Method for simultaneously determining multiple important components in heart-nourishing and palpitation-calming capsule based on LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry)

Abstract

The invention relates to the technical field of medicine quality control, in particular to a method for simultaneously determining multiple important components in a heart-nourishing and palpitation-calming capsule based on LC-MS / MS. The method can be used for synchronously determining the contents of five components such as liquiritigenin, liquiritin, rehmannia glutinosa liboside D, ginsenoside Re and ginsenoside Rg1 in the heart-nourishing and palpitation-calming capsule. In the detection process, extraction conditions, chromatographic conditions, ionization modes and the like are optimized, so that chromatographic peaks of LC-MS / MS in the detection method are good in linear relation (r is greater than or equal to 0.9992) in respective ranges, the average sample adding recovery rate is 98.91%-100.55%, and RSD is 1.37%-1.78%. The methodological investigation proves that the method disclosed by the invention is good in repeatability and stability, has the characteristics of accuracy, sensitivity, strong specificity and the like, and can be used for controlling the quality of the heart-nourishing and palpitation-calming capsules.

Description

Technical Field

[0001] The invention relates to the technical field of drug quality control, and in particular to a method for simultaneously determining multiple important components in Yangxin Dingji Capsules based on LC-MS / MS. Background Art

[0002] Yangxin Dingji Capsule is a modified version of the classic prescription "Zhi Gancao Decoction". It has the effects of nourishing blood and replenishing qi, restoring pulse and calming palpitations. It is used to treat arrhythmia, palpitations, qi deficiency and blood deficiency, etc. It is widely used in the clinic to treat various types of arrhythmias and their complications, and can effectively improve symptoms and cardiac function, etc. It has the characteristics of few adverse reactions and high safety. The drug composition of Yangxin Dingji Capsule includes Zhi Gancao, Shengdihuang, Red Ginseng, Guizhi, Ophiopogon japonicus, ginger, donkey-hide gelatin, jujube, and black sesame. Among them, Zhi Gancao is the main drug, which plays a major therapeutic role. It can replenish qi and restore pulse, and has a significant effect in the clinical treatment of arrhythmias. Shengdihuang is used in the largest amount, which has the function of nourishing heart yin and nourishing heart blood, and can strengthen the heart and increase myocardial blood flow. Red ginseng, which is also a ministerial drug, has the function of greatly replenishing vital energy and benefiting heart qi, and can protect cardiovascular, improve myocardial structure, prevent and treat myocardial ischemia and other pharmacological effects. Therefore, Zhi Gancao, Shengdihuang, and Red Ginseng are the core medicinal components in Yangxin Dingji Capsule.

[0003] Studies have shown that licorice in licorice has a certain effect on right heart remodeling in PAH rats; licorice glycoside has antioxidant, anti-inflammatory, and myocardial damage, and has a significant protective effect on important organs such as the human heart and brain. It can also reduce pressure overload-induced myocardial hypertrophy in mice and AngⅡ-induced myocardial cell hypertrophy. At the same time, licorice glycoside and licorice glycoside can synergistically inhibit myocardial fibrosis. Rehmannia glutinosa glycoside D in Rehmannia glutinosa nourishes yin and blood, and can significantly improve the blood index of mice with yin deficiency and blood deficiency models. Ginsenoside Re in red ginseng can improve mitochondrial function, enhance the antioxidant stress capacity of H9c2 cells, and reduce H9c2 cell apoptosis; ginsenoside Rg1 regulates autophagy through the miR-155 / Notch1 / Hes1 pathway to reduce H / R injury in HL-1 cardiomyocytes; both can increase the expression of tight junction proteins and inhibit the mRNA expression of genes related to inflammation and apoptosis. In summary, the pharmacological activities of the above five components, including glycyrrhizin, glycyrrhizin, rehmannia glutinosa D, ginsenoside Re and ginsenoside Rg1, are closely related to the clinical application of Yangxin Dingji Capsules. Their contents are the key factors in evaluating the quality of the medicinal components of roasted licorice, raw rehmannia root and red ginseng, and are also an important factor in evaluating the efficacy of Yangxin Dingji Capsules.

