CN113325095B - UPLC fingerprint construction method and detection method of garden burnet medicinal material - Google Patents
UPLC fingerprint construction method and detection method of garden burnet medicinal material Download PDFInfo
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Abstract
The invention relates to a UPLC fingerprint construction method and a detection method of a garden burnet medicinal material, which are used for detecting and analyzing garden burnet samples in batches by adopting an UPLC method, searching characteristic peaks of the garden burnet samples, constructing a fingerprint, and laying a foundation for 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.
Description
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.) Nees Li, which is a Rosaceae plant. The latter is commonly called "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 fingerprint spectrum of the garden burnet root medicinal material constructed by the method can be used for evaluating the quality of the garden burnet root medicinal material, and the accuracy and the stability of the detection of the garden burnet root medicinal material are improved.
A method for constructing a fingerprint of a garden burnet medicinal material comprises the following steps:
preparing gallic acid reference substance solution, radix Sangusorbae reference medicinal material solution and multiple batches of radix Sangusorbae test 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 comparison medicinal material; identifying common peaks in the chromatogram of each sanguisorba officinalis test sample, selecting peaks consistent with the retention time of the sanguisorba officinalis reference medicinal material from the common peaks as characteristic peaks, and corresponding the characteristic peaks 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 from 3% to 6%, and the B phase is from 97% to 94%;6-8min, wherein the A phase is 6-11%, and the B phase is 94-89%; 8-20min, 11% for phase A and 89% for phase B; 20-25min, 11-42% of phase A and 89-58% of phase B; 25-30min, wherein the phase A is 42 percent, and the phase B is 58 percent; 30-35min, 42-55% of phase A and 58-45% of phase B; 35-40min, wherein the A phase is 55-97%, and the B phase is 45-3%; 40-45min, 97% of phase A and 3% of phase B; 45-46min, wherein the A phase is 97-3 percent, and the B phase is 3-97 percent; 46-60min, 3 percent of phase A and 97 percent of phase B.
In some of these embodiments, the flow rate is 0.1mL/min to 0.3mL/min and the detection wavelength is 270nm.
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 30.37-30.71min.
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;
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 having the following mass spectral information: the m/z in the positive ion mode is 291.0861 +/-3 ppm, or the m/z in the negative ion mode is 289.0720 +/-3 ppm;
the fourth peak is a peak having 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 an m/z of 171.0288 ± 3ppm has at least the following fragment ion peaks at m/z: 153.0183 +/-3 ppm, 127.0391 +/-3 ppm, 125.0235 +/-3 ppm, 109.0288 +/-3 ppm, 107.0132 +/-3 ppm and 81.0341 +/-3 ppm; or
A second order mass spectrum of peaks having a m/z of 169.0132 ± 3ppm of the first peak has at least fragment ion peaks at the following m/z: 125.0232 +/-3 ppm; or
A secondary mass spectrum of a peak having a second peak m/z of 315.0710 ± 3ppm has at least the following fragment ion peaks at m/z: 153.0182 +/-3 ppm; or
A second mass spectrum of a peak having an m/z of 345.0829 ± 3ppm for the second peak has fragment ion peaks at least at the following m/z: 239.0594 +/-3 ppm, 148.0155 +/-3 ppm, 223.0280 +/-3 ppm and 87.0438 +/-3 ppm; or
A second order mass spectrum of a peak having a third peak m/z of 291.0861 ± 3ppm has at least a fragment ion peak at the following m/z: 139.0390 +/-3 ppm, 123.0442 +/-3 ppm and 147.0441 +/-3 ppm; or
A second 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 and 151.0390 +/-3 ppm; or
A second order mass spectrum of a peak having a fourth peak with an m/z of 300.9991 ± 3ppm has at least fragment ion peaks at the following 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 1.2mg/mL.
In some embodiments, the concentration of the sanguisorbae officinalis test solution is 3mg/mL to 6mg/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 to 0.12 percent of formic acid aqueous solution;
the elution procedure was: 0-6min, wherein the A phase is from 3% to 6%, and the B phase is from 97% to 94%;6-8min, wherein the A phase is 6-11%, and the B phase is 94-89%; 8-20min, 11% for phase A and 89% for phase B; 20-25min, 11-42% of phase A and 89-58% of phase B; 25-30min, wherein the phase A is 42 percent, and the phase B is 58 percent; 30-35min, 42-55% of phase A and 58-45% of phase B; 35-40min, wherein the A phase is 55-97%, and the B phase is 45-3%; 40-45min, wherein the A phase accounts for 97 percent and the B phase accounts for 3 percent; 45-46min, wherein the A phase is 97-3 percent, and the B phase is 3-97 percent; 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 acquisition 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.
