Method for detecting chemical components in rehmannia drink preparation based on UPLC-Q-TOF-MS / MS technology

Abstract

The invention discloses a method for detecting chemical components in a rehmannia root potion preparation based on a UPLC-Q-TOF-MS / MS technology, and belongs to the technical field of analysis and detection. Comprising the following steps: crushing medicinal components into coarse grains; slicing the fresh ginger and breaking the Chinese dates; adding water into the coarse grains, ginger, jujube and mint, decocting, filtering, centrifuging, and re-filtering to obtain a test solution; preparing a single decoction piece sample and a reference substance solution; the UPLC-Q-TOF-MS / MS technology is used for detection. Chromatographic conditions are as follows: acetonitrile-0. 1% formic acid is used as a mobile phase, and gradient elution is performed; the mass spectrum conditions are as follows: the operation is carried out in positive and negative ion modes, the dry gas temperature is 250-400 DEG C, the dry gas flow velocity is 5-15L / min, the atomized gas pressure is 25-40psi, the sheath gas temperature is 300-400 DEG C, and the sheath gas flow velocity is 7-15L / min. According to the method, 49 components can be successfully separated, detected and determined at one time, and the traditional Chinese medicine flavor of the rehmannia root potion preparation can be comprehensively reflected.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology of chemical components in traditional Chinese medicine compositions, and in particular relates to a method for detecting chemical components in Rehmannia glutinosa decoction preparations based on UPLC-Q-TOF-MS / MS technology. Background Technology

[0002] Rehmannia Decoction is a traditional classic formula from Liu Hejian's *Suwen* (Plain Questions) of the Song Dynasty. It consists of 15 herbs: Rehmannia glutinosa (processed), Cornus officinalis, Schisandra chinensis, Morinda officinalis, Ophiopogon japonicus, Mentha haplocalyx, Acorus tatarinowii, Dendrobium nobile, Polygala tenuifolia, Aconitum carmichaelii, Cistanche deserticola, Poria cocos, Cinnamomum cassia, Ziziphus jujuba, and Zingiber officinale. It nourishes kidney yin, tonifies kidney yang, and clears the orifices and resolves phlegm. It is primarily used to treat symptoms such as aphasia, aphasia, paralysis of the feet, dry mouth, cold feet, flushed face, and a weak, thready pulse. Rehmannia Decoction is widely used clinically, often for treating Alzheimer's disease, diabetes, cardiovascular diseases, and stroke, among other geriatric neurological disorders.

[0003] In 2016, Hu Benquan, Lian Jiangping, and others used RP-HPLC to simultaneously determine the contents of echinacoside, verbascoside, and loganin in Rehmannia Decoction. This method established an HPLC system for the simultaneous determination of echinacoside from Cistanche deserticola, verbascoside from Cistanche deserticola and Rehmannia glutinosa, and loganin from Cornus officinalis. In 2019, Zhang Xiaoyan, Zhang Hujuan, and others used high-performance liquid chromatography (HPLC) with a mobile phase of methanol-acetonitrile-water (volume ratio 45:30:25) and a flow rate of 1.0 mL / min to determine the contents of schisandrin A and B in Rehmannia Decoction granules. This method can only determine the contents of schisandrin A and B in Rehmannia Decoction granules, and the detection target is only Rehmannia Decoction granules. However, the components in Rehmannia Decoction are complex, and these two methods only detected the contents of three components from three out of 15 herbs. For the overall quality evaluation of Rehmannia Decoction, there are few indicators, and the overall chemical composition of Rehmannia Decoction is not revealed.

[0004] In 2020, Guo Siqun and Chi Xiaoxue used HPLC to study the fingerprint spectrum of Rehmannia glutinosa decoction. Using the HPLC-DAD method with gradient elution, they determined the fingerprint spectra of 10 batches of self-prepared Rehmannia glutinosa decoction, identifying and confirming common peaks. This study identified 18 common peaks, and by comparing their retention times and UV spectra with those of reference standards, they identified one component, loganin. However, this method identified a limited number of components, and the number of components confirmed through comparison was also relatively small.

