mode) of the m/z 315.0710 peak;

FIG. 17 is a secondary mass spectrum (negative ion mode) of the m/z 345.0829 peak;

FIG. 18 is a first order mass spectrometry scan of peak number 3 (positive ion mode);

FIG. 19 is a first order mass spectrometry scan of peak number 3 (negative ion mode);

FIG. 20 is a secondary mass spectrum (positive ion mode) of the m/z 291.0861 peak;

FIG. 21 is a secondary mass spectrum (negative ion mode) of the m/z 289.0720 peak;

FIG. 22 is a mirror image of a secondary ion fragment mass spectrum (positive ion mode) with the upper m/z 291.0861 peak secondary fragment; catechin in the database is arranged below;

FIG. 23 is a mirror image of a secondary ion fragment mass spectrum (negative ion mode) with the upper m/z 289.0720 peak secondary fragment; next catechins in the database);

FIG. 24 shows the structural formula of catechin₁₅H₁₄O₆)；

FIG. 25 shows the possible fragmentation patterns (positive ion mode) for the m/z 290.0861 peak;

FIG. 26 shows possible cleavage patterns (negative ion mode) for the m/z 289.022 peak;

FIG. 27 is a first mass spectrometry scan of peak 4 (negative ion mode);

FIG. 28 is a secondary mass spectrum of the m/z 300.9991 peak;

FIG. 29 is a secondary ion fragment mass spectrometry mirror image (negative ion mode) with the upper m/z 300.9991 peak secondary fragment; next, ellagic acid secondary fragments in the database are obtained;

FIG. 30 shows ellagic acid structural formula (C)₁₄H₆O₈)。

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The embodiment of the invention provides a method for constructing a fingerprint of a garden burnet medicinal material, which comprises the following steps:

s101: preparing gallic acid reference solution, radix Sangusorbae reference medicinal material solution and multiple batches of radix Sangusorbae sample solutions.

In some of these embodiments, the concentration of the gallic acid control solution is between 0.8mg/mL and 1.2 mg/mL; further, the concentration of the gallic acid control solution is 0.90mg/mL, 0.95mg/mL, 1.00mg/mL, 1.05mg/mL, 1.10mg/mL, 1.15mg/mL, or 1.2 mg/mL.

In some embodiments, the solvent of the gallic acid control solution is methanol.

In some embodiments, the concentration of the sanguisorbae test solution is 3mg/mL to 6 mg/mL; further, the concentration of the sanguisorbae radix sample solution is 3.5mg/mL, 3.8mg/mL, 3.9mg/mL, 4.0mg/mL, 4.1mg/mL, 4.2mg/mL, 4.5mg/mL, 5.0mg/mL, 5.5mg/mL, or 6 mg/mL.

In some embodiments, the concentration of the sanguisorba officinalis control drug solution is 3mg/mL to 6 mg/mL; furthermore, the concentration of the sanguisorba officinalis reference medicinal material solution is 3.5mg/mL, 3.8mg/mL, 3.9mg/mL, 4.0mg/mL, 4.1mg/mL, 4.2mg/mL, 4.5mg/mL, 5.0mg/mL, 5.5mg/mL or 6 mg/mL.

In some embodiments, the sanguisorba officinalis reference medicinal material solution and the sanguisorba officinalis test sample solution are prepared by the following steps:

(1) weighing a predetermined amount of sanguisorba officinalis reference medicinal material or a sanguisorba officinalis test sample respectively;

(2) respectively placing radix Sangusorbae reference medicinal material and radix Sangusorbae sample in solvent, heating and refluxing, cooling, metering volume to desired volume, filtering, and collecting filtrate.

In some embodiments, the solvent of the sanguisorba officinalis reference medicinal material solution and the sanguisorba officinalis test sample solution is 10-40% of methanol; further, the solvent is 25% -35% of methanol; further, the solvent was 30% methanol.

In some embodiments, in step S101, the number of lots of sanguisorba officinalis test sample is at least 15; further, at least 20 batches.

In some embodiments, the sanguisorba officinalis test samples of different producing areas are obtained from different sanguisorba officinalis test samples of different producing areas.

S102: detecting gallic acid reference solution, radix Sangusorbae reference medicinal material solution and each lot of radix Sangusorbae sample solution by ultra high liquid chromatography to obtain corresponding chromatogram.

