CN107422052B - Green extraction and determination method for various compounds in Chinese herbal compound Naoxintong - Google Patents

Green extraction and determination method for various compounds in Chinese herbal compound Naoxintong Download PDF

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CN107422052B
CN107422052B CN201710513981.3A CN201710513981A CN107422052B CN 107422052 B CN107422052 B CN 107422052B CN 201710513981 A CN201710513981 A CN 201710513981A CN 107422052 B CN107422052 B CN 107422052B
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naoxintong
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adsorbent
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CN107422052A (en
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常艳旭
王慧琳
李晋
何俊
王晖
王虹
陈璐
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Tianjin University of Traditional Chinese Medicine
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
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Abstract

The embodiment of the invention provides a green extraction and determination method of various compounds in a traditional Chinese medicine compound Naoxintong, which comprises the following steps: (1) obtaining an extract of a Naoxintong sample by a matrix solid-phase dispersion extraction method; wherein, the mass of the Naoxintong sample is 10-50 mg; the adsorbent is magnesium silicate, and the mass ratio of the Naoxintong sample to the adsorbent is (2: 1) - (1: 5); the eluent is 60-100% of methanol and contains 0-0.2% of formic acid by volume percentage; the ratio of the mass of the Naoxintong sample to the elution volume is 25-42 mg/mL; grinding the Naoxintong sample and the adsorbent for 0-8 minutes; (2) and analyzing the extract liquid of the Naoxintong sample by using ultra-high performance liquid chromatography to determine the content of the compound in the traditional Chinese medicine compound Naoxintong sample. The determination method provided by the invention can provide a theoretical basis for the research of the effectiveness of the traditional Chinese medicine and provide technical support for the quality control of the Naoxintong.

Description

Green extraction and determination method for various compounds in Chinese herbal compound Naoxintong
Technical Field
The invention relates to the technical field of quality control of traditional Chinese medicines, in particular to a green extraction and determination method for various compounds in a traditional Chinese medicine compound Naoxintong.
Background
The Naoxintong is a Chinese medicinal compound preparation consisting of 16 Chinese medicaments, wherein the Chinese medicinal compound preparation comprises 11 plant medicaments: radix astragali, radix Paeoniae Rubra, Saviae Miltiorrhizae radix, radix Angelicae sinensis, rhizoma Ligustici Chuanxiong, semen Persicae, Carthami flos, caulis Spatholobi, Achyranthis radix, ramulus Cinnamomi, and ramulus Mori; 2, resin traditional Chinese medicines: olibanum (preparata), Myrrha (preparata); and 3 animal drugs: earthworm, scorpion and leech. According to the record of the ancient book of Qing dynasty, Yi Lin staggered Gao, Nao Xin Tong is originated from Bu Yang Huan Wu Tang which has the efficacies of tonifying qi, activating blood, removing blood stasis and dredging collaterals, and is mainly used for treating apoplexy, hemiplegia, limb numbness, facial distortion, stiff tongue, angina pectoris, cerebral infarction, coronary heart disease, angina pectoris and the like caused by qi deficiency, blood stagnation and vein stasis. In recent years, with the increasing fatality rate of cardiovascular diseases, the research on the components of the effective substances of the Naoxintong is increasing. The current research shows that the Naoxintong can inhibit platelet aggregation through reducing blood fat and preventing the oxidation of H9c2 cells so as to play a role in resisting arteriosclerosis, wherein gallic acid, paeoniflorin, salvianolic acid B and ligustilide play a certain role in cardiovascular and cerebrovascular diseases to different degrees. According to the literature report, the quantitative analysis component of HPLC-UV or UPLC of Naoxintong is too single, and the quantitative analysis component is mainly focused on main chemical components, such as hydroxysafflorin A, paeoniflorin, salvianolic acid B, ferulic acid, alkannic acid and the like, and the article for simultaneously measuring the content of phenolic acids, flavonoids, lactones, monoterpenes, phenanthrenequinones and furans in Naoxintong is not reported. The quantitative analysis of the compounds also has important effects on the research of the effectiveness of the traditional Chinese medicine and the quality control of the Naoxintong.
Matrix solid-phase dispersion extraction (MSPD), Barker et al, 1989, first proposed a pretreatment method for viscous, semi-solid and solid samples or animal tissues, which is of great interest because of its rapid, efficient and simple characteristics. MSPD has been reported in a large number of documents for pretreatment in many fields, such as determination of the content of pesticide residues in vegetables and fruits, determination of the content of preservatives in skin care products, determination of the content of additives in foods, determination of the content of heavy metals in drinking water, and the like. Compared with the traditional pretreatment method (ultrasonic extraction and Soxhlet extraction), the MSPD has the characteristics of small amount of extraction reagent and short extraction time, and in addition, the pretreatment process also has the unique advantages of flexible operation, mild conditions and simple equipment (A.L.Capriotti, C.Cavalire, A.Lagan, S.Piovensa, R.Samperri, Recentrendas in matrix solid-phase dispersion. Trends in Analytical Chemistry,43(2013) 53-66; M.Garcia-Lopez, P.Canosa, I.Rodriguez, Trends and simulations of matrix solid-phase dispersion. Analytical and biological Analytical Chemistry, 2008 (97391) 391 4), and can avoid the measurement error caused by the heating of the analyte. But the dosage of the traditional MSPD sample is 0.5-1 g, the dosage of the adsorbent is 1-4 g, the elution volume is 5-20 mL, the dosage of the extraction reagent is large, and the time is long.
