A method for determining the content of multiple components in Guyinjian based on UPLC-MS and its application

Abstract

The present invention provides a method for determining the content of multiple components of Guyin decoction based on UPLC-MS, belonging to the technical field of pharmaceutical component analysis, wherein the method uses UPLC-TQD-SIR technology to simultaneously detect the contents of 14 components in Guyin decoction, including rehmannoside D, morroniside, loganin, polygala tenuifolia ketone III, liquiritin, hyperoside, verbascoside, 3',6-dicerinoylsucrose, ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1, polygala tenuifolia saponin, glycyrrhizic acid, and schisandrin A. The method for determining the content of multiple components of Guyin decoction established by the present invention has the advantages of high sensitivity, high selectivity, rapid analysis, etc., and can provide a reference for quality control research and development research of Guyin decoction.

Description

Technical Field

[0001] The present invention relates to the technical field of drug component content determination, and in particular to a method for determining the content of multiple components of Guyin decoction based on UPLC-MS and application thereof. Background Art

[0002] Guyin Decoction comes from Volume 51 of Jingyue Complete Works written by Zhang Jingyue in the Ming Dynasty (1640 AD). The original book states that "Guyin Decoction is composed of ginseng (Panax ginseng CA Mey.), Rehmannia glutinosa Libosch., Chinese yam (Dioscorea opposita Thunb.fried), Cornus officinalis (Shanzhuyu Sieb.et Zucc.), Polygalatenuifolia Willd.fried, Glycyrrhiza uralensis Fisch., Schisandra chinensis (Turcz.) Baill., Cuscuta chinensis Lam.fried, and has the effects of tonifying the liver and kidney, nourishing yin and benefiting essence." In a clinical observation experiment, 60 patients with immune infertility were treated with Guyin Decoction and compared with 25 patients treated with Western medicine as a control group. The results showed that 19 cases were cured, 32 cases were improved, and 9 cases were ineffective in the treatment group; 6 cases were cured, 8 cases were improved, and 11 cases were ineffective in the control group. The data showed that Guyin Decoction had a significant effect on serum IgA and IgG. In another study, the effect of Guyin Decoction plus and minus therapy on ovarian reserve function in patients with kidney deficiency syndrome, 90 patients were randomly divided into a control group (45 cases) and an observation group (45 cases), and both groups of patients were given antagonists. On this basis, the observation patients received Guyin Decoction plus and minus therapy. It was found that Guyin Decoction plus and minus therapy can increase ovarian blood supply, improve ovarian high reserve function, reduce Gn consumption, increase the number of eggs obtained, relieve kidney yin deficiency symptoms, and improve ovarian responsiveness and pregnancy outcomes. In addition, Guyin Decoction can also be used to treat gynecological diseases such as polycystic ovary syndrome ovulation disorders, kidney deficiency type menorrhagia, menopausal syndrome, intermenstrual bleeding, adolescent uterine bleeding, etc., and has a good therapeutic effect. At the same time, Guyin Decoction was selected as the 64th in the "Catalogue of Ancient Classics (First Batch)" issued by the State Administration of Traditional Chinese Medicine. Article 30 of the Traditional Chinese Medicine Law of the People's Republic of China: When applying to produce compound Chinese medicine preparations that meet the conditions stipulated by the state, only non-clinical safety research data can be provided. Drug approval number.

[0003] However, the lack of research on the quality control of Guyin Decoction has severely restricted its development and wider clinical application. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for determining the content of multiple components in Guyin Jian based on UPLC-MS, which can simultaneously detect the contents of multiple components in Guyin Jian.

[0005] The technical scheme of the present invention is: a method for determining the content of multiple components of Guyin Decoction based on UPLC-MS, which uses UPLC-TQD-SIR technology to simultaneously detect the contents of 14 components in Guyin Decoction, including rehmannoside D, morroniside, loganin, polygala tenuifolia ketone III, liquiritin, hyperoside, verbascoside, 3',6-diesinapoylsucrose, ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1, polygala tenuifolia saponin, glycyrrhizic acid, and schisandrin A;

[0006] The method includes the steps of using UPLC and triple quadrupole mass spectrometry system to detect standard solution and sample solution. The conditions of UPLC and triple quadrupole mass spectrometry system are as follows: the model of UPLC is Waters Acquity TM UPLC, the separation of each solution used ACQUITY UPLC HSS T3 column, the column model is: 2.1×100mm, 1.8µm; the mobile phase composition used in the system is: acetonitrile + 0.1% formic acid (A) and water + 0.1% formic acid (B), the flow rate is 0.4 mL / min, the column temperature is 35℃, and the injection volume is 1µL; the gradient elution in the positive ion mode is as follows: 0-3min 55-55%A, 3-5min 55-99%A, and the re-equilibrium time is 4min; the gradient elution in the negative ion mode is as follows: 0-2min 5-15%A, 2-6min 15-15%A, 6-15min 15-50%A, 15-18min50-99%A, and the re-equilibrium time is 5min.