[0004] At present, the research on Yangxin Dingji Capsule is mainly focused on clinical research, lacking quantitative analysis and quality control of its ingredients. There is no report in the prior art on the chemical composition detection method of Chinese herbal compound preparations with similar formula to Yangxin Dingji Capsule, and even if the compound preparations with similar formulas are combined with medicinal materials, the proportion of medicinal materials is adjusted, and the preparation process is changed, the content of the active ingredients in the preparations will be different. Therefore, the detection method of specific chemical components in compound preparations with similar formulas cannot be applied to Yangxin Dingji Capsule; and the detection of components in simple medicinal materials cannot be applied to compound preparations with complex ingredients.

[0005] LC-MS / MS is liquid chromatography-tandem mass spectrometry, a tool used in analytical chemistry to separate, quantitatively and qualitatively analyze components in complex mixtures. It combines the ability of liquid chromatography (LC) to separate mixtures with the ability of mass spectrometry (MS) to detect and analyze the structure of individual compounds. The present invention is intended to establish an LC-MS / MS method to simultaneously determine the contents of liquiritigenin, liquiritin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in Yangxin Dingji Capsules, so as to effectively evaluate the quality of Yangxin Dingji Capsules and conduct quality control. Summary of the invention

[0006] 1. Technical issues to be resolved In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a method for simultaneously determining multiple important components in Yangxin Dingji Capsules based on LC-MS / MS. The method can synchronously and quantitatively determine the contents of five important chemical components in Yangxin Dingji Capsules, namely, liquiritigenin, liquiritigenin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1. The mobile phase used in the method can obtain chromatographic peaks with good peak shape and high separation. The detection method is accurate, sensitive, and highly specific, and can be used for quality control of Yangxin Dingji Capsules.

[0007] (II) Technical solution A method for simultaneously determining multiple important components in Yangxin Dingji Capsule based on LC-MS / MS, comprising the following steps: S1. Prepare the working solution of the reference substance Weigh appropriate amounts of rehmannia glutinosa D, ginsenoside Re, ginsenoside Rg1, liquiritigenin, and liquiritigenin reference substance, accurately weigh, dissolve in pure methanol, and vortex to mix, to prepare 5 stock solutions respectively; Each stock solution was diluted stepwise with 0.1% formic acid in methanol to obtain rehmannia glutinosa D working solution, ginsenoside Re working solution, ginsenoside Rg1 working solution, liquiritigenin working solution, and liquiritin working solution with a series of gradient concentrations, respectively; S2, the working solution of rehmannia glutinosa D, the working solution of ginsenoside Re, the working solution of ginsenoside Rg1, the working solution of liquiritigenin, and the working solution of liquiritigenin with a series of gradient concentrations are tested on a machine according to the following chromatographic conditions and mass spectrometry conditions, and the peak area is calculated by the mass spectrum target component intensity of the target component within the retention time of the target component, and thereby a working curve of the mass concentration and peak area of ​​the reference substance is obtained; Chromatographic conditions: Chromatographic column: C18 chromatographic column; column temperature 40℃; mobile phase: A is 0.1% formic acid-1mM ammonium acetate aqueous solution, B is acetonitrile; the gradient elution program includes positive ion gradient elution mode and negative ion gradient elution mode, and the positive ion gradient elution mode is switched to the negative ion gradient elution mode after the positive ion gradient elution mode ends; in the positive ion elution mode, the injection volume is 5μL and the volume flow rate is 0.3mL / min; in the negative ion elution mode, the injection volume is 2μL and the volume flow rate is 0.4mL / min; The positive ion gradient elution mode was: 0-0.1min, 5% B; 0.1-2.0min, 30% B; 2.0-4.0min, 65% B; 4.0-6.0min, 65% B; 6.0-6.01min, 5% B; 6.01-8.0min, 5% B; the negative ion gradient elution mode was: 0-0.1min, 15% B; 0.1-3.0min, 30% B; 3.0-4.0min, 65% B; 4.0-5.0min, 65% B; 5.0-5.01min, 15% B; 5.01-9.0min, 15% B; Mass spectrometry conditions: electrospray ionization source ESI, multiple reaction ion monitoring (MRM) scanning mode, electrospray voltage positive ion mode 5500V, negative ion mode -4500V; the gas used was dry nitrogen; S3. Take a sample of Yangxin Dingji Capsule, accurately weigh it, dissolve it in 50% methanol, vortex mix it, centrifuge it, take the supernatant, and prepare a sample solution with a mass concentration of 20-100 mg / mL. After filtering through the membrane, the test sample is obtained; the test sample is tested on the machine according to the detection conditions of S2 to obtain the peak area of ​​the test sample, and the content of glycyrrhizin, glycyrrhizin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in the test sample is obtained according to the working curve in S2.