In some embodiments, the mobile phase a of Ultra Performance Liquid Chromatography (UPLC) is methanol and the phase B is 0.08% to 0.12% aqueous phosphoric acid.

In some embodiments, the column is a waters CORTECS T3(150mm x 2.1mm,1.6 μm) column.

In some embodiments, the flow rate of the UPLC is 0.1mL/min to 0.3 mL/min; further, the flow rate was 0.25 mL/min.

In some embodiments, the elution procedure is: 0-6min, the A phase is 3-6%, and the B phase is 97-94%; 6-8min, the A phase is 6-11%, and the B phase is 94-89%; 8-20min, 11% of phase A and 89% of phase B; 20-25min, 11-42% of phase A and 89-58% of phase B; 25-30min, 42% for phase A and 58% for phase B; 30-35min, the A phase accounts for 42-55%, and the B phase accounts for 58-45%; 35-40min, wherein the A phase is 55-97%, and the B phase is 45-3%; for 40-45min, the A phase accounts for 97%, and the B phase accounts for 3%; 45-46min, the A phase is 97-3%, and the B phase is 3-97%; 46-60min, 3% of phase A and 97% of phase B.

S103: establishing a sanguisorba officinalis medicinal material comparison characteristic map according to the chromatogram of the sanguisorba officinalis reference medicinal material; identifying common peaks in chromatogram of each radix Sangusorbae sample, selecting peaks consistent with retention time of radix Sangusorbae reference medicinal material from common peaks as characteristic peaks, and making the characteristic peaks correspond to corresponding characteristic peaks of radix Sangusorbae reference characteristic chromatogram to construct radix Sangusorbae medicinal material fingerprint chromatogram.

Further, in step S103, the sanguisorba officinalis medicinal material comparison characteristic spectrum is established by the following method: and (3) calculating the relative retention time and the relative peak area of each characteristic peak of the sanguisorba officinalis reference medicinal material by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system and taking the peak corresponding to gallic acid as a reference peak, generating a reference spectrum, and establishing the sanguisorba officinalis reference characteristic spectrum.

Furthermore, a traditional Chinese medicine chromatogram fingerprint similarity evaluation system is adopted to generate a reference map according to an average method (or a median method), so that a sanguisorba officinalis medicinal material reference characteristic map can be established.

It is understood that the term "system for evaluating the similarity of chromatographic fingerprint of traditional Chinese medicine" as used herein is understood to mean a system having the same or similar functions as the system, as long as the functions of the present invention can be achieved without departing from the object of the present invention, and is understood to be within the scope of the present invention.

In some embodiments, the characteristic peaks comprise at least three of the first peak, the second peak, the third peak, and the fourth peak.

In some embodiments, the retention time of the first peak is 4.49 to 4.67min, the retention time of the second peak is 13.28 to 13.50min, the retention time of the third peak is 17.53 to 17.70min, and the retention time of the fourth peak is 30.37 to 30.71 min.

In some embodiments, the first peak is a peak having the following mass spectral information: the m/z in the positive ion mode was 171.0288. + -.3 ppm, and the m/z in the negative ion mode was 169.0132. + -.3 ppm.

In some embodiments, a second mass spectrum of a peak having a first peak with an m/z of 171.0288 ± 3ppm has fragment ion peaks at least at the following m/z: 153.0183 + -3 ppm, 127.0391 + -3 ppm, 109.0288 + -3 ppm;

further, a second mass spectrum of a peak having a first peak m/z of 171.0288 ± 3ppm has fragment ion peaks at least at the following m/z: 153.0183 + -3 ppm, 127.0391 + -3 ppm, 125.0235 + -3 ppm, 109.0288 + -3 ppm, 107.0132 + -3 ppm, 81.0341 + -3 ppm;

further, a second mass spectrum of a peak having a first peak m/z of 171.0288 ± 3ppm has fragment ion peaks at least at the following m/z: 153.0183 + -3 ppm, 127.0391 + -3 ppm, 125.0235 + -3 ppm, 109.0288 + -3 ppm, 107.0132 + -3 ppm, 81.0341 + -3 ppm.

Further, a second mass spectrum of a peak having a first peak m/z of 169.0132 ± 3ppm has fragment ion peaks at least at the following m/z: 125.0232. + -. 3 ppm.

In some embodiments, the first peak is a peak corresponding to gallic acid.