[0007] Furthermore, the method comprises the following steps:

[0008] Preparation of S1 standard solution: using methanol solution as solvent, a mixed standard solution containing 14 components was prepared;

[0009] Preparation of S2 sample solution: first weigh ginseng, prepared rehmannia root, stir-fried yam, cornus officinalis, processed polygala root, roasted licorice root, schisandra chinensis, and stir-fried dodder seed according to the prescription of Guyin decoction to prepare Guyin decoction freeze-dried powder, then add a predetermined amount of methanol solution to the Guyin decoction freeze-dried powder, perform ultrasonic extraction, and obtain a sample solution;

[0010] S3 uses UPLC and triple quadrupole mass spectrometry system to detect standard solutions and sample solutions, and then uses ESI-MS spectrum to analyze and obtain the contents of 14 components.

[0011] Furthermore, the concentration of the methanol solution used in the preparation of the standard solution in step S1 and the preparation of the sample solution in step S2 is both 50 wt %.

[0012] Furthermore, in step S1, the preparation of the standard solution is to first prepare a mixed standard stock solution using a methanol solution and then prepare a mixed standard solution, wherein the concentrations of rehmannia glutinosa D, morroniside, loganin, polygala tenuifolia ketone III, licorice glycoside, hyperoside, verbascoside, 3',6-dicerinoylsucrose, ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1, polygala tenuifolia saponin, glycyrrhizic acid, and schisandrin A in the mixed standard stock solution are 20.410µg / ml, 98.450µg / ml, 52.975µg / ml, and 1. 9650µg / ml, 24.063µg / ml, 29.888µg / ml, 4.7850µg / ml, 33.575µg / ml, 5.3675µg / ml, 2.9850µg / ml, 6.7000µg / ml, 3.5380µg / ml, 37.300µg / ml, 8.9600µg / ml; the mixed standard solution includes working solutions of different concentrations prepared by dilution with methanol solution, and the working solutions of different concentrations contain rehmannoside D, morroniside, loganin, and polygala tenuifolia. The concentration ranges of ginsenoside III, liquiritin, hyperoside, verbascoside, 3',6-diesinapoylsucrose, ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1, polygala tenuifolia saponin, glycyrrhizic acid, and schisandrin A were 0.4082-10.205µg / ml, 1.9690-49.225µg / ml, 1.0595-26.488µg / ml, 0.0393-0.9825µg / ml, 0.4813-12.031µg / ml, and 0.5978-1 4.944µg / ml, 0.0957-2.3925µg / ml, 0.6715-16.788µg / ml, 0.1074-2.6838µg / ml, 0.0597-1.4925µg / ml, 0.1340-3.3500µg / ml, 0.0708-1.7690µg / ml, 0.7460-18.650µg / ml, 0.1792-4.4800µg / ml; each solution was filtered with a 0.22µm filter membrane before sampling and detection.

[0013] Furthermore, in the preparation of the sample solution in step S2, the contents of the ingredients in the prescription of Guyin Decoction are 7.46 parts by weight of ginseng, 14.92 parts by weight of Rehmannia root, 7.46 parts by weight of stir-fried yam, 5.60 parts by weight of Cornus officinalis, 2.61 parts by weight of processed Polygala tenuifolia, 5.60 parts by weight of roasted Licorice root, 2.00 parts by weight of Schisandra chinensis, and 9.33 parts by weight of stir-fried Cuscuta australis.

[0014] Furthermore, in step S2, the freeze-dried powder is prepared by soaking the ingredients of the prescription amount of Guyin Decoction in water and then boiling them, filtering them and freeze-drying them.

[0015] Furthermore, in step S2, the ultrasonic conditions during ultrasonic extraction are 250W, 40KHz, and the ultrasonic time is 45min. The sample solution is passed through a 0.22μm microporous membrane before sampling and detection.

[0016] Furthermore, in step S3, ESI-MS spectra were obtained in SIR mode in positive and negative ion modes; the ESI-MS analysis conditions were as follows: capillary voltage: 3 kv / -3 kv; cone voltage: 30 v / -30 v; extraction voltage: 3 v / -3 v; desolvation temperature: 350 °C; desolvation gas flow rate: 650 L / hr; cone gas flow rate: 50 L / hr; ion source temperature: 150 °C; Masslynx V4.2 software (Waters, USA) was used for data acquisition and processing.

[0017] Furthermore, the standard curves of the substances in this method are: rehmannia glycoside D y=1856.2x+162.47 (r=0.9999); morroniside y=5663.3x+9521 (r=0.9999); loganin y=5837.6x+3430.9 (r=0.9994); polygala tenuifolia ketone III y=12217x+139.22 (r=0.9999); Liquorice glycoside y=11344x+3901.7 (r=0.9988); Hyperoside y=11878x+9390.3 (r=0.9960); Verbascoside y=19206x+1084.2 (r=0.9993); 3',6-dicansinoyl y=8154.2x+2317.8 (r=0.9997); Ginsenoside Re y=12765x+325.7 (r=0.9998); Ginsenoside Rg1y=16044x+175.01 (r=0.9998); Ginsenoside Rb1 y=15886x+203.89 (r=0.9999); Polygala tenuifolia saponin y=24931x+1218.6 (r=0.9988); Glycyrrhizic acid y=41117x+28611 (r=0.9986); Schisandrin A y=46610x+26670 (r=0.9966).

