Analyzing and identifying method for chemical components of calculus removing agent

Abstract

The invention provides a method for analyzing and identifying chemical components of a calculus-eliminating prescription, which comprises the following steps: S1, preparing a test solution: performing ultrasonic treatment on a calculus-eliminating prescription extract by using methanol, centrifuging, taking supernate, diluting, filtering, and taking subsequent filtrate to obtain the test solution; s2, analyzing the test sample solution by adopting UHPLC-QE-MS (Ultra High Performance Liquid Chromatography-Quantitative Enhanced-Mass Spectrometry), wherein the detection conditions of the UHPLC-QE-MS are as follows: a chromatographic column is an octadecylsilane chemically bonded silica chromatographic column; the column temperature is 20-40 DEG C; the flow rate is 0.1 to 0.5 ml / min; the mobile phase A is 0.03-0.20% v / v formic acid aqueous solution, the mobile phase B is acetonitrile, gradient elution is carried out, and the gradient elution procedure is 0-1 min, 5% B-> 5% B; in 1-14 minutes, 5% B is changed into 60% B; and after 14-15 minutes, 60% B is converted into 95% B. The detection method disclosed by the invention can identify 68 compounds in the calculus eliminating sample at one time, and is rapid, simple, convenient, high in sensitivity and good in specificity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine ingredient identification, and particularly relates to a method for rapid analysis and identification of chemical components of Paishi Decoction. BACKGROUND

[0002] Paishi Decoction is a clinical experience prescription for treating kidney stones, which is mainly composed of Tianqingdibai (medicinal material derived from Gnaphalium japonicum Thunb., Latin name: Gnaphalium japonicum Thunb.) and Gubozuye (medicinal material derived from Ilex cornuta Lindl. ex Paxt., Latin name: Ilex cornuta Lindl. ex Paxt.), and has the effects of clearing heat and reducing dampness, soothing liver and tonifying kidney, and removing stone. However, there is currently a lack of comprehensive analysis and identification of the chemical components of Paishi Decoction. Component analysis is the basis for revealing the material basis for efficacy, quality control indicators and mechanism research, and has guiding significance for clinical application. Therefore, it is necessary to analyze and identify the chemical components of Paishi Decoction by using modern technology.

[0003] In recent years, with the development of modern analysis technology, liquid chromatography-mass spectrometry technology has become a powerful means for rapid separation and identification of active ingredients in complex natural medicine systems. Ultrahigh performance liquid chromatography-Q-Exactive Orbitrap mass spectrometry (UHPLC-QE-MS) technology has good selectivity and high separation capacity, and can efficiently separate and rapidly identify various compounds in a complex system in one experiment, and is widely used in the chemical component research of traditional Chinese medicines. In the characterization research of traditional Chinese medicine compounds, high-resolution mass spectrometry has obvious advantages, not only in small sample requirements, but also in providing accurate molecular weight, corresponding retention time and effective characteristic structure fragment information, which provides a powerful analysis means for the qualitative and quantitative analysis of complex traditional Chinese medicine systems.

[0004] Currently, there is no report on the analysis and identification of the chemical components of Paishi Decoction at home and abroad. Therefore, it is necessary to establish a rapid and efficient analysis method for comprehensively elucidating the chemical components of Paishi Decoction. The present application uses UHPLC-QE-MS technology to qualitatively identify the chemical components in the extract of Paishi Decoction, and lays a solid foundation for the quality control and material basis research of Paishi Decoction. SUMMARY

[0005] To achieve the above-mentioned purpose, the present application provides a method for analyzing and identifying the chemical components of Paishi Decoction, comprising the following steps:

[0006] S1, Preparation of the test solution: the extract of Paishi Fang is treated with methanol by ultrasonic, centrifuged, the supernatant is diluted, filtered, and the filtrate is obtained, which is the test solution; preferably, the concentration of methanol is 25% to 75%, the ultrasonic frequency is 50 to 60 kHz, the ultrasonic time is 10 to 60 min, the centrifugal speed is 12000 r / min, the centrifugal time is 5 to 30 min, and the concentration of the test solution is 10 to 100 mg / ml;

[0007] S2, the test solution is analyzed by UHPLC-QE-MS, and the detection conditions of the UHPLC-QE-MS are as follows:

[0008] The chromatographic column is an octadecylsilane-bonded silica gel chromatographic column; preferably, the octadecylsilane-bonded silica gel chromatographic column is a Waters HSS T3 chromatographic column (2.1 mm x 100 mm, 2.5 μm) or a chromatographic column with equivalent performance;

[0009] The column temperature is 20 to 40℃, and the flow rate is 0.1 to 0.5 ml / min;

[0010] The mobile phase A is 0.03 to 0.20% v / v formic acid aqueous solution, and the mobile phase B is acetonitrile, and gradient elution is carried out, and the gradient elution program is as follows: 0 to 1 min, 5% B→5% B; 1 to 14 min, 5% B→60% B; 14 to 15 min, 60% B→95% B.