In some embodiments, the second peak is a peak having the following mass spectral information: m/z in positive ion mode is 315.0710 + -3 ppm, or m/z in negative ion mode is 345.0829 + -3 ppm;

further, a second mass spectrum of a peak having a second peak m/z of 315.0710 ± 3ppm has fragment ion peaks at least at the following m/z: 153.0182. + -. 3 ppm.

Further, a second mass spectrum of a peak having a second peak m/z of 345.0829 ± 3ppm has fragment ion peaks at least at the following m/z: 239.0594. + -. 3 ppm.

Further, a second mass spectrum of a peak having a second peak m/z of 345.0829 ± 3ppm has fragment ion peaks at least at the following m/z: 239.0594 + -3 ppm, 148.0155 + -3 ppm.

Further, a second mass spectrum of a peak having a second peak m/z of 345.0829 ± 3ppm has fragment ion peaks at least at the following m/z: 239.0594 + -3 ppm, 148.0155 + -3 ppm, 223.0280 + -3 ppm, 87.0438 + -3 ppm.

In some embodiments, the third peak is a peak having the following mass spectral information: the m/z in the positive ion mode was 291.0861. + -.3 ppm, and the m/z in the negative ion mode was 289.0720. + -.3 ppm.

Further, a secondary mass spectrum of a peak having a third peak m/z of 291.0861 ± 3ppm has fragment ion peaks at least at the following m/z: 139.0390 + -3 ppm, 123.0442 + -3 ppm.

Further, a secondary mass spectrum of a peak having a third peak m/z of 291.0861 ± 3ppm has fragment ion peaks at least at the following m/z: 139.0390 + -3 ppm, 123.0442 + -3 ppm, 147.0441 + -3 ppm.

Further, a secondary mass spectrum of a peak having a third peak m/z of 289.0720 ± 3ppm has fragment ion peaks at least at the following m/z: 109.0282 + -3 ppm or 125.0232 + -3 ppm.

Further, a secondary mass spectrum of a peak having a third peak m/z of 289.0720 ± 3ppm has fragment ion peaks at least at the following m/z: 109.0282 + -3 ppm, 125.0232 + -3 ppm, 151.0390 + -3 ppm.

Further, a secondary mass spectrum of a peak having a third peak m/z of 289.0720 ± 3ppm has fragment ion peaks at least at the following m/z: 109.0282 + -3 ppm, 125.0232 + -3 ppm, 123.0440 + -3 ppm, 151.0390 + -3 ppm.

Further, the third peak is a peak corresponding to catechin or epicatechin (isomer).

In some embodiments, the fourth peak is a peak with the following mass spectral information: the m/z in the negative ion mode was 300.9991. + -.3 ppm.

Further, a secondary mass spectrum of a peak having a fourth peak with m/z of 300.9991 ± 3ppm has fragment ion peaks at least at the following m/z: 229.0138. + -. 3 ppm.

Further, the fourth peak is a peak corresponding to ellagic acid.

The technical personnel of the invention creatively selects at least three of the four peaks as characteristic peaks to construct the fingerprint of the garden burnet medicinal material, so that the overall situation of the garden burnet medicinal material can be comprehensively reflected, and when the fingerprint of the garden burnet medicinal material is used for detecting the quality of the garden burnet medicinal material, the information of the garden burnet medicinal material can be more comprehensively detected, thereby improving the accuracy of quality control.

The invention provides a method for detecting sanguisorba officinalis medicinal materials, which comprises the following steps:

s210: preparing a test solution of the elm medicinal material;

step S101 of preparing the sample solution in step S210 is not described herein again.

S220: detecting the test solution by adopting an ultra-high performance liquid chromatography-mass spectrometry combined technology to obtain a corresponding spectrogram;

in some embodiments, the UPLC-QTOF/MS method is employed.

In some embodiments, the mobile phase a of Ultra Performance Liquid Chromatography (UPLC) is methanol and the phase B is 0.08% to 0.12% formic acid solution.

In some embodiments, the mass spectrometry conditions are: HESI ion source, the sheath gas flow rate is 30-40arb, the auxiliary gas flow rate is 8-12arb, the spraying voltage is 3-4KV, the S-lens voltage is 45-55V, the heating temperature is 340-360 ℃, the capillary temperature is 340-360 ℃, the scanning collection is respectively carried out in positive and negative ion modes, the scanning mass range is m/z 100-1200, and the normalized collision energy is 20, 40 and 60eV respectively. .