Disclosure of Invention
The embodiment of the invention aims to provide a green extraction and determination method for various compounds in traditional Chinese medicine compound Naoxintong, so as to realize quantitative analysis of various compounds in traditional Chinese medicine compound Naoxintong. The specific technical scheme is as follows:
the invention firstly provides a green extraction and determination method of various compounds in the traditional Chinese medicine compound Naoxintong, wherein the compounds comprise: gallic acid, 5-hydroxymethylfurfural, chlorogenic acid, paeoniflorin, ferulic acid, 3, 5-O-dicaffeoylquinic acid, 1, 5-O-dicaffeoylquinic acid, kaempferol-3-O-rutinoside, rosmarinic acid, alkannic acid, salvianolic acid B, calycosin, formononetin, ligustilide, butenyl phthalide and cryptotanshinone; the method comprises the following steps:
(1) obtaining an extract of a Naoxintong sample by a matrix solid-phase dispersion extraction method; wherein, the mass of the Naoxintong sample is 10-50 mg; the adsorbent is magnesium silicate, and the mass ratio of the Naoxintong sample to the adsorbent is (2: 1) - (1: 5); the eluent is 60-100% of methanol and contains 0-0.2% of formic acid by volume percentage; the ratio of the mass of the Naoxintong sample to the elution volume is 25-42 mg/mL; grinding the Naoxintong sample and the adsorbent for 0-8 minutes;
(2) and analyzing the extract liquid of the Naoxintong sample by using ultra-high performance liquid chromatography to determine the content of the compound in the traditional Chinese medicine compound Naoxintong sample.
Optionally, step (1) specifically includes:
mixing the Naoxintong sample with an adsorbent, and grinding;
placing the ground mixture in a solid phase extraction device, and eluting with an eluant to obtain an eluent;
and (4) centrifuging the eluent, wherein the centrifuged supernatant is the extract of the Naoxintong sample.
Optionally, the Naoxintong sample has a mass of 20-40 mg; the mass ratio of the Naoxintong sample to the adjuvant is (1: 1) - (1: 4); the eluent is 75% of methanol, and the eluent contains 0.05% of formic acid by volume percentage; the ratio of the mass of the Naoxintong sample to the elution volume is 25 mg/mL; the grinding time of the Naoxintong sample and the adsorbent is 2-6 minutes.
Optionally, the Naoxintong sample has a mass of 25 mg; the mass ratio of the Naoxintong sample to the adjuvant is 1: 4; the grinding time of the Naoxintong sample and the adsorbent was 4 minutes.
Optionally, the step (2) is specifically:
preparing mixed standard solutions of different concentrations of the compound;
determining the areas of the ultra-performance liquid chromatography peaks corresponding to the standard substances of the compounds with different concentrations through ultra-performance liquid chromatography;
determining a standard curve corresponding to each compound according to the concentration of each compound standard substance and the peak area corresponding to the concentration;
and determining the content of each compound in the traditional Chinese medicine compound Naoxintong sample according to the standard curve.
Optionally, the determining the content of each compound in the traditional Chinese medicine compound Naoxintong sample according to the standard curve includes:
determining the corresponding ultra performance liquid chromatography peak area of the compound in the extraction liquid by ultra performance liquid chromatography; and determining the concentration C of each compound according to the established standard curve of each compoundConcentrationRespectively calculating the content of each compound in the traditional Chinese medicine compound Naoxintong sample according to the following formula;
compound content ═ CConcentration*V/M；
Wherein, CConcentrationIs the concentration of each compound in the extract; v is the volume of the extraction liquid; m is the quality of the traditional Chinese medicine compound Naoxintong sample.
Optionally, the chromatographic conditions of the ultra-high performance liquid chromatography are: a chromatographic column: octadecylsilane chemically bonded silica; phase A is 0.1-1% phosphoric acid-water; phase B is (40-60%) acetonitrile- (60-40%) methanol (v/v); flow rate: 0.2-2.0 mL/min; column temperature: 30-40 ℃; sample introduction amount: 2-10 mu L; detection wavelength: 230nm, 280nm and 324 nm.
Alternatively, the gradient elution procedure for chromatographic conditions is as follows: 0-2min, 13% B; 2-7min, 13% -22% of B; 7-25min, 22% -40% B; 25-27min, 40% -55% B; 27-30min, 55% B; 30-39min, 55% -90% B; 39-40min, 90% -5% B.
Alternatively, the compound comprises: gallic acid, 5-hydroxymethyl furfural, chlorogenic acid, paeoniflorin, ferulic acid, 1, 5-O-dicaffeoylquinic acid, kaempferol-3-O-rutinoside, rosmarinic acid, alkannic acid, salvianolic acid B, calycosin, formononetin, ligustilide, butenyl phthalide and cryptotanshinone.
Optionally, the traditional Chinese medicine compound Naoxintong is in the dosage form of tablets, capsules, powder, granules, pastilles, pills, solutions, suspensions, emulsions, syrups, powders, fine granules or pellets.
The green extraction and determination method for various compounds in the traditional Chinese medicine compound Naoxintong can overcome the problems of large sample dosage, adsorbent dosage and elution volume of the traditional MSPD method, and can complete MSPD extraction by adopting less sample dosage, adsorbent dosage and the like; experiments prove that the determination method provided by the invention can effectively determine the content of each compound in the traditional Chinese medicine compound Naoxintong; therefore, theoretical basis can be provided for the research of the effectiveness of the traditional Chinese medicine, and technical support can be provided for the quality control of the Naoxintong.
Furthermore, when the Naoxintong is pretreated, the use amount of the organic solvent used as the eluent is greatly reduced, the potential harm to the environment is reduced, and the method is more environment-friendly and has better application prospect compared with the traditional MSPD method due to the adoption of the matrix solid-phase dispersion extraction method.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a UPLC spectrum of a control solution, wherein the detection wavelength of panel A is 230 nm; the detection wavelength of the B picture is 280 nm; the detection wavelength of the C plot is 324 nm.