[0011] Further, in step S2, the numerical processing mode of the UHPLC-QE-MS is as follows: according to the accurate compound relative molecular weight information of the primary mass spectrum, the error between the measured value and the theoretical value of the mass-to-charge ratio of the primary quasi-molecular ion peak is less than 5ppm, the molecules are matched with the chemical composition database information of Paishi Fang, the compound information is preliminarily speculated, the chromatographic peak and the secondary fragment ion information of the target compound are further extracted, the target compound is identified and confirmed, and the chemical composition identification result is obtained.

[0012] Further, in step S1, the preparation method of the extract of Paishi Fang comprises the following steps: weighing each component of Paishi Fang, soaking after adding water, heating to micro-boiling to extract twice, combining the filtrates after filtering the two extract solutions, reducing pressure to concentrate, drying, and preferably vacuum drying to obtain the extract of Paishi Fang. Preferably, the soaking time of Paishi Fang is 0 to 2 h; the water addition multiple is 8 to 20 times, the heating and decoction time is 0.5 to 2 h, the pressure reduction concentration is to 1.05 to 1.20 g / ml, the pressure reduction concentration temperature is 40 to 80℃, and the vacuum drying temperature is 60 to 90℃.

[0013] Further, in step S2, the mass spectrum conditions of the UHPLC-QE-MS are as follows:

[0014] The heating electrospray ion source (H-ESI) is used, the positive and negative ion Full MS / dd-MS2 scanning mode, the Full MS resolution is 60000-80000, the dd-MS2 resolution is 10000-20000, high-purity nitrogen is used as atomization ion gas, the spray voltage is 3.8 (+) / 3.2 (-)|kV|, the sheath gas flow is 20-40 arb, the auxiliary gas flow is 8-12 arb, the auxiliary heater temperature is 280-320 DEG C, the capillary temperature is 310-330 DEG C, the S-lens radio frequency level is 50-60, the acquisition range is 80-2000 m / z, and the collision energy is 5-60 eV. Preferably, the mass spectrum condition is that the Full MS resolution is 70000, the dd-MS2 resolution is 17500, the sheath gas flow is 30 arb, the auxiliary gas flow is 10 arb, the auxiliary heater temperature is 300 DEG C, the capillary temperature is 320 DEG C, the S-lens radio frequency level is 55, the acquisition range is 100-1500 m / z, the collision energy is 10, 30, 50 eV.

[0015] The chemical component analysis and identification method provided by the application identifies 68 kinds of compounds, including D-trehalose, benzoquinone, sorbic acid, cyclic adenosine phosphate, nicotinamide, catechol, protocatechuic acid, ethyl gallate, 5-deoxy-5-methylthioadenosine, chlorogenic acid, icariside II, p-cymene, saponin, quercetin-7-glucoside, vanillin, rutin, syringaldehyde, quercetin-3-O-(6"-O-malonyl)-beta-D-glucoside, methyl cinnamate, luteolin, 1,5-dicaffeoylquinic acid, homoeriodictyol, 5-O-methylvisamminol, baringenol, plumbagin, scirpusanin, 11-keto-beta-boswellic acid, nandrolone, umbelliferone, hesperetin, alpha-linolenic acid, isoquercitrin, steviol, 1-hexadecene, 6-gingerol, coniferyl alcohol, 3,4-methylenedioxymethamphetamine, N-fructosyl pyroglutamate, ascorbic acid, benzoic acid, isovanillin acid, leonuriside a, N6-succinyl adenosine, p-hydroxyacetophenone, 3,4-dihydroxybenzoic acid ethyl ester, aesculin, aesculetin, caffeic acid, NP-000062 (6), praeroside II, iso-typhane, emodin-8-O-beta-aspidoside, isoquercitrin, isoheteroside, n-nonanoic acid, preussin, isochlorogenic acid A, astrin, emodin-6-glucoside, azelaic acid, quercetin, hydroxyemodin, scirpusanin, emodin, isorhamnetin, dodecanedioic acid, myristic acid, asiatic acid. Among them, 18 are flavonoids, 15 are phenols, 8 are acids, 5 are coumarins, 4 are terpenes, 4 are anthraquinones, 3 are nucleotides, 2 are glycosides, 2 are amino acids, 1 is a phenylpropanoid, 1 is a phenylpropanamine, 1 is a benzoquinone, 1 is an unsaturated aliphatic hydrocarbon, 1 is a steroid, 1 is an acetylenic ketone, and 1 is a sugar. The application lays a foundation for further clarifying the chemical substance basis of Pishifang, the research on active ingredients and quality control. It is conducive to the follow-up development and utilization of Pishifang.