Further, the mass spectrometry conditions were: HESI ion source, the sheath gas flow rate is 35arb, the auxiliary gas flow rate is 10arb, the spraying voltage is 3.80KV, the S-lens voltage is 50V, the heating temperature is 350 ℃, and the capillary temperature is 360 ℃.

Further, step S220 includes a step of obtaining a mass spectrum of each characteristic peak by using an ultra performance liquid chromatography-mass spectrometry technique, where mass spectrum information of each characteristic peak is as described above, and is not described herein again.

S230: the fingerprint spectrum of the garden burnet medicinal material constructed by the method is used for analyzing the spectrogram.

The method for constructing the sanguisorba officinalis medicinal material fingerprint in the step S230 is as described above, and is not repeated herein.

It is understood that, in step S230, different detection indexes (such as retention time and/or peak area, etc.) can be determined according to needs (such as qualitative and/or quantitative), and are not particularly limited herein, and should be understood as falling within the scope of the present invention.

It is understood that the quantification in step S230 can be performed by using existing analytical methods, such as internal standard or external standard, which are understood to be within the scope of the present invention.

In addition, the method can also comprise a step of detecting mass spectrum information of each peak.

The present invention will be described below with reference to specific examples.

The following examples are given below for the instrument and reagent sources:

the instrument comprises the following steps: thermo high performance liquid chromatograph (Vanqish, Therro corporation); waters high performance liquid chromatograph (H-Class, Waters corporation); waters CORTECS T3(150 mm. times.2.1 mm,1.6 μm) column; one-ten-thousandth analytical balance (ME204E, mettler-toledo); parts per million analytical balance (XP26, mettler-toledo); a numerical control ultrasonic cleaner (KQ500D, ultrasonic instruments Co., Ltd., Kunshan); a constant temperature water bath (model HWS28, model shanghai-constant technology ltd); ultrapure water systems (Milli-Q Direct, Merck, Inc.).

Reagent: ethanol (analytical purity, Fuyu fine chemical Co., Tianjin); methanol (analytical purity, Fuyu Fine chemical Co., Ltd., Tianjin); phosphoric acid (Tianjin, Mimi European chemical reagent Co., Ltd., chromatographic purity); acetonitrile (merck gmbh, chromatographically pure); the water was ultrapure water (self-made in the laboratory).

Reagent testing: gallic acid (China institute for testing and drug products, content: 90.8%, batch No. 110831-201204); ellagic acid (China institute for testing and testing of food and drug, content: 88.8%, batch No. 111959-; sanguisorba officinalis reference medicinal material (China institute for food and drug assay, lot number: 121286-; 23 lot of Sanguisorba officinalis was identified as dry root of Sanguisorba officinalis l. of Rosaceae by Weimei Master Yam pharmacist, Guangdong Fang pharmaceutical Co. The origin and the number are shown in Table 1.

TABLE 1 sample information Table

Serial number	Producing area	Serial number	Producing area
				S1	Longxi county of Deng xi city of Gansu province	S13	Rizhao City, Shandong Province
S2	Longxi county of Deng xi city of Gansu province	S14	Rizhao City, Shandong Province
				S3	Longxi county of Deng xi city of Gansu province	S15	Linyi City, Shandong Province
S4	Longxi county of Deng xi city of Gansu province	S16	Linyi City, Shandong Province
				S5	Longxi county of Deng xi city of Gansu province	S17	Rizhao City, Shandong Province
S6	Longxi county of Deng xi city of Gansu province	S18	Gansu province Longxi county
				S7	Longxi county of Deng xi city of Gansu province	S19	Gansu province Longxi county
S8	Longxi county of Deng xi city of Gansu province	S20	Gansu province Longxi county
				S9	Longxi county of Deng xi city of Gansu province	S21	Gansu province, Ping Liang City
S10	Chenzhou, Hunan province, Guiyang	S22	Gansu province, Ping Liang City
				S11	Chenzhou, Hunan province, Guiyang	S23	Gansu province, Ping Liang City
S12	Chenzhou of Hunan province

Example 1

1.1 chromatographic conditions A waters CORTECS T3(150 mm. times.2.1 mm,1.6 μm) column was used; methanol is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the flow rate was 0.25mL per minute; the column temperature was 35 ℃; the detection wavelength was 270 nm.

1.2 preparation of control solutions A proper amount of gallic acid and ellagic acid are respectively taken, precisely weighed, and added with methanol to prepare a solution containing 1mg per 1 mL.