[0005] The composition of Rehmannia glutinosa decoctions is complex, and the components interfere with each other, making them difficult to separate and detect. While some studies exist on the detection of Rehmannia glutinosa decoctions, the range of analyzable components is limited and relatively incomplete, failing to comprehensively analyze the main components in Rehmannia glutinosa decoctions. Therefore, further research is needed to determine the main components contained in Rehmannia glutinosa decoctions.

[0006] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] To address the limitations of existing technologies and methods in analyzing the limited variety and comprehensiveness of complex components in Rehmannia glutinosa decoction, this invention provides a method for detecting chemical components in Rehmannia glutinosa decoction preparations based on UPLC-Q-TOF-MS / MS (ultra-high performance liquid chromatography coupled with time-of-flight mass spectrometry).

[0008] To achieve the above objectives, this invention provides a method for detecting chemical components in Rehmannia glutinosa decoction preparations based on UPLC-Q-TOF-MS / MS technology, comprising the following steps:

[0009] (1) Solution preparation:

[0010] Preparation of the test solution: Prepared Rehmannia glutinosa, Morinda officinalis, Cornus officinalis, Dendrobium nobile, Cistanche deserticola, Aconitum carmichaelii, Schisandra chinensis, Cinnamomum cassia, Poria cocos, Ophiopogon japonicus, Acorus tatarinowii, and Polygala tenuifolia are crushed into coarse particles; ginger is sliced ​​and jujubes are broken open; the coarse particles, the processed ginger, jujubes, and mint are placed in a clay pot with water, decocted, filtered, centrifuged, filtered again, and the filtrate is collected to obtain the test solution;

[0011] Preparation of single-herb decoction sample solution: Prepared Rehmannia glutinosa slices, Morinda officinalis slices, Cornus officinalis slices, Dendrobium nobile slices, Cistanche deserticola slices, Aconitum carmichaelii slices, Schisandra chinensis slices, Cinnamomum cassia slices, Poria cocos slices, Ophiopogon japonicus slices, Acorus tatarinowii slices, Polygala tenuifolia slices, Mentha haplocalyx slices, and Ziziphus jujuba slices are crushed and sieved. Fresh ginger slices are cut into small pieces. They are decocted in water, cooled, and the supernatant is filtered. The filtrate is then collected to obtain the single-herb decoction sample solution.

[0012] Preparation of reference solutions: Take appropriate amounts of mononoside, loganin, and schisandrol A, dissolve and dilute them separately in 80% methanol to prepare reference solutions with a concentration of approximately 20 μg / ml; Take an appropriate amount of 3,6'-disinoyl sucrose, dissolve and dilute it in methanol to prepare reference solutions with a concentration of approximately 20 μg / ml; Polygala tenuifolia Take an appropriate amount of ketone III, dissolve and dilute it in methanol to prepare a reference solution with a concentration of approximately 30 μg / ml.

[0013] (2) The solution obtained in step (1) was detected using ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS).

[0014] Chromatographic conditions: Acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B; gradient elution was employed, and the elution conditions for the gradient elution are shown in the table below. This elution method can achieve good separation of the components in the test sample.

[0015]

[0016] Mass spectrometry conditions: Performed in positive and negative ion modes, with a drying gas temperature of 250-400℃, a drying gas flow rate of 5-15 L / min, a nebulizing gas pressure of 25-40 psi, a sheath gas temperature of 300-400℃, and a sheath gas flow rate of 7-15 L / min; the capillary voltage in positive ion mode is 3000-5000 V, and the voltage in negative ion mode is 2500-4500 V.

[0017] Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS) technology, by employing quadrupole time-of-flight mass spectrometry, can provide higher precision and accuracy in mass analysis, while also possessing high sensitivity and high resolution, thereby improving the detection and analysis capabilities of the complex system of 15 Chinese herbal medicines from Rehmannia Decoction.

[0018] The test solution, individual herbal decoction sample solutions, and reference solution of the Rehmannia glutinosa decoction were analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) to determine the types of components in the Rehmannia glutinosa decoction and their corresponding Chinese herbal medicine sources. The chromatographic and mass spectrometric results of the test solution were compared with those of the individual herbal decoction sample solutions and the reference solution, and also compared with literature data to determine the types of components in the Rehmannia glutinosa decoction and their corresponding Chinese herbal medicine sources. Alternatively, the chromatographic and mass spectrometric results of the test solution were compared with the information recorded in Table 1 of this invention to determine the types of components in the Rehmannia glutinosa decoction and their corresponding Chinese herbal medicine sources.