[0016] The advantages of the present application are: 1, the traditional liquid phase analysis method has the disadvantages of low sensitivity, poor specificity, less effective information, and cannot obtain exact structure information, etc. The UHPLC method of the present application can effectively separate a plurality of components in the Pai Shi Fang sample, and further using the above mass spectrometry condition can obtain accurate molecular weight, accurately deduce the molecular formula of the compound, the detection sensitivity is high, and the multi-level information of the obtained components is rich, which can realize accurate analysis and identification of the components. 2, the chromatographic condition of the present application can realize the rapid separation of 68 kinds of compounds in 15 minutes, the detection method is fast and simple, the sensitivity is high, and the specificity is good. 3, the chemical components in the Pai Shi Fang can be comprehensively analyzed. Using the UHPLC-QE-MS detection method of the present application, 68 kinds of compounds in the Pai Shi Fang sample can be identified at one time. Including 18 flavonoids, 15 phenols, 8 acids, 5 coumarins, 4 terpenes, 4 anthraquinones, 3 nucleotides, 2 glycosides, 2 amino acids, 1 phenylpropanoid, 1 benzene, 1 quinone, 1 unsaturated aliphatic hydrocarbon, 1 sterol, 1 alkyne ketone and 1 sugar. It can provide a reliable method for further elucidating the efficacy material basis of Pai Shi Fang and its quality standard research. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The positive and negative ion flow diagrams of the Pai Shi Fang UHPLC-QE-MS are shown in (a) and (b), respectively.

[0018] Figure 2 The secondary mass spectrum of compound 16 of the Pai Shi Fang is shown in the figure.

[0019] Figure 3 The secondary mass spectrum of compound 29 of the Pai Shi Fang is shown in the figure.

[0020] Figure 4 The secondary mass spectrum of compound 50 of the Pai Shi Fang is shown in the figure.

[0021] Figure 5 The secondary mass spectrum of compound 60 of the Pai Shi Fang is shown in the figure. DETAILED DESCRIPTION

[0022] The present application will be further described in detail below in conjunction with specific examples, which are not used to limit the present application, but only to illustrate the present application. Unless otherwise specified, the experimental methods used in the following examples are generally carried out under conventional conditions. Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained from commercial channels.

[0023] Example 1

[0024] 1. Materials

[0025] 1.1 Instruments

[0026] Vanquish UHPLC (Thermo Fisher Scientific, USA); Q Exactive PLUS Mass Spectrometer (Thermo Fisher Scientific, USA); electronic balance of 1 / 10,000 (Sartorius Scientific Instruments (Beijing) Co., Ltd.)

[0027] 1.2 Reagents

[0028] Methanol (ROE SCIENTIFIC INC, USA), chromatographically pure; formic acid, acetonitrile, (Thermo Fisher Scientific, USA), chromatographically pure; water is ultrapure water; other reagents are all analytically pure.

[0029] 2. Methods

[0030] 2.1 Establishment of chemical composition database: refer to the chemical compositions of Radix Isatidis and Folium Randiae that have been isolated and identified, and establish a chemical composition database, which includes chemical name, molecular formula, molecular weight and chemical structure information.

[0031] 2.2 Preparation of sample solution

[0032] 2.2.1 Preparation of Pai Shi Fang extract: mix Radix Isatidis 12 g and Folium Randiae 8 g, soak in 2 L water for 1 h, then heat to near-boiling to decoct and extract for 1 h, filter to obtain the first filtrate and dregs, then add 2 L water to the dregs, heat to near-boiling to decoct and extract for 1 h, filter to obtain the second filtrate. Combine the two filtrates. Concentrate the filtrate under reduced pressure to a relative density of 1.05-1.10 (with water as the reference substance), and dry under vacuum at 80°C to obtain the Pai Shi Fang extract.

[0033] 2.2 Preparation of test solution: accurately weigh 1 g of Pai Shi Fang extract, dissolve in 10 ml of 50% methanol, weigh, ultrasonicate (power 220 W, frequency 50-60 kHz) for 30 min, remove, cool, re-weigh, make up the weight loss with 50% methanol, shake the solution, centrifuge (12000 r / min, 15 min), take the supernatant, dilute with 50% methanol to 50 mg / ml of test solution, filter through a 0.22 μm filter membrane, and take the filtrate to obtain the test solution.