[0018] The invention discloses an application of a multi-component content determination method of Guyin Decoction based on UPLC-MS, wherein the method is used to simultaneously detect the contents of 14 components in Guyin Decoction, including rehmannoside D, morroniside, loganin, polygala tenuifolia ketone III, liquiritigenin, hyperoside, verbascoside, 3',6-diesinapoylsucrose, ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1, polygala tenuifolia saponin, glycyrrhizic acid, and schisandrin A.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The method for determining the multi-component content of Guyin Decoction established in the present invention has the advantages of high sensitivity, high selectivity, rapid analysis, etc., and can provide a reference for the quality control research and development research of Guyin Decoction.

[0021] In the selection of indicator compounds in the content detection method of the present invention, the inventors consulted the literature and found out the main components of each medicinal material that exert pharmacological activity, and referred to the quality control components in the "Pharmacopoeia of the People's Republic of China" (2020 edition), and finally determined to select ginsenoside Re, ginsenoside Rg1, and ginsenoside Rb1 from ginseng; rehmannia glutinosa D and verbascoside from Rehmannia glutinosa; morroniside and loganin from Cornus officinalis; 3',6-dicansinoylsucrose, polygala tenuifolia ketone III, and polygala tenuifolia saponin from Polygala tenuifolia; hyperoside from Cuscuta chinensis; schisandrin A from Schisandra chinensis; liquiritigenin and glycyrrhizic acid from Glycyrrhiza uralensis as the quality control components of Guyin Decoction sample.

[0022] The UPLC-MS technology used in the present invention is a powerful tool for the modernization of traditional Chinese medicine. It is widely used in qualitative and quantitative analysis due to its excellent sensitivity and resolution. Triple quadrupole mass spectrometry is widely used in quantitative research due to its high accuracy. The selected ion recording (SIR) mode is a selected ion quantitative analysis mode that allows the simultaneous quantitative analysis of multiple components using only one quadrupole. Therefore, based on the UPLC-TQD-SIR technology, this study successfully established a rapid, simple, and efficient simultaneous quantitative analysis method for multiple components of Guyin Decoction, and carried out methodological verification, which provides a reference for the quality control, formula development, and other related research of Guyin Decoction.

[0023] Based on UPLC-TQD-SIR technology, the present invention successfully developed a method for determining the content of 14 components in Guyinjian samples with good specificity, linearity (r=0.9960-0.9999), precision (RSD%≤3.18%), stability (RSD%≤2.64%) and accuracy (recovery rate 88.64%-107.43%, RSD% between 2.82%-6.22%). Through statistical analysis of the content determination results of 15 batches of Guyinjian samples, it was found that the compounds that had a greater impact on inter-batch stability were mainly rehmannoside D, loganin, morroniside, ginsenoside Re and 3',6-dicansinoylsucrose. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0025] Figure 1 The structural information of 14 key compounds in Example 1;

[0026] Figure 2are representative chromatograms in Example 1, wherein (A) the total ion chromatogram (TIC) of the mixed standard is compared with the Guyinjian sample, and (B) the chromatogram of the individual extracts of each compound in the Guyinjian sample;

[0027] Figure 3 The cluster analysis and multivariate statistical analysis results of the content determination of 15 batches of Guyin Decoction samples in Example 1. (A) Cluster analysis heat map; (B) PCA analysis results; (C) OPLS-DA analysis results; (D) VIP score map;

[0028] Figure 4 The chromatograms are compared under different column temperature conditions in Example 1;

[0029] Figure 5 It is a comparative chromatogram under different flow rate conditions in Example 1;

[0030] Figure 6 The chromatograms are compared under different injection volume conditions in Example 1. DETAILED DESCRIPTION

[0031] The technical scheme in the embodiment of the present invention is described clearly and completely below in conjunction with the accompanying drawings in the embodiment of the present invention. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. Example 1

[0032] 1. The method of this embodiment includes the following:

[0033] This example establishes a methodologically validated UPLC-TQD-SIR method for the simultaneous determination of 14 components in Guyin Decoction samples, including rehmannoside D, morroniside, loganin, polygala quinone III, liquiritin, hyperoside, verbascoside, 3',6-dicerinoylsucrose, ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1, polygala tenuifolia saponin, glycyrrhizic acid, and schisandrin A. This example also performs cluster analysis and multivariate statistical analysis on the content determination results of the above components in 15 batches of Guyin Decoction samples. The details are as follows:

[0034] 2. Materials and Methods

[0035] 2.1 Reagents and Materials

[0036] Chemical standards of loganin, polygala tenuifolia ketone III, glycyrrhizin, hyperoside, ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1, polygala tenuifolia saponin, and schisandrae alcohol A were purchased from the Beijing Institute of Food and Drug Control, China; morroniside, verbascoside, and 3',6-dicansinoylsucrose were provided by Chengdu Ruifen Biotechnology Co., Ltd. (Chengdu, China); rehmannoside D was purchased from Chengdu Ruifen Biotechnology Co., Ltd. (Chengdu, China); and glycyrrhizic acid was purchased from Sichuan Weikeqi Biotechnology Co., Ltd. (Chengdu, China). The purity and batch number (or product number) are shown in Table 1. The purity of the above standards can be used for content determination studies. The structures of the 14 compounds are as follows: Figure 1 As shown. HPLC-grade methanol and acetonitrile were provided by Fisher Scientific (Fair Lawn, NJ, USA), and chromatographic-grade formic acid was purchased from Tianjin Komiou Chemical Reagent Co., Ltd. (Tianjin, China). The water used in the entire process was prepared by a Unique-R202 multifunctional ultrapure water system (Xiamen, China). Other chemicals and solvents were of analytical grade. The Chinese herbal medicines used to prepare 15 batches of Guyinjian samples (S1-S15) were all from authentic production areas and main production areas. The origin and batch number information are shown in Table 2. All decoction pieces comply with the provisions of Section 1 of the Chinese Herbal Medicine Decoction Pieces of the Pharmacopoeia of the People's Republic of China (2020 Edition), including properties, identification, inspection, content determination, and processing.

[0037] Table 1 Purity and batch number information of each standard product

[0038]

[0039] Table 2 Origin and batch number information of the ingredients of 15 batches of Guyin Decoction reference samples (S1-S15)

[0040]

[0041] 2.2 Preparation of standard solution

[0042] The preparation of the mixed standard stock solution (in 50%wt methanol) contained rehmannoside D (1), morroniside (2), loganin (3), polygala tenuifolia ketone III (4), liquiritin (5), hyperoside (6), verbascoside (7), 3',6-dicercosylsucrose (8), ginsenoside Re (9), ginsenoside Rg1 (10), ginsenoside Rb1 (11), polygala tenuifolia saponin (12), glycyrrhizic acid (13), and schisandrin A (14) at concentrations of 20.410µg / ml (1), 98.450µg / ml (2), respectively. l(2), 52.975µg / ml(3), 1.9650µg / ml(4), 24.063µg / ml(5), 29.888µg / ml(6), 4.7850µg / ml(7), 33.575µg / ml(8), 5.3675µg / ml(9), 2.9850µg / ml(10), 6.7000µg / ml(11), 3.5380µg / ml(12), 37.300µg / ml(13), 8.9600µg / ml(14). The mixed standard solution was diluted with 50wt% methanol to obtain working solutions of different concentrations: 0.4082-10.205µg / ml (1); 1.9690-49.225µg / ml (2); 1.0595-26.488µg / ml (3); 0.0393-0.9825µg / ml (4); 0.4813-12.031µg / ml (5); 0.5978-14.944µg / ml (6); 0.0957-2.39 25µg / ml(7); 0.6715-16.788µg / ml(8); 0.1074-2.6838µg / ml(9); 0.0597-1.4925µg / ml(10); 0.1340-3.3500µg / ml(11); 0.0708-1.7690µg / ml(12); 0.7460-18.650µg / ml(13); 0.1792-4.4800µg / ml(14). All standard solutions were stored at 4℃ and filtered with a 0.22µm filter membrane before injection.

[0043] 2.3 Preparation of sample solution

[0044] Daily dose of Guyin Decoction: Take the daily dose of Guyin Decoction pieces (7.46g of ginseng, 14.92g of Rehmannia root, 7.46g of stir-fried yam, 5.60g of Cornus officinalis, 2.61g of processed Polygala tenuifolia, 5.60g of roasted Licorice root, 2.00g of Schisandra chinensis, and 9.33g of stir-fried Cuscuta australis), put them in a Supor decoction casserole, add 400mL of water, soak for 60min, use a Joyoung electric ceramic stove to boil for 10min on high heat (1800W), then use low heat (400W) to decoct for 75min, filter the liquid while hot (120-mesh filter cloth), and get about 280ml of decoction. The decoction was then frozen and rotated in a low-temperature anhydrous ethanol bath (-60°C) to evenly spread the inner wall of the freeze-dried bottle until it was completely solid. It was then stored at -80°C for 24 hours and freeze-dried in a freeze dryer for 18 hours to obtain the corresponding substance of the Guyin Decoction benchmark sample (the freeze-dried powder was a yellow-brown loose powder with a fragrant smell and a slightly sweet taste that turned bitter). Accurately weigh 0.4g of freeze-dried powder, place it in a 50ml conical bottle with a stopper, accurately add 50ml of 50%wt methanol solution, weigh it, and ultrasonically extract it for 45min (250W, 40KHz). After ultrasonic treatment, place it at room temperature, weigh it again, make up for the weight loss with 50% methanol solution, and take an appropriate amount of extract to pass through a 0.22μm microporous membrane for UPLC-MS analysis.