[0008] According to a preferred embodiment of the present invention, in S2, in the mass spectrometry conditions, the curtain gas, nebulizing gas (atomizer gas) and heating gas (drying gas) are 35 psi, 55 psi and 55 psi respectively; the dry nitrogen temperature is 500°C; the collision gas flow rate is 0.4L / min; and the vacuum interface temperature is 150°C.

[0009] According to a preferred embodiment of the present invention, in S2, the chromatographic retention times of the components to be tested, including liquiritigenin, liquiritigenin, rehmannoside D, ginsenoside Re, and ginsenoside Rg1, are: 4.242 min for liquiritigenin, 2.489 min for liquiritigenin, 1.405 min for rehmannoside D, 4.087 min for ginsenoside Re, and 4.094 min for ginsenoside Rg1.

[0010] According to a preferred embodiment of the present invention, in S2, the mass spectrometry parameters of the five components to be tested, namely, liquiritigenin, liquiritigenin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1, are as follows: .

[0011] According to a preferred embodiment of the present invention, the method for preparing the working fluid in S1 comprises: The stock solution of rehmannia glycoside D was diluted stepwise with 0.1% formic acid in methanol to prepare working solutions with mass concentrations of 200 μg / mL, 150 μg / mL, 100 μg / mL, 50 μg / mL, 20 μg / mL, 10 μg / mL, 4 μg / mL, and 2 μg / mL of rehmannia glycoside D; The stock solutions of ginsenoside Re, ginsenoside Rg1, liquiritigenin, and liquiritin were respectively diluted stepwise with 0.1% formic acid in methanol to prepare working solutions with gradient mass concentrations of 100 μg / mL, 75 μg / mL, 50 μg / mL, 25 μg / mL, 10 μg / mL, 5 μg / mL, 2 μg / mL, and 1 μg / mL.

[0012] According to a preferred embodiment of the present invention, in S3, a sample of Yangxin Dingji Capsule is dissolved in 50% methanol, vortexed and mixed, centrifuged, and the supernatant is taken to prepare a sample solution with a mass concentration of 50 mg / mL.

[0013] According to a preferred embodiment of the present invention, in S2, during the mass spectrometry detection process, the quantitative ion of glycyrrhizin is [M-CH2OH-C6H3O2] - ; The quantitative ion of liquiritin is [M-CH2OH-C 12 H 12 O6] - ; The quantitative ion of rehmannia glycoside D is [M+HC 18 H 33 O 16 -2H2O] + ; The quantitative ion of ginsenoside Re is [M+HCOO-C2H6O-C6H 10 O5] - ; [M+HCOO-C2H6O-2(C6H 10 O5)] - .

[0014] (III) Beneficial effects The present invention synchronously determines the contents of liquiritigenin, liquiritin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in Yangxin Dingji capsules by liquid chromatography-tandem mass spectrometry. The five components have a good linear relationship within their respective ranges (r≥0.9992), with an average sample recovery rate of 98.91%-100.55% and an RSD of 1.37%-1.78%. Methodological investigations have shown that the quantitative method of the present invention has good repeatability and stability, and has the characteristics of accuracy, sensitivity, strong specificity, etc., and can be used for quality control of Yangxin Dingji capsules.