[0019] In some preferred embodiments, in the preparation of the test solution, the particle size of the coarse particles is 2-5 mm; preferably, the particle size of the coarse particles is 3-5 mm; most preferably, the particle size of the coarse particles is 3-4 mm.

[0020] In some preferred embodiments, the decoction method in the preparation of the test solution is as follows: first, heat with an open flame, cover and decoct, bring to a boil over high heat, and then simmer over low heat; preferably, the simmering time is 20-50 minutes; most preferably, the simmering time is 30 minutes.

[0021] In some preferred embodiments, the decoction time with water in the preparation of the single herb sample solution is 20-50 minutes; most preferably, the decoction time with water is 30 minutes.

[0022] In some preferred embodiments, the above chromatographic conditions use a Waters ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm) equipped with an online filter; the detection wavelength is 230 nm-250 nm, the column temperature is 28-33 °C, and the flow rate is 0.2-0.5 ml / min; preferably, the detection wavelength is 240 nm, the column temperature is 30 °C, the flow rate is 0.3 ml / min, and the injection volume is 2 μl. At a wavelength of 240 nm, the response values ​​of each chromatographic peak are also relatively moderate, the peak information content is large, and the baseline is relatively stable, thus the detection wavelength is determined to be 240 nm.

[0023] In some preferred embodiments, the above-mentioned mass spectrometry conditions are achieved by using a combination of primary mass spectrometry analysis and secondary mass spectrometry scanning to obtain ion fragment information data of the chemical components in the test sample solution.

[0024] In some preferred embodiments, the first-stage mass spectrometer uses MS mode with a mass scan range of 100-1700 m / z; the second-stage mass spectrometer uses Auto-MS / MS mode with a mass scan range of 100-1300 m / z; and the collision voltage is 5 eV-35 eV, preferably 10 eV, 15 eV, 20 eV, or 30 eV.

[0025] In some preferred embodiments, the aforementioned ion fragment information data is acquired by Agilent Mass Hunter Qualitative Analysis software, and data processing is performed using Agilent software Qualitative Navigator (B.08.00) and Qualitative Workflows (B.08.00).

[0026] In some preferred embodiments, the mass spectrometry conditions described above are as follows: drying gas temperature: 350°C; drying gas flow rate: 10 L / min; nebulizing gas pressure: 35 psi; sheath gas temperature: 350°C; sheath gas flow rate: 12 L / min.

[0027] In some preferred embodiments, the Rehmannia glutinosa decoction preparation described above contains 49 chemical components, including 13 glycolipids, 11 iridoids, 4 flavonoids, and... Four ketone compounds, three alkaloid compounds, and 14 other compounds were found.

[0028] Among them, the glycolipid compounds are Sibiricose A5 (peak 13), Polygalatenoside (peak 14), Sibiricose A6 (peak 15), Echinacoside (peak 21), Globose A (peak 24), Polygalactoside B (peak 31), 3,6'-diglucosinolate (peak 36), Arillanin A (peak 38), Polygalactoside A (peak 40), Tenuifoliose J (peak 42), Tenuifoliose B (peak 43), Tenuifoliose H (peak 44), and Tenuifoliose A (peak 45).

[0029] Iridoid ethers and terpenoids: crystallizing glycoside (peak 1), deacetylated cypermethrin (peak 3), rehmannia glycoside A (peak 6), leonurin (peak 7), genipin (peak 8), 8-epiogenic acid (peak 9), strychnine (peak 10), dihydrocornein (peak 11), monoglobin (peak 12), loganin (peak 18), and neocornein (peak 34).

[0030] Flavonoids: rutin (peak 28), myricetin-3-O-rutinoside (peak 32), hesperidin (peak 35), and robinin-7-O-neohesperidin (peak 41);

[0031] Ketone compounds: Sibiricaxanthon A (peak 22), Polygalaxanthone III (peak 23), Polygalaxanthone XI (peak 26), Polygalaxanthone VIII (peak 27);

[0032] The alkaloid compounds are aconitine (peak 19), magnoflorine (peak 20), and benzoylneoprothiolane (peak 39);