A detection method of sanguisorba officinalis medicinal materials comprises the following steps:
(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 sample 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 test solution;
FIG. 6 is a first order mass spectrometry scan of peak number 1 (positive ion mode);
FIG. 7 is a first mass spectrometric scan of peak number 1 (negative ion mode);
FIG. 8 is a m/z 171.0288 peak secondary mass spectrum (positive ion mode);
FIG. 9 is a m/z 169.0132 peak secondary mass spectrum (negative ion mode);
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 pattern (positive ion mode) of the m/z 171.0288 peak;
FIG. 13 shows a possible fragmentation 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 mass spectrometric scan of peak number 3 (negative ion mode);
FIG. 20 is a second order 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 secondary ion fragment mass spectrometry (positive ion mode) with a m/z 291.0861 peak secondary fragment above; next, catechin in the database;
FIG. 23 is a secondary ion fragment mass spectrometry mirror image (negative ion mode) with the m/z 289.0720 peak secondary fragment above; next catechins in the database);
FIG. 24 shows the structural formula of catechin 15 H 14 O 6 )；
FIG. 25 shows a possible cleavage pattern (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 number 4 peak-to-peak primary mass spectrometry scan (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 is ellagic acidStructural formula (C) 14 H 6 O 8 )。
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 1.2mg/mL; further, the concentration of the gallic acid control solution was 0.90mg/mL, 0.95mg/mL, 1.00mg/mL, 1.05mg/mL, 1.10mg/mL, 1.15mg/mL, or 1.2mg/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 6mg/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 6mg/mL.
In some embodiments, the concentration of the sanguisorba officinalis control drug solution is 3mg/mL to 6mg/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 6mg/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 (150 mm. Times.2.1mm, 1.6 μm) column.
In some embodiments, the flow rate of the UPLC is between 0.1mL/min and 0.3mL/min; further, the flow rate was 0.25mL/min.
In some embodiments, the elution procedure is: 0-6min, the A phase is from 3% to 6%, and the B phase is from 97% to 94%;6-8min, the A phase is 6-11%, and the B phase is 94-89%; 8-20min, 11% for phase A and 89% for phase B; 20-25min, 11-42% of phase A and 89-58% of phase B; 25-30min, 42% of phase A and 58% of phase B; 30-35min, 42-55% of phase A and 58-45% of phase B; 35-40min, wherein the A phase is 55-97%, and the B phase is 45-3%; 40-45min, 97% of phase A and 3% of phase B; 45-46min, 97-3% of phase A and 3-97% of phase B; 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 comparison 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: adopting a traditional Chinese medicine chromatogram fingerprint spectrum similarity evaluation system, taking a peak corresponding to gallic acid as a reference peak, calculating relative retention time and relative peak area of each characteristic peak of the sanguisorba officinalis reference medicinal material, 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 30.71min.
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. + -. 3ppm, and the m/z in the negative ion mode was 169.0132. + -. 3ppm.
In some 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 and 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 and 81.0341 +/-3 ppm;
further, the second mass spectrum of the peak with the first peak having an m/z of 171.0288 ± 3ppm has at least the fragment ion peaks 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 and 81.0341 +/-3 ppm.
Further, a second mass spectrum of a peak having a first peak with an 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 at least the following fragment ion peaks at m/z: 239.0594 +/-3 ppm.
Further, a secondary 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 and 148.0155 +/-3 ppm.
Further, a second mass spectrum of a peak having a second peak m/z of 345.0829 ± 3ppm has at least the following fragment ion peaks at m/z: 239.0594 +/-3 ppm, 148.0155 +/-3 ppm, 223.0280 +/-3 ppm and 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 is 291.0861 +/-3 ppm, and the m/z in the negative ion mode is 289.0720 +/-3 ppm.
Further, a second 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 and 123.0442 +/-3 ppm.
Further, a second 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 and 147.0441 +/-3 ppm.
Further, the second mass spectrum of the peak with the third peak having m/z of 289.0720 ± 3ppm has fragment ion peaks at least at the following m/z: 109.0282. + -. 3ppm or 125.0232. + -. 3ppm.
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 and 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 and 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. + -. 3ppm.
Further, a second mass spectrum of a peak having a fourth peak with an 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 detection method of 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, a UPLC-QTOF/MS approach 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 column is a waters CORTECS T3 (150 mm. Times.2.1mm, 1.6 μm) column.
In some embodiments, the flow rate of the UPLC is 0.1mL/min to 0.3mL/min; further, the flow rate was 0.25mL/min.
In some embodiments, the elution procedure is: 0-6min, wherein the A phase is from 3% to 6%, and the B phase is from 97% to 94%;6-8min, the A phase is 6-11%, and the B phase is 94-89%; 8-20min, 11% for phase A and 89% for phase B; 20-25min, 11-42% of phase A and 89-58% of phase B; 25-30min, wherein the phase A is 42 percent, and the phase B is 58 percent; 30-35min, 42-55% of phase A and 58-45% of phase B; 35-40min, wherein the A phase is 55-97%, and the B phase is 45-3%; 40-45min, 97% of phase A and 3% of phase B; 45-46min, wherein the A phase is 97-3 percent, and the B phase is 3-97 percent; 46-60min, 3% of phase A and 97% of phase B.