[0045] 2.4 Equipment and conditions

[0046] All samples were analyzed using UPLC (Waters Acquity™ UPLC, USA) coupled with a triple quadrupole mass spectrometer (Waters Synapt TM TQD, USA). Separation was performed using an ACQUITY UPLC HSS T3 column (2.1×100 mm, 1.8 µm) (Waters, USA). The mobile phase consisted of acetonitrile + 0.1% formic acid (A) and water + 0.1% formic acid (B) at a flow rate of 0.4 mL / min. The column temperature was 35 °C and the injection volume was 1 µL. The gradient elution in the positive ion mode was as follows: 0-3 min 55-55% A, 3-5 min 55-99% A, and the re-equilibrium time was 4 min; the gradient elution in the negative ion mode was as follows: 0-2 min 5-15% A, 2-6 min 15-15% A, 6-15 min 15-50% A, 15-18 min 50-99% A, and the re-equilibrium time was 5 min.

[0047] ESI-MS spectra were acquired in SIR mode in positive and negative ion modes. The ESI-MS analysis conditions were as follows: capillary voltage: 3 kv / -3 kv; cone voltage: 30 v / -30 v; extraction voltage: 3 v / -3 v; desolvation temperature: 350 °C; desolvation gas flow rate: 650 L / hr; cone gas flow rate: 50 L / hr; ion source temperature: 150 °C; Masslynx V4.2 software (Waters, USA) was used for data acquisition and processing. XSE105 dual-range analytical balance (Mettler Toledo, Switzerland); MODULYO freeze dryer (Thermo Fisher, USA); pipettes (0.5ml, 1ml, 2ml, 2.5ml, 3ml, 4ml, 5ml, BRAND, Germany); KQ-250DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., China); TB18A1 Supor soup casserole (Zhejiang Supor Co., Ltd., China); H22-X1 Joyoung electric ceramic stove (Joyoung Co., Ltd., China).

[0048] 2.5 Method Validation

[0049] Specificity, instrument precision, linearity, repeatability, intermediate precision, stability and accuracy were studied in the method validation of this embodiment. Each negative control solution was prepared according to the method for preparing the specificity study sample solution in 2.3. After the instrument was collected, the chromatograms of the standard and the solid Yin decoction samples were compared and analyzed to observe whether there were other chromatographic peaks interfering with the determination of specific components in the sample by this method, and to evaluate whether the method specificity met the determination requirements. Relative standard deviation (RSD) was used to measure precision, stability and repeatability. Instrument precision was calculated by continuously collecting six mixed standard solutions and calculating the RSD% of the peak area of ​​each component. For the calibration curve, six different concentrations of the standard solution were analyzed in triplicate. The calibration curve was obtained by plotting the peak area and concentration of each compound. To confirm repeatability, the same sample was collected and analyzed six times. The operation of the intermediate precision experiment was the same as the repeatability experiment, and the operator and operation date were changed. The intermediate precision experiment results and the repeatability results were combined to calculate the RSD%, indicating whether the precision of the method met the content determination requirements. For stability testing, the same samples were kept in the sample chamber and obtained by repeated analysis at 0, 2, 4, 6, 8, 10 and 12 hours. A recovery test was performed to evaluate the accuracy of the method. A known amount of a standard (6 samples in parallel) was added to a certain amount of sample, and then the samples were extracted and analyzed according to the established method. The recovery of each compound was calculated by the following formula: Recovery = (detected amount - original amount) / spiked amount × 100%.

[0050] 2.5.1 Optimization of sample extraction conditions

[0051] In order to obtain the best quantitative extraction, the variables involved in the extraction process, such as the extraction method, extraction solvent, extraction time, and sampling volume, were optimized. The extraction methods included ultrasonic extraction and heating reflux. The extraction efficiency of each component under different extraction conditions was compared. It was found that the ultrasonic extraction and heating reflux extraction effects were not much different, so the more convenient ultrasonic extraction method was selected. The extraction solvents examined were 50% methanol, 75% methanol, and 100% methanol. The content of most components did not change much under the conditions of 50% methanol and 75% methanol, but because rehmannia glutinosa glycoside D has good water solubility and is an important indicator component of the monarch drug Rehmannia glutinosa, 50% methanol was selected. The extraction time was examined for 15min, 30min, 45min, and 60min, respectively, and it was found that the content of each compound was the highest under the condition of 45min of ultrasound. The extraction solvent was fixed at 50ml, and the sampling volume was 0.2g, 0.4g, and 0.8g, respectively. The unit mass extraction results of 0.2g and 0.4g were slightly different, and 0.2g was better, but considering that the sampling volume was too small, the measurement error would increase. In summary, the final sample extraction conditions were to accurately weigh 0.4 g, accurately add 50 ml of 50% methanol, and perform ultrasonic extraction for 45 min.

[0052] The optimization of the above sample extraction conditions took the Guyin Decoction sample S1 group as the experimental sample. The single factor experimental results under different extraction conditions are shown in Table 3:

[0053] Table 3 Contents of various substances in the Guyin Decoction reference sample (S1) under different extraction conditions

[0054]

[0055] The extraction conditions used for the control in Table 1 are the optimal extraction conditions of the present invention in 2.3 above, and all the detection conditions in Table 1 are the optimal equipment and conditions of the present invention in 2.4 above.