FIG. 2 shows the peak areas of the respective compounds corresponding to the different adsorbents.
FIG. 3 is the peak area of each compound for different sample to adsorbent ratios.
FIG. 4 shows the peak areas of the respective compounds for different polishing times.
FIG. 5 shows the peak areas of the compounds for different eluents.
FIG. 6 shows the peak areas of the compounds corresponding to different concentrations of methanol as eluent.
FIG. 7 is the peak area of each compound for different formic acid contents in the eluent.
FIG. 8 shows the sample recovery rate of each compound for different elution volumes.
Detailed Description
The invention provides a green extraction and determination method for various compounds in traditional Chinese medicine compound Naoxintong, which is mainly used for determining the following compounds contained in the traditional Chinese medicine compound Naoxintong: gallic acid, 5-hydroxymethylfurfural, chlorogenic acid, paeoniflorin, ferulic acid, 3, 5-O-dicaffeoylquinic acid, 1, 5-O-dicaffeoylquinic acid, kaempferol-3-O-rutinoside, rosmarinic acid, alkannic acid, salvianolic acid B, calycosin, formononetin, ligustilide, butenyl phthalide and cryptotanshinone; the compounds are reported to be contained in a traditional Chinese medicine compound Naoxintong, such as Naoxintong capsules, and the compounds also have an important effect on the clinical curative effect of Naoxintong. The method comprises the following steps:
(1) obtaining an extract of a Naoxintong sample by a matrix solid-phase dispersion extraction method; wherein, the mass of the Naoxintong sample is 10-50 mg; the adsorbent is magnesium silicate, and the mass ratio of the Naoxintong sample to the adsorbent is (2: 1) - (1: 5); the eluent is 60-100% of methanol and contains 0-0.2% of formic acid by volume percentage; the ratio of the mass of the Naoxintong sample to the elution volume is 25-42 mg/mL; grinding the Naoxintong sample and the adsorbent for 0-8 minutes; the inventor finds that by adopting the parameters of the matrix solid-phase dispersion extraction, each compound in the Naoxintong sample can be effectively extracted; in order to ensure that each compound is more sufficiently extracted into the extract liquid of the obtained Naoxintong sample, in a preferred embodiment of the invention, the mass of the Naoxintong sample is 20-40 mg; the mass ratio of the Naoxintong sample to the adjuvant is (1: 1) - (1: 4); the eluent is 75% of methanol, and the eluent contains 0.05% of formic acid by volume percentage; the ratio of the mass of the Naoxintong sample to the elution volume is 25 mg/mL; the grinding time of the Naoxintong sample and the adsorbent is 2-6 minutes. In a preferred embodiment of the invention, the Naoxintong sample has a mass of 25 mg; the mass ratio of the Naoxintong sample to the adjuvant is 1: 4; the grinding time of the Naoxintong sample and the adsorbent was 4 minutes. The term "60% to 100% methanol" has the ordinary meaning in the art, and is understood to mean an aqueous methanol solution having a methanol volume percentage of 60% to 100%, which is obtained by mixing methanol and water by volume, and it is understood that the 100% aqueous methanol solution is anhydrous methanol. As mentioned above, the matrix solid phase dispersion extraction method is a common method for sample pretreatment, so those skilled in the art will not know the specific operation process of the method, and the detailed description of the invention is omitted here. In a specific embodiment of the present invention, the step (1) is specifically:
mixing the Naoxintong sample with an adsorbent, and grinding;
placing the ground mixture in a solid phase extraction device, and eluting with an eluant to obtain an eluent;
and (4) centrifuging the eluent, wherein the centrifuged supernatant is the extract of the Naoxintong sample.
The solid phase extraction apparatus used in the present invention may be any one commonly used in the matrix solid phase dispersion extraction method, and the present invention is not limited thereto.
(2) And analyzing the extract liquid of the Naoxintong sample by using ultra-high performance liquid chromatography to determine the content of the compound in the traditional Chinese medicine compound Naoxintong sample.
Ultra Performance Liquid Chromatography (UPLC) is a common method for analyzing chemical components in liquid samples in the field of biochemistry, and it is known to those skilled in the art that the key point for UPLC is the determination of chromatographic conditions; in particular, the present invention requires that the peaks of the 16 compounds be separated from each other and that a suitable chromatographic condition be determined. The inventors have found, by intensive studies and without being bound to any theory, that the solution of the present invention can be achieved by separating 16 compounds from each other using the following chromatographic conditions. Specifically, the chromatographic conditions of the ultra-high performance liquid chromatography adopted by the invention are as follows: a chromatographic column: octadecylsilane chemically bonded silica; mobile phase: phase A is 0.1-1% phosphoric acid-water; phase B is (40-60%) acetonitrile- (60-40%) methanol (v/v); flow rate: 0.2-2.0 mL/min; column temperature: 30-40 ℃; sample introduction amount: 2-10 mu L; detection wavelength: 230nm, 280nm and 324 nm. Preferably, the mobile phase: phase A is 0.1% phosphoric acid-water; phase B is 50% acetonitrile-50% methanol (v/v); flow rate: 0.2 mL/min; column temperature: 40 ℃; sample introduction amount: 2 μ L. In one embodiment of the invention, the gradient elution procedure for chromatographic conditions is as follows: 0-2min, 13% B; 2-7min, 13% -22% of B; 7-25min, 22% -40% B; 25-27min, 40% -55% B; 27-30min, 55% B; 30-39min, 55% -90% B; 39-40min, 90% -5% B.