In some embodiments, the mass spectrometry conditions are: HESI ion source, the flow rate of sheath gas is 30-40arb, the flow rate of auxiliary gas is 8-12arb, the voltage of spraying is 3-4KV, the voltage of S-lens is 45-55V, the heating temperature is 340-360 ℃, the temperature of capillary tube is 340-360 ℃, scanning and collecting are 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, sheath gas flow rate 35arb, auxiliary gas flow rate 10arb, spray voltage 3.80KV, S-lens voltage 50V, heating temperature 350 deg.C, capillary temperature 360 deg.C.
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 root 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, and should be 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: thermo high performance liquid chromatograph (Vanqish, therro corporation); waters high performance liquid chromatograph (H-Class, waters corporation); waters CORTECS T3 (150 mm. Times.2.1mm, 1.6 μm) column; one-ten-thousandth analytical balance (ME 204E, mettler-toledo); one part per million analytical balance (XP 26, mettler-toledo); a numerical control ultrasonic cleaner (KQ 500D, ultrasonic instruments ltd, kunshan); a constant temperature water bath (model HWS28, model shanghai-changshiki ltd); ultrapure water systems (Milli-Q Direct, merck Ltd.).
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 preparation: gallic acid (content: 90.8% by China institute for food and drug assay, batch No. 110831-201204); ellagic acid (China institute for food and drug testing, content: 88.8%, batch No. 111959-201903); sanguisorba officinalis reference medicinal material (China institute for food and drug inspection, lot number: 121286-201703); 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 Dingxi 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 Dingxi City of Gansu province | S19 | Gansu province Longxi county |
| S8 | Longxi county of Dingxi City of Gansu province | S20 | Gansu province Longxi county |
| S9 | Longxi county of Dingxi City of Gansu province | S21 | Cool market of Gansu province |
| S10 | Chenzhou Guiyang from Hunan province | 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.2.1mm, 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 is 35 ℃; the detection wavelength was 270nm.
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 taken, the test solution was prepared according to the method under item "1.3", the sample injection was repeated 6 times according to the chromatographic conditions under 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 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 samples of Ulmus pumila (S1) were taken, 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 the 4 common peaks and the 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 according to the chromatographic condition of "1.1" for 0,2,4,6,8, 12, 24h, and the relative retention time of 4 common peaks and RSD of the relative peak area were calculated using gallic acid as reference peak. 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 sample is stable within 24 hours.
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 Pattern
Taking sanguisorba officinalis reference medicinal material (batch number: 121286-201703) and 23 batches of sanguisorba officinalis medicinal material, preparing a test solution determined under chromatographic conditions under the item of 1.1 and the item of 1.3, carrying out sample injection measurement to obtain a sanguisorba officinalis reference medicinal material characteristic spectrum as shown in figure 2, calculating relative retention time and relative peak area of each characteristic peak by using a gallic acid peak as a reference peak by using traditional Chinese medicine chromatogram fingerprint similarity evaluation software, generating a reference spectrum by using an average method (or a median method), and establishing a sanguisorba officinalis medicinal material reference characteristic spectrum (as shown in figure 3); identifying common peaks for each test article (see fig. 4); 4 common peaks consistent with the retention time of sanguisorba officinalis control medicinal materials were selected as characteristic peaks, namely, a first peak (peak No. 1, see 1 (S) in fig. 4), a second peak (peak No. 2, see 2 in fig. 4), a third peak (peak No. 3, see 3 in fig. 4) and a fourth peak (peak No. 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 characteristic spectrum of ulmus pumila (relative retention time)
TABLE 3 characteristics of lot sanguisorba officinalis medicinal materials (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 4 measurement results of the content of 23 batches of sanguisorba officinalis medicinal materials
Analysis and discussion of results:
the result shows 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 all meet the content requirement of sanguisorba officinalis decoction pieces in 2020 edition 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 content is similar to that of the corresponding raw medicinal materials.