[0056] 2.5.2 Optimization of UPLC-MS / MS conditions

[0057] The sample solution was first subjected to full scan analysis in MS Scan mode. It was found that among the 14 monitored components, only Schisandrin A had no response in negative ion mode, so Schisandrin A was selected for positive ion mode monitoring, and the remaining 13 compounds had good response values ​​in negative ion mode. The SIR parameters of the 14 compounds were optimized individually to achieve the highest sensitivity and resolution. The optimal cone voltage was determined by comparing the peak area of ​​each compound under different cone voltages. Taking Schisandrin as an example, the cone voltages were set to 20v, 25v, 30v, 35v, 40v and 45v, respectively. It was found that the peak area of ​​the compound first increased and then decreased with the increase of cone voltage, and the peak area was the largest when the cone voltage of Schisandrin was 35v. The summary results of the optimal cone voltage are shown in Table 4. In order to obtain a chromatogram with good separation effect, the elution gradient was investigated. After optimization, Schisandrin A appeared in 2.07min in positive ion mode, and the 13 components were successfully separated in 14min in negative ion mode. Then the column temperature (30℃, 35℃, 40℃) was investigated, and the chromatograms were compared. Figure 4 As shown), flow rate (0.3ml / min, 0.4ml / min, 0.5ml / min, compare the chromatogram as shown Figure 5 As shown), injection volume (5μL, 2μL, 1μL, compare the chromatogram as shown Figure 6 The separation effect was best when the column temperature was 35°C, the flow rate was 0.4ml / min, and the injection volume was 1µL. The representative chromatogram is shown in Figure 2 shown.

[0058] Table 4 Summary of SIR mode mass spectrometry parameters of 14 compounds

[0059]

[0060] 2.5.3 Validation of the UP LC-MS method

[0061] The results of the specificity experiment showed that the method was used for the determination of 14 components such as rehmannoside D, loganin, and morroniside, without interference from other compounds, and had good specificity. By analyzing a series of concentration standard solutions, the calibration curves of each component were drawn by peak area (y) and concentration (x: in µg / ml), and it was found that each component showed a good linear regression relationship within the linear range (r=0.9960-0.9999). The results of the instrument precision survey showed that the peak area RSD% of each component was less than or equal to 3.49%, indicating that the precision of the instrument used in this study met the experimental requirements. Repeatability, intermediate precision and the comprehensive calculation of the two showed that the RSD% of each component was between 0.68% and 3.18%, proving that the method established above has better precision. The results of the stability survey showed that each compound was relatively stable within 12 hours of storage in the sample chamber (RSD%≤2.64%). The accuracy test showed that the average recovery rate of each component was 88.64%-107.43%, and the RSD% was 2.82%-6.22%, which met the recovery rate requirements of the Pharmacopoeia of the People's Republic of China (2020 edition). All summary results are listed in Table 5.

[0062] Table 5 Summary of UP LC-MS method validation results

[0063]

[0064] 2.5.4 Simultaneous Quantification of Fourteen Compounds in Guyin Decoction Samples

[0065] The contents of the above 14 key compounds in 15 batches of Guyinjian samples were calculated using the newly established method and the accompanying mixed standard method. Two parallel samples were prepared for each batch, and each sample was collected twice. The content results calculated using the peak area and concentration ratio are shown in Table 6. Obviously, due to the source of the medicinal materials, some components such as rehmannia glutinosa glycoside D, polygala quinone III, liquiritin, verbascoside, ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1 and glycyrrhizic acid have large differences in content between batches. Morroniside, loganin, hyperoside, 3',6-dicansinoylsucrose, hyperoside, and schisandrin A are relatively stable between batches.

[0066] Table 6 Content determination results of 15 batches of samples of Guyin Decoction

[0067]

[0068] 2.6 Cluster analysis and multivariate statistical analysis

[0069] Cluster analysis is often used for preliminary exploratory analysis of data, which can make data conclusions more concise and intuitive. Principal component analysis (PCA) is an algorithm for simplifying data sets, often used to visualize similarities or differences in multivariate data, which is an unsupervised pattern recognition technique. Orthogonal partial least squares-discriminant analysis (OPLS-DA) is a supervised model that reduces the dimensionality of data and helps to screen differential variables that contribute significantly to grouping. The content results of 14 key components in 15 batches of Guyinjian samples (S1-S15) determined by the above method were imported into Metware Cloud (online data analysis platform, https: / / cloud.metware.cn / # / tools / tool-form?toolId=169) for advanced cluster analysis, advanced PCA and OPLS-DA analysis.