[0034] 2.3 Detection conditions

[0035] The ultra-high performance liquid chromatograph is Vanquish UHPLC, and the chromatographic column is Waters HSS T3 column (2.1 mm x 100 mm, 2.5 μm). Mass spectrometer was Q Exactive PLUS Mass Spectrometer, and the first and second mass spectrometry data acquisition was Xcalibur (Thermo Fisher Scientific, USA).

[0036] 2.3.1 Chromatographic conditions

[0037] Chromatographic column: Waters HSS T3 column (2.1 mm x 100 mm, 2.5 μm), mobile phase 0.1% formic acid (A)-acetonitrile (B), elution gradient: 0-1 min, 5% B→5% B; 1-14 min, 5% B→60% B; 14-15 min, 60% B→95% B, the running time was 15 min, the post running time was 5 min, and the flow rate was 0.4 ml / min. The column temperature was 30℃, and the injection volume was 1 μL.

[0038] Table 1 Chromatographic gradient elution conditions

[0039]

[0040]

[0041] 2.3.2 Mass spectrometry conditions

[0042] Full MS / dd-MS2 scan mode was used with heating electrospray ion source (H-ESI), positive and negative ions, Full MS resolution 70000, dd-MS2 resolution 17500, high-purity nitrogen gas as atomization ionization gas, spray voltage 3.8(+) / 3.2(-)|kV|, sheath gas flow 30 arb, auxiliary gas flow 10 arb, auxiliary heater temperature 300℃, capillary temperature 320℃, S-lens radio frequency level 55, acquisition range 100-1500 m / z, collision energy 10, 30, 50 eV. Before use, the instrument was calibrated with a tuning liquid, and reference liquid was continuously added for quality calibration during use.

[0043] 2.4 Data processing

[0044] According to the mass spectrometry accurate relative molecular weight information, TraceFinder TM5.1 The software selects molecules with a mass-to-charge ratio error of less than 5 ppm between the measured and theoretical values ​​of the primary quasi-molecular ion peak. This data is then matched with information from the Paishifang chemical composition database to preliminarily infer compound information. Xcalibur 4.5 software is used to further extract the chromatographic peak and secondary fragment ion information of the target compound. The target compound is then identified and confirmed by combining the Paishifang chemical composition database and literature information. The obtained fragment ions are compared with the chemical structure information in the chemical composition database to further determine the chemical composition of the mass spectrometry molecular ion peak.

[0045] 3. Test Results

[0046] The positive and negative ion chromatograms of the purging stone formula sample obtained by UHPLC-QE-MS are attached. Figure 1 As shown, based on the fragmentation pattern, the obtained fragment ions are compared with the chemical structure information in the chemical composition database to determine the chemical composition of the molecular ion peak in the mass spectrometry.

[0047] For example:

[0048] The quasi-molecular ion of compound 16 in positive ion mode is m / z 377.08392 [M+Na]. + The predicted molecular formula is C 16 H 18 O9 (RDB = 8). Comparison with the chemical composition database of the stone-dissolving formula showed that the mass of chlorogenic acid was consistent, with a m / z of 215.0526 [M+Na-C9H6O3] in the secondary mass spectrometry. + The ion with the highest relative abundance is the quinic acid ion after the loss of the caffeoyl group, therefore compound 16 is chlorogenic acid.

[0049] Compound 29 has a quasi-molecular ion of m / z 611.1618 [M+H] in positive ion mode. + The mass spectrometry software calculated the precise molecular formula C. 27 H 30 O 16 The mass of rutin was consistent with that in the database of chemical components for treating kidney stones. Typical fragment ion peaks of rutin were observed in the secondary mass spectrum at m / z 303.0497, 465.1024, and 153.0186, with m / z 303.0497 being the aglycone ion peak. Consistent with the database and literature reports, compound 29 is presumed to be rutin.

[0050] Compound 50 was first obtained as a quasi-molecular ion with m / z 301.0356 [MH] in negative ion mode. - The mass spectrometry software calculated the precise molecular formula C. 15 H 10O7, then compared with the chemical composition database of Pailu Fang, the mass of quercetin is consistent with one, so it is initially speculated that it is quercetin, and according to the secondary fragment ions 178.9978, 151.0027, 107.0162, which are consistent with the database and literature reports, it is thus speculated to be quercetin.