1.3 preparation of test solution A preparation method of the test solution comprises the steps of taking about 0.1g of garden burnet powder (screened by a No. four sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 30% methanol, weighing, heating and refluxing for 60 minutes, cooling, weighing again, supplementing the weight loss by 30% methanol, shaking uniformly, filtering, and taking a subsequent filtrate.

1.4 methodological considerations

1.4.1 precision test the same lot of Ulmus pumila samples (S1) were sampled and tested according to the method under item "1.3", and the sample solution was repeatedly injected and measured for 6 times under the chromatographic condition of item "1.1", and the relative retention time of 4 common peaks and the RSD of the relative peak area were calculated using gallic acid as reference peak. The results show that the relative retention time and the relative peak area RSD of 4 chromatographic peaks are less than 3 percent, which indicates that the precision is good together.

1.4.2 reproducibility test the same lot of Ulmus pumila samples (S1) were sampled, 6 test solutions were prepared in parallel according to the method under item "1.3", the sample injection was repeated 6 times under the chromatographic condition of item "1.1", gallic acid was used as reference peak, and the relative retention time of 4 common peaks and RSD of the relative peak area were calculated. The result shows that the relative retention time and the relative peak area RSD of the 4 chromatographic peaks are less than 3 percent, which indicates that the method has good reproducibility.

1.4.3 stability test the same sample solution was sampled at 0, 2, 4, 6, 8, 12, 24h according to "1.1" chromatographic conditions, gallic acid was used as reference peak, and the relative retention time of 4 common peaks and RSD of the relative peak area were calculated. The result shows that the relative retention time and the relative peak area RSD of the 4 chromatographic peaks are less than 3 percent, which indicates that the test article is stable within 24 h.

1.4.4 investigation on specificity the gallic acid reference solution, ellagic acid reference solution and radix Sangusorbae test solution are injected into a liquid chromatograph under the chromatographic conditions of '1.1', and chromatogram is recorded. The result shows that the retention time of characteristic pattern gallic acid peak and ellagic acid peak in sanguisorba is consistent with that of reference solution (see figure 1), and the method has good specificity.

1.5 creation of feature map

1.5.1 establishment of fingerprint common mode

Taking sanguisorba officinalis reference medicinal material (batch number: 121286-; identifying common peaks for each test article (see fig. 4); the 4 common peaks consistent with the retention time of sanguisorba officinalis control medicinal material are selected as characteristic peaks, namely a first peak (peak 1, see 1(S) in fig. 4), a second peak (peak 2, see 2 in fig. 4), a third peak (peak 3, see 3 in fig. 4) and a fourth peak (peak 4, see 4 in fig. 4), respectively.

1.5.2 characteristic spectrum determination results the characteristic spectrum of 23 batches of sanguisorba officinalis medicinal materials is analyzed, the gallic acid chromatographic peak is taken as a reference peak S, the relative retention time and the relative peak area of each characteristic peak and the S peak are calculated, the RSD value is calculated, and the experimental results are shown in tables 2 and 3.

TABLE 223 lot radix elm medicinal material feature map (relative retention time)

TABLE 323 lot radix Sanguisorbae medicinal material characteristic (relative peak area)

1.6 measurement of content

1.6.3 content determination results A garden burnet root medicinal material sample is taken, a test solution is prepared according to item 1.3, determination analysis is carried out according to chromatographic conditions under item 1.1, and the content of gallic acid and tannin in the garden burnet root medicinal material is calculated by adopting an external standard method (see table 4). The determination of the content of tannin is carried out according to the determination method of the content of tannin (general rule 2202) in the 'Chinese pharmacopoeia' 2020 edition, and the results are as follows:

TABLE 423 content determination results of Ulmus pumila

Analysis and discussion of results:

the results show that the gallic acid content (1.6-3.8%) and the tannin content (8.9-23.6%) of 23 batches of sanguisorba officinalis decoction pieces meet the content requirements of sanguisorba officinalis decoction pieces in 2020 version of Chinese pharmacopoeia (the gallic acid content is not less than 0.6%, and the tannin content is not less than 2.0%), and the contents are similar to the contents of corresponding raw medicinal materials.