[0033] Other compounds include 1-deoxy-1-L-leucine-D-fructose (peak 2), cyclic adenosine monophosphate (peak 4), gallic acid (peak 5), Procyanidin B1 / Procyanidin B2 (peak 16), dehydrodipineol (peak 17), (2E,6E)-3,7-dimethyl-8-hydroxyoctadien-1-O-β-D-glucoside (peak 25), Kankanoside O (peak 29), 1-(3,4-dihydroxyphenyl)-3-[2-(3,4-dihydroxyphenyl)-1-hydroxy-1,2-dihydronaphthalene-2-carboxylic acid (peak 30), Norboldine (peak 33), rosmarinic acid (peak 37), 6-shogaol (peak 46), schisandrol A (peak 47), α-asarone (peak 48), and 1-deoxy-6-shogaol (peak 49).

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The detection method used in this invention can successfully separate, detect and characterize the above 49 components in one go. Compared with the prior art, it can more comprehensively reflect the medicinal taste in the Rehmannia Decoction preparation. It solves the problems of inaccurate dosage of medicinal slices and processing methods and insufficient analysis of complex components of Rehmannia Decoction in the prior art. It can be used for quality control in the production process of Rehmannia Decoction preparation.

[0036] (2) This invention uses ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS) to detect and analyze Rehmannia glutinosa decoction preparations by adjusting the mass spectrometry and chromatographic conditions. By comparing and analyzing the liquid chromatography-mass spectrometry results of the test solution of Rehmannia glutinosa decoction preparations and the solutions of each single herb, the medicinal source of 49 components and the qualitative analysis of the main chromatographic peaks are clarified. Knowing the medicinal source of each component is beneficial to judging the pharmacological activity and safety of Rehmannia glutinosa decoction preparations and can make an overall quality evaluation of Rehmannia glutinosa decoction preparations.

[0037] (3) The present invention uses ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS) to analyze and detect Rehmannia glutinosa decoction preparations. This method has high accuracy and good stability. Even when there are many active ingredients, it can achieve good separation effect and can truly reflect the chemical components in Rehmannia glutinosa decoction preparations. Attached Figure Description

[0038] Figure 1 These are the UPLC-UV chromatograms and UPLC-TOF-MS total ion chromatograms (positive and negative modes) of the Rehmannia glutinosa decoction composition. Detailed Implementation

[0039] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0040] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0041] The reagents used in the following examples and their sources are as follows:

[0042] Example 1: Preparation of test solution of Rehmannia glutinosa decoction

[0043] The preparation steps include: Grinding Rehmannia glutinosa, Morinda officinalis, Cornus officinalis, Dendrobium nobile, Cistanche deserticola, Aconitum carmichaelii, Schisandra chinensis, Cinnamomum cassia, Poria cocos, Ophiopogon japonicus, Acorus tatarinowii, and Polygala tenuifolia into coarse particles of 2-5 mm, weighing 1.03 g of each. Separately, take 5 g of sliced ​​ginger, 3 g of jujube (broken open), and 1 g of peppermint. Place these ingredients in a clay pot (750 ml capacity, 13.5 cm inner diameter, 16.0 cm outer diameter, 6.0 cm height, and 1 cm thickness), add 450 ml of water, heat over an open flame (gas stove), cover and simmer. After bringing to a boil over high heat, reduce to a simmer and reduce to approximately 240 ml (simmering for 30 minutes). While hot, filter through a 300-mesh filter cloth. Take an appropriate amount of filtrate, centrifuge (5000 rpm) for 5 minutes, filter again, and collect the filtrate to obtain the test solution of the Rehmannia glutinosa decoction.

[0044] Example 2: Preparation of single-herb medicinal slice sample solution

[0045] The preparation method is as follows: Take 0.2g of each of the following herbs: Rehmannia glutinosa (processed), Morinda officinalis (processed), Cornus officinalis (processed), Dendrobium nobile (processed), Cistanche deserticola (processed), Aconitum carmichaelii (processed), Schisandra chinensis (processed), Cinnamomum cassia (processed), Poria cocos (processed), Ophiopogon japonicus (processed), Acorus tatarinowii (processed), Polygala tenuifolia (processed), Mentha haplocalyx (processed), Ziziphus jujuba (processed), and Zingiber officinale (processed). Except for Zingiber officinale, all other herbs are pulverized and passed through a No. 3 sieve. Zingiber officinale is cut into small pieces, accurately weighed, and placed in conical flasks. Add 40ml of water, decoct for 30 minutes, cool, filter the supernatant, and collect the filtrate to obtain the sample solution of each herb.