Since UPLC is a common method in the biochemical field, after the chromatographic conditions are given in the present invention, the specific operation steps of analyzing the extract of the naoxintong sample by the ultra performance liquid chromatography are easily realized by those skilled in the art. In a specific embodiment of the present invention, the step (2) is specifically:
preparing mixed standard solutions of different concentrations of the compound;
determining the areas of the ultra-performance liquid chromatography peaks corresponding to the standard substances of the compounds with different concentrations through ultra-performance liquid chromatography;
determining a standard curve corresponding to each compound according to the concentration of each compound standard substance and the peak area corresponding to the concentration;
and determining the content of each compound in the traditional Chinese medicine compound Naoxintong sample according to the standard curve.
More specifically, the determining the content of each compound in the traditional Chinese medicine compound Naoxintong sample according to the standard curve comprises the following steps:
determining the corresponding ultra performance liquid chromatography peak area of the compound in the extraction liquid by ultra performance liquid chromatography; and determining the concentration C of each compound according to the established standard curve of each compoundConcentrationRespectively calculating the content of each compound in the traditional Chinese medicine compound Naoxintong sample according to the following formula;
compound content ═ CConcentration*V/M；
Wherein, CConcentrationIs the concentration of each compound in the extract; v is the volume of the extraction liquid; m is the quality of the traditional Chinese medicine compound Naoxintong sample.
In one embodiment of the present invention, the method provided by the present invention can be used to determine the fraction of the 16 compounds as needed, for example, specifically, the following 15 compounds other than 3, 5-O-dicaffeoylquinic acid can be determined:
gallic acid, 5-hydroxymethyl furfural, chlorogenic acid, paeoniflorin, ferulic acid, 1, 5-O-dicaffeoylquinic acid, kaempferol-3-O-rutinoside, rosmarinic acid, alkannic acid, salvianolic acid B, calycosin, formononetin, ligustilide, butenyl phthalide and cryptotanshinone.
The dosage form of the traditional Chinese medicine compound Naoxintong can be tablets, capsules, powder, granules, pastilles, pills, solutions, suspensions, emulsions, syrups, powders, fine granules or pellets.
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The technical scheme of the invention is explained by taking the Naoxintong capsule as an example and through a specific embodiment.
It should be noted that, in this context, the 16 compounds mentioned above are sometimes represented by arabic numerals: specifically, "1" represents gallic acid, "2" represents 5-hydroxymethylfurfural, "3" represents chlorogenic acid, "4" represents paeoniflorin, "5" represents ferulic acid, "6" represents 3, 5-O-dicaffeoylquinic acid, "7" represents 1, 5-O-dicaffeoylquinic acid, "8" represents kaempferol-3-O-rutinoside, "9" represents rosmarinic acid, "10" represents lithospermic acid, "11" represents salvianolic acid B, "12" represents verbascoflavone, "13" represents formononetin, "14" represents ligustilide, "15" represents butenylphthalide, and "16" represents cryptotanshinone.
1 materials and reagents
1.1 instruments
Waters ACQUITY UPLC (Waters, Milford, MA, USA) ultra high performance liquid chromatograph; WatersACQUITY UPLC PDA Detector (Waters, Milford, MA, USA) Detector; ultra pure water devices (Millipore Corp., Mill-QII type); vacuum pump (BUCHI Labortechnik AG, V-700) centrifuge (Sigma, Germany); a one-tenth-of-ten-thousandth electronic balance (METTLER TOLEDO); vortex mixer (Shanghai West analytical instruments, XW-80A)
1.2 reagent
And (3) standard substance: gallic acid, 5-hydroxymethylfurfural, chlorogenic acid, paeoniflorin, ferulic acid, 3, 5-O-dicaffeoylquinic acid, 1, 5-O-dicaffeoylquinic acid, kaempferol-3-O-rutinoside, rosmarinic acid, alkannic acid, salvianolic acid B, calycosin, formononetin, ligustilide, butenyl phthalide and cryptotanshinone are all purchased from Kwangsite Biotech GmbH and China biosample laboratory. The experimental water is obtained by a Milli-Q ultrapure water system; acetonitrile, methanol and formic acid are chromatographically pure (Merck), phosphoric acid is chromatographically pure (Tianjin Guang select chemical research institute), and the rest of the reagents are chromatographically pure. 20 batches of Naoxintong capsules (20 batches, batch numbers 1609109, 1609110, 1609111, 1609112, 1609114, 1609115, 1609116, 1609126, 1609127, 1609128, 1609129, 1609130, 1609131, 1609132, 1609139, 1609140, 1609141, 1609146, 1609149, 1609150) were provided by Shanxi stepsize pharmaceuticals, Inc. (0.4 g/capsule).
1.3 UPLC chromatographic conditions
A chromatographic column: ACQUITY UPLC BEH C18(2.1X 100mm,1.7 μm); protection: waters C18(2.1X 12.5mm,5 μm); mobile phase: 0.1% phosphoric acid-water (a) -50% acetonitrile-50% methanol (v/v) (B); flow rate: 0.2 mL/min; column temperature: 40 ℃; sample introduction volume: 2 mu L of the solution; detection wavelength: 230nm, 280nm and 324 nm. The gradient elution procedure was as follows: 0-2min, 13% B; 2-7min, 13% -22% of B; 7-25min, 22% -40% of B; 25-27min, 40% -55% B; 27-30min, 55% B; 30-39min, 55% -90% B; 39-40min, 90% -5% B.