2 UPLC-QTOF/MS method and results

2.1 conditions of liquid phase-Mass Spectrometry

A chromatographic column: waters ACQUITY UPLC BEH C18(2.1 mm. times.100 mm, 1.7 μm); gradient elution was performed as specified in table 5 using methanol as mobile phase a and 0.1% formic acid solution as mobile phase B; the flow rate was 0.25mL per minute; the column temperature was 35 ℃; the detection wavelength was 270 nm. The mass spectrometer ion source and scan parameter settings are detailed in table 6.

TABLE 5 gradient elution Table

TABLE 6 Mass Spectrometry parameters Table

2.2 preparation of test article the preparation of test article is as under item "1.3".

2.3 results of assignment

2.3.1 detecting the sample solution by adopting the liquid chromatography and mass spectrometry analysis conditions to obtain a corresponding mass spectrogram and an ultraviolet absorption chart, which are shown in figure 5.

(1) Peak number 1

Extracting the first chromatogram of peak 1 for 4.49-4.67 min. The results are shown in FIGS. 6-7.

As can be seen from FIGS. 6 and 7, in the primary chromatogram extraction chart of peak No. 1, the peak M/z 171.0288 in positive ion mode has the highest response value, namely [ M + H ]]⁺A peak; has M/z 169.0132 peak under the negative ion mode, and is M-H]^-A peak; presumably C from its exact molecular weight₉H₁₁NO₂(ii) a A secondary plot of peak collision energy 40 for m/z 171.0288 was extracted and showed 153.0183, 127.0391, 125.0235, 109.0288, 107.0132, 81.0341 as detailed results in figure 8. A secondary plot of the m/z 169.0132 peak collision energy of 40 was extracted, and the results showed 125.0232, etc., of the major ion fragments, with detailed results shown in FIG. 9.

The accurate molecular weight and secondary ion fragment information of the signal peak are matched with the compound spectrum in the geological spectrum database, and the result shows that the compound with the highest m/z 171.0288 and m/z 169.0132 peak matching degree is gallic acid, the matching degree is 94.62%, the matching condition of the secondary fragment is shown in figure 10, and the structural formula of the gallic acid is shown in figure 11. The possible cracking modes of gallic acid are shown in fig. 12-fig. 13, and the compound is suggested to be gallic acid according to the fragment ion comparison result and the cracking inference analysis of the structural formula.

(2) Peak No. 2

Extracting the first chromatogram of peak 2 in 13.28-13.50 min. The results are shown in FIGS. 14-15.

As can be seen from FIGS. 14-15, in the primary chromatogram extraction of peak No. 2, there is a peak m/z 315.0710 in the positive ion mode and a peak m/z 345.0829 in the negative ion mode; a secondary mass spectrum with a peak collision energy of m/z 315.0710 of 40 in the positive ion mode is extracted, and the result shows that 153.0182 and the like exist in main ion fragments, and the detailed result is shown in FIG. 16. The secondary mass spectrum with m/z 345.0829 peak collision energy of 40 in the negative ion mode was extracted, and the results showed that the main ion fragments were 239.0594, 148.0155, 223.0280, 87.0438, etc., and the detailed results are shown in fig. 17.

(3) Peak No. 3

Extracting the first-order chromatogram of peak No. 3, wherein the time period is 17.53-17.70 min. The results are shown in FIGS. 18-19.

As can be seen from FIGS. 18 to 19, in the primary chromatogram extraction of peak No. 3, there is a peak of M/z 291.0861 in positive ion mode, which is [ M + H ]]⁺Extracting a secondary mass spectrogram with the collision energy of m/z 291.0861 peak of 40, wherein the result shows that main ion fragments comprise 139.0390, 123.0442, 147.0441 and the like, and the result is shown in figure 20; has M/z 289.0720 peak under the negative ion mode, and is M-H]^-Peak, secondary mass spectrum with m/z 289.0720 peak collision energy of 40 was extracted, and the results showed that there were 109.0282, 125.0232, 123.0440, 151.0390, etc. of the major ion fragments, and the results are shown in fig. 21. Presumably C from its exact molecular weight₁₅H₁₄O₆。

The accurate molecular weight of the signal peak and the secondary ion fragment information are matched with the compound spectrum in the geological spectrum database, and the result shows that the compound with the highest matching degree of m/z 291.0861 and m/z 289.0720 peaks is epicatechin or catechin (isomer), the matching degree is 94.24%, the matching condition of the secondary fragments is shown in fig. 22-23, the structural formula of the catechin is shown in fig. 24, and the possible cracking fragments are shown in fig. 25-26. The compound was suggested to be either catechin or epicatechin (isomers) based on the fragment ion comparison and the lytic inference analysis of the structural formula.