2 UPLC-QTOF/MS method and results
2.1 conditions of liquid-Mass Spectrometry
A chromatographic column: waters ACQUITY UPLC BEH C18 (2.1 mm. Times.100mm, 1.7 μm); methanol was used as mobile phase a, and 0.1% formic acid solution was used as mobile phase B, and gradient elution was performed as specified in table 5; the flow rate was 0.25mL per minute; the column temperature was 35 ℃; the detection wavelength was 270nm. 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 4.49-4.67min. 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; from which the molecule is precisely determinedQuantitatively presuming that its molecular formula may be C 9 H 11 NO 2 (ii) a A secondary plot with a peak collision energy of 40 at m/z 171.0288 was extracted, and the results showed 153.0183, 127.0391, 125.0235, 109.0288, 107.0132, and 81.0341 for the major fragment ions, and the detailed results are shown in FIG. 8. A secondary plot of m/z 169.0132 peak collision energy of 40 was extracted, and the results showed that the major ion fragments were 125.0232, etc., and the detailed results are 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 matching degree of m/z 171.0288 and m/z 169.0132 peaks is gallic acid, the matching degree is 94.62%, the matching condition of the secondary fragments 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 13.28-13.50min. 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 are m/z 315.0710 peak in the positive ion mode and m/z 345.0829 peak in the negative ion mode; the secondary mass spectrum with the peak collision energy of m/z 315.0710 of 40 in the positive ion mode is extracted, and the result shows that the main ion fragments are 153.0182 and the like, and the detailed result is shown in figure 16. The secondary mass spectrum with m/z 345.0829 peak collision energy of 40 in the negative ion mode is extracted, and the result shows that the main ion fragments are 239.0594, 148.0155, 223.0280, 87.0438 and the like, and the detailed result is shown in figure 17.
(3) Peak No. 3
Extracting the first-order chromatogram of peak No. 3, wherein the time period is 17.53-17.70min. The results are shown in FIGS. 18-19.
As can be seen from FIGS. 18 to 19, in the first chromatogram extract of peak No. 3, there is a peak M/z 291.0861 in positive ion mode, which is [ M + H ]] + The peak, extracted secondary mass spectrum with m/z 291.0861 peak collision energy of 40, shows that the main ion fragments are 139.0390, 123.0442, 147.0441 and the like, and the results are shown in the figure20; has M/z 289.0720 peak in negative ion mode, which is [ M-H] - The secondary mass spectrum of the peak with the collision energy of 40 of m/z 289.0720 peak is extracted, and the result shows that the main ion fragments are 109.0282, 125.0232, 123.0440, 151.0390 and the like, and the result is shown in figure 21. Presumably C from its exact molecular weight 15 H 14 O 6 。
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 secondary fragment matching condition is shown in figures 22-23, the catechin structural formula is shown in figure 24, and the possible cracking fragments are shown in figures 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 30.37-30.71min. 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 of M/z 300.9991 in the negative ion mode, which is [ M-H ]] - Peak, extract m/z 300.9991 peak collision energy of 40 secondary mass spectrum, results show, main ion fragment 300.9991, the results are shown in figure 28. Presumably C from its exact molecular weight 14 H 6 O 8 。
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.
All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present disclosure 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 specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
Claims (4)
1. A method for constructing a fingerprint of a garden burnet medicinal material is characterized by comprising the following steps:
preparing gallic acid reference substance solution, radix Sangusorbae reference medicinal material solution and multiple batches of radix Sangusorbae test 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;
the preparation method of the sanguisorba officinalis reference medicinal material solution and the sanguisorba officinalis test sample solution comprises 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, fixing volume to desired volume, and filtering to obtain filtrate;
the solvents of the sanguisorba reference medicinal material solution and the sanguisorba test sample solution are 10-40% of methanol;
the mobile phase A of the ultra-high performance liquid chromatography is methanol, and the phase B of the ultra-high performance liquid chromatography is 0.08 to 0.12 percent of phosphoric acid aqueous solution;
the elution procedure of the ultra-high performance liquid chromatography is as follows: 0-6min, the A phase is from 3% to 6%, and the B phase is from 97% to 94%;6-8min, wherein the A phase is 6-11%, and the B phase is 94-89%; 8-20min, 11% for phase A and 89% for phase B; 20-25min, 11-42% of phase A and 89-58% of phase B; 25-30min, 42% of phase A and 58% of phase B; 30-35min, 42-55% of phase A and 58-45% of phase B; 35-40min, wherein the A phase is 55-97%, and the B phase is 45-3%; 40-45min, 97% of phase A and 3% of phase B; 45-46min, wherein the A phase is 97-3 percent, and the B phase is 3-97 percent; 46-60min, 3% of phase A and 97% of phase B;
the chromatographic column of the ultra-high performance liquid chromatography is waters CORTECS T3 mm multiplied by 2.1mm,1.6 mu m;
the characteristic peaks comprise 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 30.37-30.71min;
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.
2. The method of claim 1, wherein the sanguisorbae officinalis medicinal material reference 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.
3. The method of claim 1, wherein the flow rate is 0.1mL/min to 0.3mL/min and the detection wavelength is 270nm.
4. The method of any of claims 1-3, wherein the concentration of the gallic acid control solution is between 0.8mg/mL and 1.2mg/mL; and/or
The concentration of the sanguisorba officinalis test solution is 3mg/mL-6mg/mL.
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