[0046] Example 3: Preparation of the reference solution

[0047] The preparation method is as follows: Take appropriate amounts of mononoside, loganin, and schisandrol A, dissolve and dilute them separately in 80% methanol to prepare a reference solution with a concentration of approximately 20 μg / ml; take an appropriate amount of 3,6'-disinoyl sucrose, dissolve and dilute it in methanol to prepare a reference solution with a concentration of approximately 20 μg / ml; Polygala tenuifolia... Take an appropriate amount of ketone III, dissolve and dilute it in methanol to prepare a reference solution with a concentration of approximately 30 μg / ml.

[0048] Example 4: Detection and Identification of Chemical Components in Rehmannia Decoction Preparations

[0049] (1) Chromatographic conditions

[0050] A Waters ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm) equipped with an online filter was used. Acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B. The detection wavelength was 240 nm, the column temperature was 30 °C, the flow rate was 0.3 mL / min, and the injection volume was 2 μL. Gradient elution was used, and the elution conditions were as follows:

[0051]

[0052] (2) Mass spectrometry conditions

[0053] Mass spectrometry was performed in both positive and negative ion modes. The drying gas temperature was 350℃, the drying gas flow rate was 10 L / min, the nebulizer gas pressure was 35 psi, the sheath gas temperature was 350℃, the sheath gas flow rate was 12 L / min, and the capillary voltage was 4000 V (positive mode) and 3500 V (negative mode). The primary mass spectrometry was performed in MS mode with a mass scan range of 100–1700 m / z. The secondary mass spectrometry was performed in Auto-MS / MS mode with a mass scan range of 100–1300 m / z and collision voltages of 10, 15, 20, and 30 eV. Data were acquired using Agilent MassHunter software.

[0054] (3) Identification

[0055] The solutions prepared in the examples were analyzed under the chromatographic and mass spectrometric detection conditions of this example to obtain the total ion chromatogram (TIC) of the test solution of Rehmannia glutinosa decoction in positive and negative ion modes, as shown in the figure. Figure 1 .

[0056] The data were acquired using Agilent Mass Hunter software and processed using Agilent Qualitative Navigator (B.08.00) and Qualitative Workflows (B.08.00). A comprehensive chemical composition database was established with reference to relevant literature. The molecular formulas of compounds were deduced by comparing the precise relative molecular masses of the main chromatographic peaks, and each chromatographic peak was identified and assigned based on mass spectrometry fragment information. A total of 49 chemical components were identified in the Rehmannia glutinosa decoction preparation, as shown in Table 1.

[0057] Table 1. UPLC-Q-TOF-MS / MS Analysis Results of Chemical Components in Rehmannia Decoction Preparation

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] The traditional Chinese medicine composition samples of Rehmannia Decoction were analyzed by primary mass spectrometry and secondary mass spectrometry to obtain the ion fragment information of the compounds. Then, the obtained compounds were identified and inferred by combining the fragmentation pattern of reference standards and literature data. A total of 49 compounds were identified and inferred, of which 5 compounds were identified by comparison with reference standards and assigned to the corresponding single herbs.

[0065] The chemical components in Rehmannia glutinosa decoctions are mainly glycolipids and iridoids, and also include flavonoids. Ketones, alkaloids, and others.

[0066] Examples 5 and 6: Detection methods for chemical components in Rehmannia glutinosa decoction preparations based on UPLC-Q-TOF-MS / MS technology

[0067] Examples 5 and 6 are methods for detecting chemical components in Rehmannia glutinosa decoction preparations based on UPLC-Q-TOF-MS / MS technology. The only differences are in a few parameters in the solvent preparation step, chromatographic conditions, and mass spectrometry conditions; otherwise, they are the same as in Examples 1-4. See Table 2 for specific methods.

[0068] Table 2 shows some parameters of the solvent preparation steps, chromatographic conditions, and mass spectrometry conditions in Examples 5 and 6.

[0069]

[0070]

[0071] The analytical results obtained in Examples 5 and 6 are basically the same as those in Example 4.