[0070] 2.6.1 Quality evaluation of cluster analysis and multivariate statistical analysis

[0071] The cluster heat map analysis results are as follows Figure 3 (A) As shown. The darker the color of the heat map, the higher the content of each component in the corresponding batch. The 15 batches of samples were divided into 2 batches, batches 1-5 as one group and batches 6-15 as another group. Batches 1-5, 6-10 and 11-15, every 5 batches of medicinal materials came from the same origin. From the classification results, the difference in the content of the target compound is related to the quality difference of medicinal materials from different origins. PCA is used to study the relationship or trend of similarities or differences between these samples. The clustering and dispersion of the samples can be observed from the score graph, such as Figure 3 As shown in (B), the results of PCA analysis are consistent with those of cluster analysis. Due to the different origins of the formula medicinal materials, the contents of some ingredients vary greatly, resulting in poor stability of Guyin Decoction samples, suggesting that the origin of the medicinal materials can be controlled or mixed batches of medicinal materials from different origins can be tried during the preparation process. Further OPLS-DA analysis was performed to obtain the VIP (Variable Importance for the Projection) value ranking results of the ingredients that have a greater impact on quality between different batches, as shown in Figure 3 As shown in (C, D), the top five compounds (VIP>1) are rehmannoside D, loganin, morroniside, ginsenoside Re and 3',6-dicansinoylsucrose. These five components are derived from Rehmannia glutinosa, Cornus officinalis, Ginseng and Polygala tenuifolia, indicating that the quality stability of these four medicinal materials has a great influence on the stability of Guyin decoction, which deserves further attention.

[0072] The present invention has developed for the first time an analytical method for simultaneous quantitative analysis of multiple components of Guyin Decoction based on UPLC-TQD-SIR technology. The method has good specificity, linearity, precision, stability and accuracy as verified by methodological research. At the same time, the present invention uses cluster analysis and PCA analysis to conduct a preliminary exploration of the content determination results of 15 batches of Guyin Decoction samples, and finds that due to the different origins of the medicinal materials of the prescription, there are large differences in the content of some components, resulting in poor sample stability, suggesting that in the preparation production process, it is possible to try to control the origin of the medicinal materials or mix and input medicinal materials from different origins. OPLS-DA analysis found that the components that have a greater impact on batch stability mainly come from rehmannia glutinosa, dogwood, ginseng and polygala root. , ginsenoside D, loganin, morroniside, ginsenoside Re and 3', 6-dicansinoyl sucrose. In summary, the simultaneous quantitative determination method of Guyin Decoction established by the present invention has the advantages of high sensitivity, high selectivity, rapid analysis, etc., and can provide a reference for the quality control research and development research of Guyin Decoction.

[0073] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for determining the content of multiple components of Guyin Jian based on UPLC-MS, characterized in that: This method uses UPLC-TQD-SIR technology to simultaneously detect the contents of 14 components in Guyin Decoction, including rehmannoside D, morroniside, loganin, polygala tenuifolia ketone III, liquiritin, hyperoside, verbascoside, 3',6-dicerinoylsucrose, ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1, polygala tenuifolia saponin, glycyrrhizic acid, and schisandrin A. The method comprises the following steps: Preparation of S1 standard solution: methanol solution was used as solvent to prepare a mixed standard solution containing 14 components; Preparation of S2 sample solution: first weigh ginseng, prepared rehmannia root, stir-fried yam, cornus officinalis, processed polygala root, roasted licorice root, schisandra chinensis, and stir-fried dodder seed according to the prescription of Guyin decoction to prepare Guyin decoction freeze-dried powder, then add a predetermined amount of methanol solution to the Guyin decoction freeze-dried powder, perform ultrasonic extraction, and obtain a sample solution; S3 uses UPLC and triple quadrupole mass spectrometry system to detect standard solutions and sample solutions, and then uses ESI-MS spectroscopy to analyze and obtain the contents of 14 components; The conditions of the UPLC and triple quadrupole mass spectrometry system in this method are as follows: the UPLC model is Waters Acquity TM UPLC, the separation of each solution uses ACQUITY UPLC HSS T3 chromatographic column, the model of the chromatographic column is: 2.1×100mm, 1.8µm; the mobile phase A used in the system is: acetonitrile + 0.1% formic acid, the mobile phase B is: water + 0.1% formic acid, the flow rate is 0.4 mL / min, the column temperature is 35℃, and the injection volume is 1µL; the gradient elution in the positive ion mode is as follows: 0-3min 55-55%A, 3-5 min 55-99%A, and the re-equilibrium time is 4min; the gradient elution in the negative ion mode is as follows: 0-2min 5-15%A, 2-6min 15-15%A, 6-15min15-50%A, 15-18min 50-99%A, and the re-equilibrium time is 5min.

2. The method for determining the content of multiple components of Guyin Jian based on UPLC-MS according to claim 1, characterized in that: The concentration of the methanol solution used in the preparation of the standard solution in step S1 and the preparation of the sample solution in step S2 is 50 wt %.