[0015] The present invention does not require pretreatment techniques such as solid phase extraction (SPE) and liquid-liquid extraction (LLE) to purify the target components of the sample to be tested. The steps are simple and the detection is fast. The determination can be completed within 5 minutes, and the analysis time is short. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 LC-MS / MS chromatograms of liquiritigenin, liquiritigenin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in the reference and test samples. DETAILED DESCRIPTION

[0017] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below in conjunction with the accompanying drawings through specific embodiments. In the following embodiments and comparative examples, unless otherwise specified, the mobile phase composition refers to volume percentage; the composition of the mixed solution refers to volume percentage, such as 50% methanol means that the volume ratio of methanol to water is 1:1; 0.1% formic acid in methanol solution means that the volume ratio of formic acid to methanol is 0.1:99.9.

[0018] The sources of instruments or materials, reagents and drugs in the following embodiments and comparative examples are as follows: Instruments or materials: High performance liquid chromatograph (CIRUI, Chengdu); 3200 QTRAP triple quadrupole linear ion trap mass spectrometer (SCIEX, USA); electronic analytical balance (Sartorius, Germany); vortex mixer (MX-F, Wuhan Savier Biotechnology Co., Ltd.).

[0019] Reagents and drugs: Yangxin Dingji Capsules (Hebei Yongfeng Pharmaceutical Co., Ltd., batch numbers 06724030141, 06724030241, 06724030341); liquiritigenin, liquiritin, rehmannia glutinosa D, ginsenoside Re, ginsenoside Rg1 (batch numbers AB0563-0020, AB0619-0020, AB0199-0020, AB0700-0020, AB1275-0020, purity all 98%); acetonitrile, formic acid, and ammonium acetate were chromatographically pure (Fisher Chemicals, USA); the rest were analytically pure; water was ultrapure water.

[0020] Example 1 This embodiment is a method for simultaneously determining multiple important components in Yangxin Dingji Capsules based on LC-MS / MS, and the contents of liquiritigenin, liquiritin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in Yangxin Dingji Capsules are simultaneously determined by liquid chromatography-tandem mass spectrometry. The specific detection steps are as follows: (1) Preparation of reference substance working solution Weigh an appropriate amount of rehmannia glycoside D reference substance, accurately weigh it, dissolve it in pure methanol, vortex mix it, prepare a stock solution with a mass concentration of 2 mg / mL, and store it at 4°C for later use. Weigh an appropriate amount of ginsenoside Re, ginsenoside Rg1, liquiritigenin, and liquiritin reference substance, accurately weigh it, dissolve it in pure methanol, vortex mix it, prepare a stock solution with a mass concentration of 1 mg / mL, and store it at 4°C for later use.

[0021] Take an appropriate amount of the stock solution of rehmannia glycoside D, dilute it step by step with 0.1% formic acid in methanol solution, and prepare working solutions with mass concentrations of 200μg / mL, 150μg / mL, 100μg / mL, 50μg / mL, 20μg / mL, 10μg / mL, 4μg / mL, and 2μg / mL of rehmannia glycoside D. Take an appropriate amount of the stock solution of ginsenoside Re, ginsenoside Rg1, liquiritigenin, and liquiritin, respectively, and dilute it step by step with 0.1% formic acid in methanol solution to obtain working solutions with gradient mass concentrations of 100μg / mL, 75μg / mL, 50μg / mL, 25μg / mL, 10μg / mL, 5μg / mL, 2μg / mL, and 1μg / mL.

[0022] (2) Preparation of test samples Weigh an appropriate amount of Yangxin Dingji Capsule sample, accurately weigh it, dissolve it in 50% methanol, vortex mix it, centrifuge it, take the supernatant, and finally prepare a sample stock solution with a mass concentration of 50 mg / mL (based on the solid mass of the Yangxin Dingji Capsule sample). After filtering through the membrane, the test sample was obtained.

[0023] (3) The working solutions of rehmannia glutinosa D, ginsenoside Re, ginsenoside Rg1, liquiritigenin and liquiritin with a series of gradient concentrations were tested on the machine under the following chromatographic and mass spectrometry conditions. The testing conditions are as follows: ①Chromatographic conditions: Chromatographic column: C18 chromatographic column; column temperature 40℃; mobile phase: A is 0.1% formic acid-1mM ammonium acetate aqueous solution, B is acetonitrile. 0.1% formic acid-1mM ammonium acetate aqueous solution means adding 0.1% formic acid and 1mM ammonium acetate to the aqueous solution.