(4) Peak No. 4

Extracting the first chromatogram of peak 4 for 30.37-30.71 min. The results are shown in FIG. 27.

As can be seen from FIG. 27, in the first chromatographic extraction of peak No. 6, there is a peak M/z 300.9991 in the negative ion mode, which is [ M-H ]]^-Extracting a secondary mass spectrum with the collision energy of m/z 300.9991 peak of 40, and displaying the result that the main mass spectrum isThe ion fragment has 300.9991, and the result is shown in FIG. 28. Presumably C from its exact molecular weight₁₄H₆O₈。

The accurate molecular weight and secondary ion fragment information of the signal peak are matched with the compound spectrum in the geological spectrum database, and the result shows that the compound with the highest m/z 300.9991 peak matching degree is ellagic acid, the matching degree is 89.24%, the secondary fragment matching condition is shown in figure 29, and the ellagic acid structural formula is shown in figure 30.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A method for constructing a fingerprint of a garden burnet medicinal material is characterized by comprising the following steps:

2. The method of claim 1, wherein the sanguisorbae officinalis medicinal material reference characteristic spectrum is established by the following method:

3. The method of claim 1, wherein the mobile phase A of the ultra high performance liquid chromatograph is methanol, and the phase B is 0.08% -0.12% phosphoric acid in water.

4. The method of claim 3, wherein the elution procedure of the ultra high performance liquid chromatograph is: 0-6min, the A phase is 3-6%, and the B phase is 97-94%; 6-8min, the A phase is 6-11%, and the B phase is 94-89%; 8-20min, 11% of phase A and 89% of phase B; 20-25min, 11-42% of phase A and 89-58% of phase B; 25-30min, 42% for phase A and 58% for phase B; 30-35min, the A phase accounts for 42-55%, and the B phase accounts for 58-45%; 35-40min, wherein the A phase is 55-97%, and the B phase is 45-3%; for 40-45min, the A phase accounts for 97%, and the B phase accounts for 3%; 45-46min, the A phase is 97-3%, and the B phase is 3-97%; 46-60min, 3% of phase A and 97% of phase B.

5. The method of claim 3, wherein the flow rate is 0.1mL/min to 0.3mL/min and the detection wavelength is 270 nm.

6. The method of any one of claims 1-5, wherein the characteristic peaks comprise at least three of a first peak, a second peak, a third peak, and a fourth peak;

7. The method of claims 1-5, wherein the characteristic peaks comprise at least three of a first peak, a second peak, a third peak, and a fourth peak;

8. The method of claim 7, wherein the first peak has a secondary mass spectrum of a peak with an m/z of 171.0288 ± 3ppm with fragment ion peaks at least at the following m/z: 153.0183 + -3 ppm, 127.0391 + -3 ppm, 125.0235 + -3 ppm, 109.0288 + -3 ppm, 107.0132 + -3 ppm, 81.0341 + -3 ppm; or

9. The method according to any one of claims 1 to 5, wherein the first peak is a peak corresponding to gallic acid, the third peak is a peak corresponding to catechin or epicatechin, and the fourth peak is a peak corresponding to ellagic acid.

10. The method of any of claims 1-5, wherein the concentration of the gallic acid control solution is between 0.8mg/mL and 1.2 mg/mL; and/or

The concentration of the sanguisorba officinalis test solution is 3mg/mL-6 mg/mL.

11. A detection method of sanguisorba officinalis medicinal materials is characterized by comprising the following steps:

preparing a test solution of a garden burnet medicinal material;

comparing and analyzing the fingerprint of the sanguisorba officinalis medicinal material constructed by the method of any one of claims 1 to 10 with the spectrogram.

12. The detection method according to claim 11, wherein the chromatographic conditions of the ultra high performance liquid chromatograph are: the phase A is methanol, and the phase B is 0.08-0.12% formic acid aqueous solution;

the mass spectrum conditions are as follows: HESI ion source, the sheath gas flow rate is 30-40arb, the auxiliary gas flow rate is 8-12arb, the spraying voltage is 3-4KV, the S-lens voltage is 45-55V, the heating temperature is 340-360 ℃, the capillary temperature is 340-360 ℃, the scanning collection is respectively carried out in positive and negative ion modes, the scanning mass range is m/z 100-1200, and the normalized collision energy is 20, 40 and 60eV respectively.