[0072] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for detecting chemical components in Dihuang Yinzi preparation based on UPLC-Q-TOF-MS / MS technology, characterized in that, The detection method comprises the following steps: (1) Preparation of a solution: Preparation of a test sample solution: Rehmanniae Radix Praeparata, Radix Morindae Officinalis, Fructus Corni, Dendrobium, Herba Cistanche, Radix et Rhizoma Aconiti, Fructus Schisandrae, Cinnamomum cassia, Poria, Ophiopogon japonicus, Acorus gramineus, Polygala, ginger slices, and Fructus Jujubae are crushed into coarse particles; the coarse particles, the treated ginger slices, and Fructus Jujubae are placed in a sand pot together with Mentha haplocalyx, water is added, decoction is performed, filtration is performed, centrifugation is performed, re-filtration is performed, and the re-filtered liquid is obtained, thereby obtaining the test sample solution; Preparation of a single-ingredient decoction piece sample solution: Rehmanniae Radix Praeparata decoction pieces, Radix Morindae Officinalis decoction pieces, Fructus Corni decoction pieces, Dendrobium decoction pieces, Herba Cistanche decoction pieces, Radix et Rhizoma Aconiti decoction pieces, Fructus Schisandrae decoction pieces, Cinnamomum cassia decoction pieces, Poria decoction pieces, Ophiopogon japonicus decoction pieces, Acorus gramineus decoction pieces, Polygala decoction pieces, Mentha haplocalyx decoction pieces, and Fructus Jujubae decoction pieces are crushed and sieved, ginger decoction pieces are cut into pieces, water is added for decoction, the supernatant is taken, filtration is performed, and the re-filtered liquid is obtained, thereby obtaining the single-ingredient decoction piece sample solution; Preparation of control solution: take appropriate amount of morroniside, loganin, schisantherin A, respectively, add 80% methanol to dissolve and dilute, and prepare the control solution with a concentration of about 20 μg / ml; take appropriate amount of 3,6'-dihydroxybenzene, add methanol to dissolve and dilute, and prepare the control solution with a concentration of about 20 μg / ml; take appropriate amount of polygalaxanthin, add methanol to dissolve and dilute, and prepare the control solution with a concentration of about 30 μg / ml. Preparation of control solution: take appropriate amount of morroniside, loganin, schisantherin A, respectively, add 80% methanol to dissolve and dilute, and prepare the control solution with a concentration of about 20 μg / ml; take appropriate amount of 3,6'-dihydroxybenzene, add methanol to dissolve and dilute, and prepare the control solution with a concentration of about 20 μg / ml; take appropriate amount of polygalaxanthin, add methanol to dissolve and dilute, and prepare the control solution with a concentration of about 30 μg / ml. (2) Detection of the solution obtained in step (1) by using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS) technology: Chromatographic conditions: acetonitrile is used as mobile phase A, and 0.1% formic acid solution is used as mobile phase B; gradient elution is used, and the elution conditions of the gradient elution are shown in the following table, which can achieve better separation of the ingredients in the test sample: Mass spectrometry conditions: analysis is performed in positive and negative ion modes, the drying gas temperature is 250-400 DEG C, the drying gas flow rate is 5-15 L / min, the atomization gas pressure is 25-40 psi, the sheath gas temperature is 300-400 DEG C, and the sheath gas flow rate is 7-15 L / min; the capillary voltage in the positive ion mode is 3000-5000 V, and the voltage in the negative ion mode is 2500-4500 V.

2. The detection method according to claim 1, characterized in that, In the preparation of the test sample solution, the particle size of the coarse particles is 2-5 mm; preferably, the particle size of the coarse particles is 3-5 mm; most preferably, the particle size of the coarse particles is 3-4 mm.

3. The method of claim 1, wherein In the preparation of the test sample solution, the decoction method is as follows: first, open fire is used for heating, covered decoction is performed, and then a gentle fire is used for decoction; preferably, the time for the gentle fire decoction is 20-50 min; most preferably, the time for the gentle fire decoction is 30 min. In the preparation of the single-ingredient decoction piece sample solution, the water decoction time is 20-50 min; most preferably, the water decoction time is 30 min.