[0024] Since the target components are ginsenoside Re and ginsenoside Rg1, which belong to the saponins; liquiritigenin and liquiritin belong to the flavonoids, both of which are compounds with relatively low polarity, a C18 chromatographic column was selected.

[0025] The gradient elution program is shown in Table 1, including positive ion gradient elution mode and negative ion gradient elution mode. After the positive ion gradient elution mode is completed, it is switched to the negative ion gradient elution mode. In the positive ion elution mode, the injection volume is 5 μL and the volume flow rate is 0.3 mL / min; in the negative ion elution mode, the injection volume is 2 μL and the volume flow rate is 0.4 mL / min.

[0026] Table 1: Gradient elution program .

[0027] ②Mass spectrometry conditions The ion source was an electrospray ionization source (ESI), and the multiple reaction ion monitoring (MRM) scanning mode was adopted. The electrospray voltage was 5500 V in positive ion mode and -4500 V in negative ion mode. The curtain gas, nebulizer gas and heating gas were 35 psi, 55 psi and 55 psi respectively, and all the gases used were dry nitrogen. The retention time and mass spectrometry parameters of the components to be measured in the chromatogram are shown in Table 2.

[0028] Table 2: Retention time and mass spectrometry parameters of each component in the chromatogram

[0029] Note: In the above table, represents quantitative ion, and # represents qualitative ion.

[0030] During the detection process, the target compound was confirmed by the qualitative ion, and the peak area was calculated according to the mass spectrometry signal intensity of the quantitative ion within the retention time corresponding to the target compound. The peak area corresponding to the different concentrations of each reference substance was calculated, and regression was performed with the mass concentration of the reference substance as the abscissa (X) and the peak area as the ordinate (Y). The working curves of the mass concentration and peak area of ​​the five reference substances are shown in Table 3.

[0031] Table 3: Linear relationship between mass concentration and peak area of ​​5 reference substances

[0032] As shown in Table 3, the regression equation of each component has r≥0.9992. r usually refers to the Pearson correlation coefficient. The closer r is to 1, the better the linear relationship of positive correlation. Therefore, the linear relationship of each target component is good within its respective range.

[0033] (4) The test sample prepared in step (2) is tested on the machine according to the injection detection conditions of the reference working solution in step (3). During the detection process, the target compound is confirmed by the qualitative ion, and the peak area of ​​the test sample is calculated by the mass spectrometry signal intensity of the quantitative ion within the retention time corresponding to the target compound. And by comparing the working curves of the mass concentration and peak area of ​​the five reference samples, substituting the peak area of ​​the test sample, the concentrations of glycyrrhizin, glycyrrhizin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in the test sample are obtained respectively. Then, according to the preparation concentration of the capsule contents during the preparation of the test sample (50 mg / mL in this embodiment), the contents of glycyrrhizin, glycyrrhizin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in the Yangxin Dingji Capsule sample are further converted (μg / mg). Figure 1 As shown, A1 and B1 correspond to the chromatographic peak of glycyrrhizin obtained by the on-machine detection of glycyrrhizin (1 μg / mL) reference substance working solution and 50 mg / L test sample (RT=4.242 min); A2 and B2 correspond to the chromatographic peak of glycyrrhizin obtained by the on-machine detection of glycyrrhizin (1 μg / mL) reference substance working solution and 50 mg / L test sample (RT=2.489 min); A3 and B3 correspond to the chromatographic peak of glycyrrhizin D obtained by the on-machine detection of glycyrrhizin (2 μg / mL) reference substance working solution and 50 mg / L test sample (RT=1.405 min); A4 and B4 correspond to the chromatographic peak of ginsenoside Re obtained by the on-machine detection of ginsenoside Re (1 μg / mL) reference substance working solution and 50 mg / L test sample (RT=4.087 min); A5 and B5 correspond to the chromatographic peak of ginsenoside Rg1 obtained after the working solution of ginsenoside Rg1 (1μg / mL) and 50mg / L test sample were tested on the machine (RT=4.094 min). Figure 1 It can be seen that the chromatographic peaks of liquiritigenin, liquiritin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in the test sample have good peak shapes and each component can be well separated without interference from other components in the capsule. This shows that the extraction conditions (i.e., the preparation methods of the reference sample and the test sample) and the injection detection conditions (chromatographic detection conditions and mass spectrometry detection conditions) designed by the present invention are reasonable and have good specificity.