1.4 preparation of control sample solution
Precisely weighing gallic acid, 5-hydroxymethylfurfural, chlorogenic acid, paeoniflorin, ferulic acid, 3, 5-O-dicaffeoylquinic acid, 1, 5-O-dicaffeoylquinic acid, kaempferol-3-O-rutinoside, rosmarinic acid, alkannic acid, salvianolic acid B, calycosin, formononetin, ligustilide, butenyl phthalide and cryptotanshinone, dissolving with methanol to obtain mother liquor with concentration of 1mg/mL,1mg/mL,5mg/mL,5mg/mL,1mg/mL,500 μ g/mL,500 μ g/mL,1mg/mL,1mg/mL,5mg/mL,1mg/mL,1mg/mL,5mg/mL,5mg/mL and 1mg/mL, 80. mu.L, 10. mu.L, 20. mu.L, 200. mu.L, 70. mu.L, 10. mu.L, 30. mu.L, 50. mu.L, 25. mu.L, 60. mu.L, 200. mu.L, 25. mu.L, 30. mu.L, 20. mu.L, 25. mu.L were separately pipetted and diluted to 1mL as a stock solution of mixed standards, and a 10-fold dilution was used as a control solution, and the UPLC test was carried out on the control solution under the chromatographic conditions of item 1.3, and the chromatogram of the standard of each compound obtained was as shown in FIG. 1A-C.
1.5 preparation method of extract liquid of Naoxintong sample
Accurately weighing 25mg of Naoxintong capsule powder and a certain amount of adsorbent, uniformly mixing in a mortar, fully grinding, transferring the mixture to a 1mLSPE tube with a sieve plate at the bottom, slightly flattening the mixture by using another sieve plate, then putting the mixture on an extraction device connected with a vacuum pump, eluting by using an eluent to obtain an eluent, centrifuging the eluent for 10min twice at 14,000rpm, and taking the supernatant as an extract of a Naoxintong sample; the extract was used for UPLC analysis.
2 determination of extraction parameters of matrix solid phase dispersion
2.1 selection of the adsorbent
The influence of different adsorbents on the extraction effect of 16 compounds is mainly considered here, and 7 different fillers, namely Alumina-A (acidic Alumina), Alumina-B (basic Alumina), Alumina-N (neutral Alumina), C18 (octadecyl bonded Silica gel), Celite AZO (diatomaceous earth AZO), Florisil PR (magnesium silicate), and Silica (Silica gel), are selected as adsorbents, and other parameters are fixed as follows: 25mg of Naoxintong sample powder, grinding for 2min, wherein the ratio of the sample powder to the adsorbent is 1:1, the eluent is methanol, and the elution volume is 1 mL. Preparing the extract of Naoxintong sample according to the method of item 1.5; then carrying out UPLC analysis on the extract according to chromatographic conditions under 1.3 items; obtaining UPLC spectrograms corresponding to different adsorbents; comparing the spectrogram with standard spectrogram of each compound shown in figure 1, and determining the corresponding peak position of each compound; determining peak areas of the compounds corresponding to different adsorbents, as shown in FIG. 2; as can be seen from FIG. 2, Florisil PR is the best extraction, where Al is less effective in extracting phenolic acid compounds, probably because it has a weaker adsorption on phenolic acid compounds, silica gel has a weaker adsorption on silica gel than Florisil PR, Celite AZO as an inert material has no adsorption, and thus the extraction is inferior to Florisil PR, and C is18The characteristic of small particle size is inferior to Florisil PR in the degree of damage to the physical structure of the sample. Therefore, Florisil PR was identified as the optimal adsorbent; and for optimized analysis of other parameters.
2.2 optimization of sample to adsorbent ratio
The change of the extraction effect of 16 compounds when the ratio of the sample to the adsorbent is 2:1,1:1,1:2,1:4 and 1:5 respectively is mainly examined. Florisil PR is used as an adsorbent, 25mg of Naoxintong sample powder is fixed, the grinding time is 2min, the eluent is methanol, and the elution volume is 1 mL. Preparing the extract of Naoxintong sample according to the method of item 1.5; then carrying out UPLC analysis on the extract according to chromatographic conditions under 1.3 items; obtaining UPLC spectrograms corresponding to different adsorbent proportions; comparing the spectrogram with standard spectrogram of each compound shown in figure 1, and determining the corresponding peak position of each compound; determining the peak area of each compound corresponding to different sample and adsorbent ratios, as shown in FIG. 3; as can be seen from fig. 3, the extraction rate of most compounds increases with the increase of Florisil PR in a certain range, which may be caused by the gradual increase of adsorption on the compounds and the improvement of extraction rate, but when the amount of the adsorbent exceeds 4 times of the sample, the extraction rate is basically unchanged or even reduced, which may be caused by the too large adsorption on the compounds by Florisil PR, so that the compounds are difficult to elute, thereby reducing the extraction rate of the sample, therefore, in order to obtain the optimal extraction conditions, 1:4 is used as the optimal ratio of the sample to the adsorbent, and is used for the optimal analysis of other factors.
2.3 optimization of grinding time
Mainly considers the change situation of 16 compound extraction effects when the grinding time is respectively 0min, 2min, 4min, 6min and 8 min. Florisil PR is used as an adsorbent, the adding amount is 100mg, the fixed Naoxintong sample powder is 25mg, the eluent is methanol, and the elution volume is 1 mL. Preparing the extract of Naoxintong sample according to the method of item 1.5; then carrying out UPLC analysis on the extract according to chromatographic conditions under 1.3 items; obtaining UPLC spectrograms corresponding to different grinding times; comparing the spectrogram with standard spectrogram of each compound shown in figure 1, and determining the corresponding peak position of each compound; and determining peak areas of the compounds corresponding to different grinding times, as shown in fig. 4; as can be seen from fig. 4, the extraction rate of the compound increases with the increase of the grinding time within a certain range, and the cause of this phenomenon may be that the damage degree to the physical structure of the sample gradually increases, and thus the extraction rate is improved, but when the grinding time exceeds 4min, the extraction rate is basically unchanged or even reduced, and the cause may be that Florisil PR adsorbs and wraps the compound too much, so that the compound is difficult to elute, and thus the extraction rate of the sample is reduced, so in order to obtain the optimal extraction condition, 4min is used as the optimal grinding time for the optimal analysis of other factors.