[0051] Compound 60 in negative ion mode Quasi-molecular ion is m / z 315.0512[M-H] - , the predicted molecular formula is C 16 H 12 O7(RDB=11). The quasi-molecular ion in the secondary mass spectrum loses CH3 to produce m / z 300.0276, further loses CO to produce m / z 272.0312; ring-opening ion m / z 151.00 1,3 A - ) It is speculated that ring A has two hydroxyl groups, so the methyl group should be connected to the hydroxyl group of ring B, so it is speculated that compound 60 is isorhamnetin.

[0052] UHPLC-QE-MS identified the retention time of each chromatographic peak of Pailu Fang, the identification results and their source attribution are as follows:

[0053] Table 4 Chemical composition of Pailu Fang identified based on UHPLC-QE-MS

[0054]

[0055]

[0056]

[0057]

Claims

1. A method for chemical composition analysis and identification of a stone-dissolving formula, comprising the following steps: S1. Preparation of the test solution: The extract of the stone-expelling formula was ultrasonically treated with methanol, centrifuged, the supernatant was diluted, filtered, and the filtrate was collected to obtain the test solution. S2. The test solution was analyzed using UHPLC-QE-MS. The UHPLC-QE-MS detection conditions were as follows: Chromatographic column: octadecylsilane-bonded silica gel column; column temperature: 20–40℃; flow rate: 0.1–0.5 ml / min; Mobile phase A is a 0.03–0.20% v / v formic acid aqueous solution, and mobile phase B is acetonitrile. Gradient elution is performed, and the gradient elution program is as follows: 0–1 min, 5% B → 5% B; 1–14 min, 5% B → 60% B; 14–15 min, 60% B → 95% B.

2. The analytical identification method as described in claim 1, characterized in that, The concentration of the test solution is 10–100 mg / ml.

3. The analytical identification method as described in claim 2, characterized in that, In step S1, the methanol concentration is 25%–75%, the ultrasonic frequency is 50–60 kHz, the ultrasonic time is 10–60 min, the centrifugation speed is 12000 r / min, and the centrifugation time is 5–30 min.

4. The analytical identification method as described in claim 1 or 2, characterized in that, In step S1, the preparation method of the stone-expelling formula extract includes the following steps: weigh each component of the stone-expelling formula, soak it in water, heat it to a gentle boil and decoct it twice, filter the two extracts and combine them, concentrate them under reduced pressure, and dry them to obtain the stone-expelling formula extract.

5. The analytical identification method as described in claim 4, characterized in that, The soaking time for the stone-expelling formula is 0-2 hours; the water addition ratio is 8-20 times; the heating and decocting time is 0.5-2 hours; the concentration under reduced pressure is 1.05-1.20 g / ml; the concentration temperature under reduced pressure is 40-80℃; and the vacuum drying temperature is 60-90℃.

6. The analytical identification method as described in claim 1 or 2, characterized in that, In step S2, the UHPLC-QE-MS numerical processing method is as follows: based on the precise relative molecular weight information of the compound from the primary mass spectrometry, molecules with a mass-to-charge ratio error of less than 5 ppm between the measured and theoretical values ​​of the primary quasi-molecular ion peak are selected and matched with the information in the chemical composition database of the stone-dissolving formula to preliminarily infer the compound information. Further extraction of the chromatographic peak of the target compound and its secondary fragment ion information is performed to identify and confirm the target compound, thereby obtaining the chemical composition identification results.

7. The analytical identification method as described in claim 6, characterized in that, In step S2, the UHPLC-QE-MS mass spectrometry conditions are as follows: A heated electrospray ionization source was used, with positive and negative ion Full MS / dd-MS2 scanning modes. The Full MS resolution was 60,000–80,000, and the dd-MS2 resolution was 10,000–20,000. High-purity nitrogen was used as the atomizing ionization gas. The spray voltage was 3.8(+) / 3.2(-)|kV|, the sheath gas flow rate was 20–40 arb, the auxiliary gas flow rate was 8–12 arb, the auxiliary heater temperature was 280–320℃, the capillary temperature was 310–330℃, the S-lens RF level was 50–60, the acquisition range was 80–2000 m / z, and the collision energy was 5–60 eV.

8. The analytical identification method as described in claim 7, characterized in that, The mass spectrometry conditions were as follows: Full MS resolution 70000, dd-MS2 resolution 17500, sheath gas flow rate 30 arb, auxiliary gas flow rate 10 arb, auxiliary heater temperature 300℃, capillary temperature 320℃, S-lens RF level 55, acquisition range 100~1500m / z, and collision energies 10, 30, and 50 eV.

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