[0034] Example 2 Take an appropriate amount of the stock solution of rehmannoside D and dilute it to 50 μg / mL with a 0.1% formic acid methanol solution. Take an appropriate amount of the stock solution of ginsenoside Re, ginsenoside Rg1, liquiritigenin, and liquiritin and dilute them to 25 μg / mL with a 0.1% formic acid methanol solution. According to the on-machine detection conditions of step (3) of Example 1, the sample was injected and measured 6 times. The peak area RSDs of liquiritigenin, liquiritigenin, rehmannoside D, ginsenoside Re, and ginsenoside Rg1 were measured to be 1.08%, 1.66%, 1.87%, 1.24%, and 1.26%, respectively, indicating that the instrument precision was good.

[0035] Example 3 Six samples of Yangxin Dingji Capsules (batch number 06724030141) were taken and prepared as test samples according to the preparation method of the test sample in Example 1. The test samples were tested on the machine according to the detection conditions of step (3) of Example 1. The RSDs of the contents of glycyrrhizin, glycyrrhizin, rehmannia glutinosa D, ginsenoside Re and ginsenoside Rg1 were 1.56%, 1.43%, 1.82%, 1.11% and 1.36%, respectively, indicating that the method had good repeatability.

[0036] Example 4 Take 6 portions of the Yangxin Dingji Capsule sample (batch number 06724030141) with known contents of each component, add appropriate amount of reference solution (2.480 μg / mg of liquiritigenin, μg / mg of liquiritin, 4.015 μg / mg of ginsenoside Re, 2.252 μg / mg of ginsenoside RG1, and 5.020 μg / mg of rehmannia glutinosa D) according to the proportion, and inject and measure according to the detection conditions of step (3) of Example 1, and calculate the recovery rate. Results The average recovery rates (RSD) of liquiritigenin, liquiritigenin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 were 99.62% (1.78%), 100.55% (1.58%), 100.28% (1.45%), 100.32% (1.37%), and 98.91% (1.47%), respectively.

[0037] Example 5 Three batches of Yangxin Dingji Capsule samples (06724030141, 06724030241, 06724030341) were taken, with 3 portions in each batch, and the samples were injected and measured according to the on-machine detection conditions of step (3) of Example 1. The average content of each batch was calculated. The results are shown in Table 4.

[0038] Table 4: Content test results of liquiritigenin, liquiritin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in three batches of samples (μg / mg, n=3)

[0039] It can be seen from the above table that the contents of glycyrrhizin, glycyrrhizin, rehmannia glutinosa D, ginsenoside Re and ginsenoside Rg1 in the three batches of Yangxin Dingji Capsule samples are relatively stable.

[0040] Example 6 Weigh an appropriate amount of Yangxin Dingji capsule sample, accurately weigh it, dissolve it in 50% methanol, vortex mix it, centrifuge it, take the supernatant, and finally prepare a sample reserve solution with a mass concentration of 50 mg / mL. After filtering the membrane, obtain the test sample. Take an appropriate amount of the test sample, place it for 0, 2h, 4h, 8h, 12h, and 24h, and then inject and test it according to the detection conditions of step (3) of Example 1. The peak area RSDs of liquiritigenin, liquiritin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in the samples placed for different times are 1.34%, 1.71%, 1.53%, 1.95%, and 1.88%, respectively, indicating that the test sample has good stability within 24 hours. In the actual production and sample inspection process, the method of the present invention has applicability.

[0041] Comparative Example 1 The conditions for preparing the reference substance stock solution in Example 1 were changed to: using 50% methanol (50% water), 70% methanol (30% water), and 100% methanol to dissolve a symmetrical amount of the reference substance, and mixing by ultrasound and vortex. When preparing the reference substance working solution, 50% methanol and 100% methanol solutions were used for stepwise dilution to prepare a series of working solutions with gradient mass concentrations. When preparing the test sample, 50% methanol (50% water), 70% methanol (30% water), and 100% methanol were used to dissolve a symmetrical amount of the contents of the Yangxin Dingji capsule.