2.4 selection of eluent
The type of eluent is another major factor affecting the extraction efficiency of matrix solid phase dispersion extraction techniques, and desorption may be closely related to the nature of the dispersant and the polarity of the compound. The change of the extraction effect of these 16 compounds when the eluents were ethanol (EtOH), n-Propanol (n-Propanol), Ethyl Acetate (EA), Acetonitrile (ACN) and methanol (MeOH) respectively was mainly examined here. And fixing other parameters: florisil PR is adsorbent, the adding amount is 100mg, the grinding time is 4min, the fixed sample powder is 25mg, and the elution volume is 1 mL; preparing the extract of Naoxintong sample according to the method of item 1.5; then carrying out UPLC analysis on the extract according to chromatographic conditions under 1.3 items; obtaining UPLC spectrograms corresponding to different eluents; comparing the spectrogram with standard spectrogram of each compound shown in figure 1, and determining the corresponding peak position of each compound; and determining peak areas of the compounds corresponding to different eluents, as shown in fig. 5; as can be seen from fig. 5, the extraction effect is best when methanol is used as the eluent, while the compounds with large polarity are not detected in the case of elution with other elution solvents, and the cause of this phenomenon may be related to the polarity of the eluent and the solubility of the compounds, so that methanol is used as the best eluent for the optimal analysis of other factors in order to obtain the best extraction conditions.
2.5 optimization of the polarity of the eluent
The change in the extraction efficiency of 16 compounds was mainly examined at methanol-water ratios (volume ratios) of 100% (dry methanol), 90% (90: 10), 75% (75: 25) and 60% (60: 40), respectively. And fixing other parameters: florisil PR is adsorbent, the adding amount is 100mg, the fixed sample powder is 25mg, grinding is carried out for 4min, and the elution volume is 1 mL; preparing the extract of Naoxintong sample according to the method of item 1.5; then carrying out UPLC analysis on the extract according to chromatographic conditions under 1.3 items; obtaining UPLC spectrograms corresponding to methanol with different concentrations; comparing the spectrogram with standard spectrogram of each compound shown in figure 1, and determining the corresponding peak position of each compound; and determining peak areas of the compounds corresponding to methanol with different concentrations, as shown in fig. 6; as can be seen from fig. 6, the extraction effect is the best when 75% methanol is used as the eluent, and the reason for this may be related to the polarity of the eluent and the solubility of the compound, so in order to obtain the best extraction conditions, 75% methanol is used as the best eluent for the optimal analysis of other factors.
2.6 optimization of acidity of eluent
In the desorption process, the influence of the acid concentration in the eluent is also a main investigation factor, and the acidity can influence the existence form and the ionic state of the compound to be detected, and further influence the interaction between the adsorption of the compound by the dispersant and the desorption of the eluent. The change of the extraction effect of 16 compounds when the content of formic acid (volume percentage content) in the eluent is respectively 0, 0.05%, 0.10% and 0.20% is mainly examined. And fixing other parameters: florisil PR is adsorbent, the adding amount is 100mg, the grinding time is 4min, the fixed sample powder is 25mg, the grinding time is 4min, and the elution volume is 1 mL; preparing the extract of Naoxintong sample according to the method of item 1.5; then carrying out UPLC analysis on the extract according to chromatographic conditions under 1.3 items; obtaining UPLC spectrograms corresponding to different formic acid contents; comparing the spectrogram with standard spectrogram of each compound shown in figure 1, and determining the corresponding peak position of each compound; determining peak areas of the compounds corresponding to different formic acid contents, as shown in FIG. 7; as can be seen from fig. 7, in a certain range, the extraction rate of the compound increases with the increase of the formic acid content, and the reason for this may be that under a certain acidic environment, the hydrogen bond, van der waals force, etc. between the eluent and the compound are enhanced, so that the desorption of the eluent is increased, and the extraction rate is further improved, but when the formic acid content is higher than 0.05%, the extraction rate is basically unchanged or even reduced, and the absorption of the dispersant on the compound is too large, so that the compound is difficult to elute, thereby reducing the extraction rate of the sample, so that in order to obtain the optimal extraction conditions, the acid content of the eluent is 0.05% as the optimal eluent for the optimal analysis of other factors.
2.7 optimization of elution volumes
The elution volume is an important factor for investigating whether the compound to be detected can be completely desorbed from the dispersing agent, so that the influence of the elution volume on the extraction effect is optimized by using the sample-adding recovery rate as a reference index in the step. Here, the change in the recovery rates of 16 compounds was mainly examined when the elution volumes were 0.6mL, 0.8mL and 1mL, respectively. And fixing other parameters: florisil PR as adsorbent, adding 100mg, fixing sample powder to 25mg, grinding for 4min, and eluting with 75% methanol containing 0.05% formic acid; preparing the extract of Naoxintong sample according to the method of item 1.5; then, the extract was subjected to sample application recovery test under the chromatographic conditions of item 1.3 (the sample application recovery test method was referred to item 3.4); and determining the sample recovery rate of each compound corresponding to different elution volumes, as shown in fig. 8; within a certain range, the recovery rate of the compound increases with the increase of the elution volume, and the sample application recovery rate of the compound to be tested is between 61.36% and 96.94% when the elution volume reaches 1mL, except that the recovery rate of 3, 5-O-dicaffeoylquinic acid is lower than 50%, which may be caused because the adsorption effect of the dispersant on the 3, 5-O-dicaffeoylquinic acid is greater than the desorption effect of the eluent, so that the 3, 5-O-dicaffeoylquinic acid cannot be completely eluted, and therefore, the optimal elution volume is 1mL for obtaining the optimal extraction conditions. The term "elution volume" as used herein is understood to mean the volume of eluent that flows from the mixture of Naoxintong and the adsorbent when the mixture is eluted with the eluent. In general, unequal volumes of liquid remain in the solid phase microextraction column during elution, and thus the elution volume is generally less than the volume of eluent employed.