3. The method for determining the content of multiple components of Guyin Jian based on UPLC-MS according to claim 1, characterized in that: Step S1: Preparation of standard solution: using methanol solution to prepare a mixed standard stock solution and then prepare a mixed standard solution. The concentrations of rehmannia glutinosa D, morroniside, loganin, polygala tenuifolia ketone III, liquiritin, hyperoside, verbascoside, 3',6-dicerinoylsucrose, ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1, polygala tenuifolia saponin, glycyrrhizic acid, and schisandrin A in the mixed standard stock solution are 20.410 µg / ml, 98.450 µg / ml, 52.975 µg / ml, and 1.965 0µg / ml, 24.063µg / ml, 29.888µg / ml, 4.7850µg / ml, 33.575µg / ml, 5.3675µg / ml, 2.9850µg / ml, 6.7000µg / ml, 3.5380µg / ml, 37.300µg / ml, 8.9600µg / ml; the mixed standard solution includes working solutions of different concentrations prepared by dilution with methanol solution, and the working solutions of different concentrations contain rehmannoside D, morroniside, loganin, and polygala tenuifolia ketone III, the concentration ranges of liquiritin, hyperoside, verbascoside, 3',6-diesinapoylsucrose, ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1, polygala tenuifolia saponin, glycyrrhizic acid, and schisandrae alcohol A were 0.4082-10.205µg / ml, 1.9690-49.225µg / ml, 1.0595-26.488µg / ml, 0.0393-0.9825µg / ml, 0.4813-12.031µg / ml, and 0.5978-14 .944µg / ml, 0.0957-2.3925µg / ml, 0.6715-16.788µg / ml, 0.1074-2.6838µg / ml, 0.0597-1.4925µg / ml, 0.1340-3.3500µg / ml, 0.0708-1.7690µg / ml, 0.7460-18.650µg / ml, 0.1792-4.4800µg / ml; each solution was filtered with a 0.22µm filter membrane before sampling and detection.

4. The method for determining the content of multiple components of Guyin Jian based on UPLC-MS according to claim 1, characterized in that: Step S2: Preparation of sample solution. The contents of the ingredients in the Guyin Decoction prescription are 7.46 parts by weight of ginseng, 14.92 parts by weight of Rehmannia root, 7.46 parts by weight of stir-fried yam, 5.60 parts by weight of Cornus officinalis, 2.61 parts by weight of processed Polygala tenuifolia, 5.60 parts by weight of roasted Licorice root, 2.00 parts by weight of Schisandra chinensis, and 9.33 parts by weight of stir-fried Cuscuta australis.

5. The method for determining the content of multiple components of Guyin Jian based on UPLC-MS according to claim 1, characterized in that: In step S2, the freeze-dried powder is prepared by soaking the ingredients of the Guyin Decoction in water and then boiling them, filtering them and freeze-drying them.

6. The method for determining the content of multiple components of Guyin Jian based on UPLC-MS according to claim 1, characterized in that: In step S2, the ultrasonic conditions during ultrasonic extraction are 250W, 40KHz, and the ultrasonic time is 45min. The sample solution is passed through a 0.22μm microporous membrane before sampling and detection.

7. The method for determining the content of multiple components of Guyin Jian based on UPLC-MS according to claim 1, characterized in that: In step S3, the ESI-MS spectrum was obtained in SIR mode in positive and negative ion modes; the ESI-MS analysis conditions were as follows: capillary voltage: 3kv / -3kv; cone voltage: 30v / -30v; extraction voltage: 3v / -3v; desolvation temperature: 350°C; desolvation gas flow rate: 650L / hr; cone gas flow rate: 50L / hr; ion source temperature: 150°C; MasslynxV4.2 software (Waters, USA) was used for data acquisition and processing.

8. The method for determining the content of multiple components of Guyin Jian based on UPLC-MS according to claim 1, characterized in that: The standard curves of the substances in this method are: rehmannia glycoside D y=1856.2x+162.47, r=0.9999; morroniside y=5663.3x+9521, r=0.9999; loganin y=5837.6x+3430.9, r=0.9994; polygala tenuifolia ketone III y=12217x+139.22, r=0.9999; Liquorice glycoside y=11344x+3901.7, r=0.9988; Hyperoside y=11878x+9390.3, r=0.9960; Verbascoside y=19206x+1084.2, r=0.9993; 3',6-dicansinoyl y=8154.2x+2317.8, r=0.9997; Ginsenoside Re y=12765x+325.7, r=0.9998; Ginsenoside Rg1 y=16044x+175.01, r=0.9998; Ginsenoside Rb1 y=15886x+203.89, r=0.9999; Polygala tenuifolia saponin y=24931x+1218.6, r=0.9988; Glycyrrhizic acid y=41117x+28611, r=0.9986; Schisandrin A y=46610x+26670, r=0.9966.

9. Application of the method for determining the content of multiple components of Guyin Decoction based on UPLC-MS according to any one of claims 1 to 8, characterized in that: This method was used to simultaneously detect the contents of 14 components in Guyin Decoction, including rehmannoside D, morroniside, loganin, polygala tenuifolia ketone III, liquiritigenin, hyperoside, verbascoside, 3',6-diesinapoylsucrose, ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1, polygala tenuifolia saponins, glycyrrhizic acid, and schisandrin A.

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