[0042] By comparing with Example 1, it is found that the efficiency of vortex mixing is higher than that of ultrasound; when pure methanol is used to prepare the reference substance, the peak number of the reference substance chromatographic peak is stable and the peak shape is better; compared with 100% or 50% methanol dilution, when 0.1% formic acid methanol solution is used to dilute the gradient mass concentration working solution step by step, the peak number of the reference substance chromatographic peak is more stable and the peak shape is better. In addition, compared with using 70% methanol and 100% methanol to prepare the test sample, when 50% methanol is used for dissolution and extraction, the peak number of the test sample chromatographic peak is stable and the peak shape is better.

[0043] Comparative Example 2 In this comparative example, the parent ions of saponins Re and Rg1 in Table 2 of Example 1 were selected as positive ions and the mass spectrometry signal response value was low in the positive ion mode. Therefore, the negative ion mode was adjusted, that is, the initial [M+H] + Change to [M+HCOO] - After (see Example 1), the mass spectrometer response value increases.

[0044] Comparative Example 3 This comparative example is in the negative ion elution mode in Example 1, and the mobile phase acetonitrile-water (0.1% formic acid-1mM ammonium acetate) in Example 1 is changed to: methanol-water (containing 0.1% formic acid, 5mM ammonium formate). The results show that under this elution condition, the chromatographic peaks of glycyrrhizin and glycyrrhizin are not well separated, and problems such as tailing and low response value occur. Even by trying to adjust the plateau time, the gradient organic phase ratio, the organic phase increase rate, etc. cannot be separated, and the peak shape is worse. Therefore, if the organic phase acetonitrile of the mobile phase in Example 1 is replaced with methanol, a chromatographic peak with high separation and good peak shape cannot be obtained. In Example 1, B is acetonitrile, and A is 0.1% formic acid-1mM ammonium acetate aqueous solution, which can separate the components well in the liquid chromatography process, and the peak shape is better.

[0045] Comparative Example 4 In this comparative example, in the positive ion elution mode of Example 1, the mobile phase composition was 90% A + 10% B at 0-0.1 min using the positive ion gradient elution mode. As a result, a double peak appeared in the chromatographic peak of the test product Rehmannia glutinosa D. After the B in the initial mobile phase was changed to 5%, the test product Rehmannia glutinosa D appeared as follows Figure 1 Therefore, the starting organic phase in the positive ion elution mode was 5%.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features therein may be replaced by equivalents. These modifications or replacements, or the technical features in the above embodiments may be combined in the manner described in the embodiments if they do not conflict with each other, and these modifications, replacements or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for simultaneously determining multiple important components in Yangxin Dingji Capsules based on LC-MS / MS, wherein the contents of liquiritigenin, liquiritin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in Yangxin Dingji Capsules are simultaneously determined by liquid chromatography-tandem mass spectrometry, characterized in that: The following steps are involved: S1. Prepare the working solution of the reference substance Weigh appropriate amounts of rehmannia glutinosa D, ginsenoside Re, ginsenoside Rg1, liquiritigenin, and liquiritigenin reference substance, accurately weigh, dissolve in pure methanol, and vortex to mix, to prepare 5 stock solutions respectively; Each stock solution was diluted stepwise with 0.1% formic acid in methanol to obtain rehmannia glutinosa D working solution, ginsenoside Re working solution, ginsenoside Rg1 working solution, liquiritigenin working solution, and liquiritin working solution with a series of gradient concentrations, respectively; S2, the rehmannia glutinosa D working solution, ginsenoside Re working solution, ginsenoside Rg1 working solution, liquiritigenin working solution, and liquiritin working solution with a series of gradient concentrations are tested on a machine according to the following chromatographic conditions and mass spectrometry conditions, and the peak area is calculated according to the mass spectrometry signal intensity of the target component within the retention time of the target component, and thereby a working curve of the reference substance mass concentration and peak area is obtained; Chromatographic conditions: Chromatographic column: C18 chromatographic column; column temperature 40°C; Mobile phase: A is 0.1% formic acid-1mM ammonium acetate aqueous solution, B is acetonitrile; the gradient elution program includes positive ion gradient elution mode and negative ion gradient elution mode, and the positive ion gradient elution mode is switched to the negative ion gradient elution mode after the positive ion gradient elution mode ends; in the positive ion elution mode, the injection volume is 5μL and the volume flow rate is 0.3mL / min; in the negative ion elution mode, the injection volume is 2μL and the volume flow rate is 0.4mL / min; The positive ion gradient elution mode was: 0-0.1 min, 5% B; 0.1-2.0 min, 30% B; 2.0-4.0min, 65%B; 4.0-6.0min, 65%B; 6.0-6.01min, 5%B; 6.01-8.0min, 5%B; negative ion gradient elution mode: 0-0.1min, 15%B; 0.1-3.0min, 30%B; 3.0-4.0min, 65%B; 4.0-5.0min, 65%B; 5.0-5.01min, 15%B; 5.01-9.0min, 15%B; Mass spectrometry conditions: electrospray ionization source ESI, multiple reaction ion monitoring (MRM) scanning mode, electrospray voltage positive ion mode 5500 V, negative ion mode -4500 V; the gas used was dry nitrogen; S3. Take a sample of Yangxin Dingji Capsule, accurately weigh it, dissolve it in 50% methanol, vortex mix it, centrifuge it, take the supernatant, and prepare a sample solution with a mass concentration of 20-100 mg / mL. After filtering through the membrane, the test sample is obtained; the test sample is tested on the machine according to the detection conditions of S2 to obtain the peak area of ​​the test sample, and the content of glycyrrhizin, glycyrrhizin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in the test sample is obtained according to the working curve in S2.