Through the optimization experiment, the optimal parameters of the matrix solid phase dispersion extraction are basically determined: the adsorbent is magnesium silicate (Florisil PR), and the mass ratio of the Naoxintong sample to the adsorbent is 1: 4; eluent is 75% methanol containing 0.05% formic acid; the ratio of the mass of the Naoxintong sample to the elution volume is 25 mg/mL; the milling time was 4 minutes. In the following examples, unless otherwise specified, the above-mentioned optimum parameters were used for matrix solid phase dispersion extraction, specifically, the Naoxintong sample mass was 25 mg; the adsorbent is magnesium silicate (Florisil PR), and the mass of the adsorbent is 100 mg; (ii) a Eluent is 75% methanol containing 0.05% formic acid; the elution volume was 1 mL; the milling time was 4 minutes.
3 methodological validation
3.1 Standard Curve, Linear Range, LOD and LOQ
Taking the mixed standard stock solution prepared under the item 1.4 to gradually dilute (the dilution times are sequentially 2, 2.5, 2 and 2.5 times) into 10 mixed standard solutions with different concentrations, carrying out UPLC analysis according to the chromatographic conditions under the item 1.3, recording the peak area, drawing a standard curve by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate, and solving a regression equation. Gallic acid, 5-hydroxymethylfurfural, chlorogenic acid, paeoniflorin, ferulic acid, 3, 5-O-dicaffeoylquinic acid, 1, 5-O-dicaffeoylquinic acid, kaempferol-3-O-rutinoside, rosmarinic acid, alkannic acid, salvianolic acid B, calycosin, formononetin, ligustilide, butenyl phthalide, and LOD and LOQ of cryptotanshinone standard curve are shown in Table 1. The results show that the 16 compounds have good linear relation in the concentration range of the prepared standard solution, and each compound can be well qualitatively and quantitatively determined.
TABLE 1 regression equation and Linear Range of Standard curves, LODs and LOQs
Figure BDA0001336222470000131
3.2 Intra-day and inter-day precision
According to the above-defined optimum parameters of matrix solid-phase dispersion extraction, the extract liquor of Naoxintong sample is prepared by the method under item 1.5, and the same extract liquor is taken as test sample solution, so that the precision and stability of the test sample solution in day and night can be examined. The intra-day precision is 6 times of continuous sample injection for the same sample solution in one day, and the inter-day precision is 6 times of continuous sample injection for 3 days, the peak area of each compound is measured according to the chromatographic conditions under 1.3, and the RSD value is calculated, wherein the intra-day precision and the inter-day precision are shown in Table 2. The test result shows that the precision of the instrument is good.
3.3 stability test
Taking the same extract as a test solution, performing UPLC analysis under the chromatographic condition of item 1.3, injecting 2 μ L of gallic acid, 5-hydroxymethyl furfural, chlorogenic acid, paeoniflorin, ferulic acid, 3, 5-O-dicaffeoylquinic acid, 1, 5-O-dicaffeoylquinic acid, kaempferol-3-O-rutinoside, rosmarinic acid, lithospermic acid, salvianolic acid B, calycosin, formononetin, ligustilide, butenylphthalide and the peak area of cryptotanshinone, and calculating the RSD value, which is shown in Table 2. The results show that the sample has good stability within 24 h. The chromatographic condition of the invention has good resolution, short separation time and high sensitivity.
TABLE 2 precision and stability of 16 Compounds in Naoxintong
Figure BDA0001336222470000141
Figure BDA0001336222470000151
3.4 sample application recovery test
Adding 50 μ L of the mixed standard stock solution prepared under item 1.4 into 12.5mg of NAOXINTONG sample, and mixing to obtain the labeled sample. Placing the standard-added sample at room temperature, standing for volatilizing the solvent, pretreating according to the method under item 1.5, grinding for 4min, eluting to 1mL with 75% methanol containing 0.05% formic acid, centrifuging, and sampling the supernatant to obtain peak area (referred to as standard-added peak area). Meanwhile, the non-labeled 12.5mg Naoxintong sample is processed and analyzed according to the same steps to determine the peak area (called sample peak area); 50 μ L of the control solution prepared under item L1.4 was similarly eluted to 1mL with 75% methanol containing 0.05% formic acid, and the peak area (referred to as mixed standard peak area) was measured. Calculating the formula: the sample recovery rate is (peak area measured by adding standard-sample peak area)/peak area of mixed standard × 100%. The results are shown in Table 3.
Recovery of 316 compounds in Table
Figure BDA0001336222470000161
The verification by the methodology described above shows that: the result of methodology investigation shows that the linear relation is good, the precision and the stability are both 95.00-105.00%, the RSD is less than 5%, the sample recovery rate is 61.36-96.94%, and except that the 3, 5-O-dicaffeoylquinic acid is less than 50%, the method shows that the method has certain feasibility for preprocessing the Naoxintong by adopting a matrix solid-phase dispersion extraction method.