4. The method of claim 1, wherein In the chromatographic conditions, a Waters ACQUITY UPLC HSS T3 chromatographic column (2.1*100 mm, 1.8 μm) is used, which is provided with an online filter; the detection wavelength is 230 nm-250 nm, the column temperature is 28-33 DEG C, and the flow rate is 0.2-0.5 ml / min; preferably, the detection wavelength is 240 nm, the column temperature is 30 DEG C, and the flow rate is 0.3 ml / min; the injection amount is 2 μl.

5. The method of claim 1, wherein In the mass spectrometry conditions, the ion fragment information data of the chemical ingredients in the test sample solution are obtained by using a combination of primary mass spectrometry analysis and secondary mass spectrometry scanning.

6. The detection method according to claim 5, characterized in that, The MS mode is selected for the primary mass spectrum, and the mass scan range is 100-1700 m / z; the Auto-MS / MS mode is selected for the secondary mass spectrum, and the mass scan range is 100-1300 m / z; the collision voltage is 5eV-35eV, preferably 10eV, 15eV, 20eV, 30eV.

7. The detection method according to claim 5, characterized in that, The ion fragment information data is collected by Agilent Mass Hunter Qualitative Analysis software, and data processing adopts Agilent software Qualitative Navigator (B.08.00) and Qualitative Workflows (B.08.00).

8. The method of claim 1, wherein, In the mass spectrum condition, the drying gas temperature is 350℃, the drying gas flow rate is 10L / min, the atomization gas pressure is 35psi, the sheath gas temperature is 350℃, and the sheath gas flow rate is 12L / min.

9. The assay of any one of claims 1-9, wherein, Forty-nine compounds were identified in the Dihuangyinzi preparation, including 13 glycolipids, 11 iridoid glycosides, 4 flavonoids, 4 ketones, 3 alkaloids, and 14 other compounds. Forty-nine compounds were identified in the Dihuangyinzi preparation, including 13 glycolipids, 11 iridoid glycosides, 4 flavonoids, 4 ketones, 3 alkaloids, and 14 other compounds.

10. The detection method according to claim 9, characterized in that, The glycolipid compounds are Sibiricose A5 (peak 13), Polygalatenoside (peak 14), Sibiricose A6 (peak 15), Echinacoside (peak 21), Globoside A (peak 24), Polygalaenoside B (peak 31), 3,6'-Dihydroxybenzylsucrose (peak 36), Arillanin A (peak 38), Polygalaenoside A (peak 40), Tenuifoliose J (peak 42), Tenuifoliose B (peak 43), Tenuifoliose H (peak 44), Tenuifoliose A (peak 45); The iridoid compounds are Yinkuanganoside (peak 1), Deacetyl-Asperospermoside (peak 3), Rehmoside A (peak 6), Leonuride (peak 7), Geniposide (peak 8), 8-Epi-loganic acid (peak 9), Loganin (peak 10), Dihydroevonymoside (peak 11), Monoikoside (peak 12), Loganin (peak 18), Evonikoside (peak 34); The flavonoids are Rutin (peak 28), Myricetin-3-O-rutinoside (peak 32), Hesperidin (peak 35), Acacetin-7-O-neohesperidoside (peak 41); The The ketone compounds are Sibiricaxanthon A (peak 22), Polygalaxanthone III (peak 23), Polygalaxanthone XI (peak 26), and Polygalaxanthone VIII (peak 27). The alkaloids are Aconitine (peak 19), Magnoflorine (peak 20), Benzoylmesaconitine (peak 39); The alkaloids are Aconitine (peak 19), Magnoflorine (peak 20), Benzoylmesaconitine (peak 39); The other compounds are 1-deoxy-1-L-leucine-D-fructose (peak 2), cyclic adenosine monophosphate (peak 4), gallic acid (peak 5), Procyanidin B1 / Procyanidin B2 (peak 16), dehydrodiconiferyl alcohol (peak 17), (2E,6E)-3,7-dimethyl-8-hydroxyoctadien-1-O-β-D-glucoside (peak 25), Kankanoside O (peak 29), 1-(3,4-dihydroxyphenyl)-3-[2-(3,4-dihydroxyphenyl)-1-hydroxy-1,2-dihydronaphthalene-2-carboxylic acid (peak 30), Norboldine (peak 33), Rosmarinic acid (peak 37), 6-Gingerol (peak 46), Schisandrol A (peak 47), a-asarone (peak 48), 1-deoxy-6-shogaol (peak 49).

Images