2. The method according to claim 1, characterized in that In S2, in the mass spectrometry conditions, the curtain gas, nebulizer gas and heating gas were 35 psi, 55 psi and 55 psi respectively; the dry nitrogen temperature was 500°C; the collision gas flow rate was 0.4 L / min; and the vacuum interface temperature was 150°C.

3. The method according to claim 1, characterized in that In S2, the chromatographic retention times of the components to be tested, including liquiritigenin, liquiritin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1, are: Licoricein 4.242min, liquiritin 2.489min, rehmannia glutinosa D 1.405min, ginsenoside Re 4.087min, ginsenoside Rg1 4.094min.

4. The method according to claim 1, characterized in that: In S2, the mass spectrometry parameters of the components to be tested, including liquiritigenin, liquiritin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1, are as follows: 。 5. The method according to claim 1, characterized in that In S3, a sample of Yangxin Dingji Capsule was dissolved in 50% methanol, vortexed and mixed, centrifuged, and the supernatant was collected to prepare a sample solution with a mass concentration of 50 mg / mL.

6. The method according to claim 1, characterized in that In S1, the method for preparing the working solution includes: taking a stock solution of rehmannia glycoside D, diluting it step by step with a 0.1% formic acid methanol solution to prepare a rehmannia glycoside D working solution with a gradient mass concentration of 200 μg / mL, 150 μg / mL, 100 μg / mL, 50 μg / mL, 20 μg / mL, 10 μg / mL, 4 μg / mL, and 2 μg / mL; The stock solutions of ginsenoside Re, ginsenoside Rg1, liquiritigenin, and liquiritin were respectively diluted stepwise with 0.1% formic acid in methanol to prepare ginsenoside Re working solutions, ginsenoside Rg1 working solutions, liquiritigenin working solutions, and liquiritin working solutions with gradient mass concentrations of 100 μg / mL, 75 μg / mL, 50 μg / mL, 25 μg / mL, 10 μg / mL, 5 μg / mL, 2 μg / mL, and 1 μg / mL.

7. The method according to claim 4, characterized in that In S2, during mass spectrometry detection, the quantitative ion of glycyrrhizin is [M-CH2OH-C6H3O2] - ; The quantitative ion of liquiritin is [M-CH2OH-C 12 H 12 O6] - ; The quantitative ion of rehmannia glycoside D is [M+HC 18 H 33 O 16 -2H2O] + ; The quantitative ion of ginsenoside Re is [M+HCOO-C2H6O-C6H 10 O5] - ; [M+HCOO-C2H6O-2(C6H 10 O5)] - .

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