4 sample determination
Treating 20 batches of Naoxintong capsule samples by using the optimal MSPD condition to obtain extract liquor of each compound; and injecting sample according to 1.3 chromatographic conditions, measuring peak areas of gallic acid, 5-hydroxymethylfurfural, chlorogenic acid, paeoniflorin, ferulic acid, 3, 5-O-dicaffeoylquinic acid, 1, 5-O-dicaffeoylquinic acid, kaempferol-3-O-rutinoside, rosmarinic acid, alkannic acid, salvianolic acid B, calycosin, formononetin, ligustilide, butenyl phthalide and cryptotanshinone, repeating the sample for 3 parts, respectively substituting the peak areas of 16 compounds according to a linear regression equation in Table 1 to determine the concentration C of each compoundConcentrationRespectively calculating the content of each compound in the traditional Chinese medicine compound Naoxintong sample according to the following formula;
compound content ═ CConcentration*V/M；
Wherein, CConcentrationThe concentration of each compound in each Naoxintong capsule sample extraction liquid is obtained; v is the volume of the extraction liquid; m is the quality of the traditional Chinese medicine compound Naoxintong sample. The results of the sample measurements are shown in tables 4 and 5.
The results show that the content difference of part of gallic acid, 5-hydroxymethyl furfural, chlorogenic acid, kaempferol-3-O-rutinoside and calycosin in different Naoxintong capsule extracts is obvious, which is probably related to the instability of the compound, the decomposition of the compound due to the influence of factors such as illumination and the like and the content (low content and large measurement error) of the compound.
Table 420 contents of 8 compounds in Naoxintong batches (mg/g, n ═ 3)
Figure BDA0001336222470000171
TABLE 520 Naoxintong groups with 8 compounds (mg/g, n ═ 3)
Figure BDA0001336222470000181
The green extraction and determination method of the compounds in the Chinese herbal compound Naoxintong provided by the invention is described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its central concept. It should be noted that it would be apparent to those skilled in the art that various changes and modifications can be made in the invention without departing from the principles of the invention, and such changes and modifications are intended to be covered by the appended claims.

Claims (5)

1. The method for measuring the compounds in the traditional Chinese medicine compound Naoxintong is characterized in that the compounds comprise: gallic acid, 5-hydroxymethylfurfural, chlorogenic acid, paeoniflorin, ferulic acid, 3, 5-O-dicaffeoylquinic acid, 1, 5-O-dicaffeoylquinic acid, kaempferol-3-O-rutinoside, rosmarinic acid, alkannic acid, salvianolic acid B, calycosin, formononetin, ligustilide, butenyl phthalide and cryptotanshinone;
the method comprises the following steps:
(1) mixing the Naoxintong sample with an adsorbent, and grinding; placing the ground mixture in a solid phase extraction device, and eluting with an eluant to obtain an eluent; centrifuging the eluent, wherein the centrifuged supernatant is the extract of the Naoxintong sample;
wherein the mass of the Naoxintong sample is 20-40 mg; the mass ratio of the Naoxintong sample to the adsorbent is (1: 1) - (1: 4); the eluent is 75% of methanol, and the eluent contains 0.05% of formic acid by volume percentage; the ratio of the mass of the Naoxintong sample to the elution volume is 25 mg/mL; grinding the Naoxintong sample and the adsorbent for 2-6 minutes; the adsorbent is Florisil PR magnesium silicate;
(2) analyzing the extract liquid of the Naoxintong sample by ultra-high performance liquid chromatography to determine the content of the compound in the traditional Chinese medicine compound Naoxintong sample;
the chromatographic conditions of the ultra-high performance liquid chromatography are as follows:
a chromatographic column: octadecylsilane chemically bonded silica;
phase A is 0.1-1% phosphoric acid-water; phase B is 40-60% acetonitrile-60-40% methanol;
flow rate: 0.2-2.0 mL/min; column temperature: 30-40 ℃; sample introduction amount: 2-10 mu L; detection wavelength: 230nm, 280nm, 324 nm;
the gradient elution procedure for chromatographic conditions was as follows: 0-2min, 13% B; 2-7min, 13% -22% of B; 7-25min, 22% -40% B; 25-27min, 40% -55% B; 27-30min, 55% B; 30-39min, 55% -90% B; 39-40min, 90% -5% B.
2. The assay of claim 1, wherein the Naoxintong sample has a mass of 25 mg; the mass ratio of the Naoxintong sample to the adjuvant is 1: 4; the grinding time of the Naoxintong sample and the adsorbent was 4 minutes.
3. The assay method according to claim 1, wherein the step (2) is specifically:
preparing mixed standard solutions of different concentrations of the compound;
determining the areas of the ultra-performance liquid chromatography peaks corresponding to the standard substances of the compounds with different concentrations through ultra-performance liquid chromatography;
determining a standard curve corresponding to each compound according to the concentration of each compound standard substance and the peak area corresponding to the concentration;
and determining the content of each compound in the traditional Chinese medicine compound Naoxintong sample according to the standard curve.
4. The determination method according to claim 3, wherein the determining the content of each compound in the sample of the compound traditional Chinese medicine Naoxintong according to the standard curve comprises:
determining the corresponding ultra performance liquid chromatography peak area of the compound in the extraction liquid by ultra performance liquid chromatography; and determining the concentration C of each compound according to the established standard curve of each compoundConcentrationRespectively calculating the content of each compound in the traditional Chinese medicine compound Naoxintong sample according to the following formula;
compound content ═ CConcentration*V/M；
Wherein, CConcentrationIs the concentration of each compound in the extract; v is the volume of the extraction liquid; m is the quality of the traditional Chinese medicine compound Naoxintong sample.
5. The method for determining according to claim 1, wherein the formulation of the compound traditional Chinese medicine Naoxintong is tablet, capsule, granule, lozenge, pill, syrup or powder.
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