A method for simultaneously determining multiple important components in Yangxin Dingji capsules based on LC-MS / MS

Abstract

The present application relates to the technical field of drug quality control, in particular to a method for simultaneously determining multiple important components in Yangxin Dingji Capsules based on LC-MS / MS, which can simultaneously determine the contents of five components in Yangxin Dingji Capsules, including glycyrrhizin, glycyrrhizin, rehmatrionoside D, ginsenoside Re and ginsenoside Rg1, etc. In the detection process, the extraction conditions, chromatographic conditions and ionization mode are optimized, so that the chromatographic peaks of the detection method LC-MS / MS have good linear relationship in their respective ranges (r >= 0.9992), the average sample recovery rate is 98.91% to 100.55%, and the RSD is 1.37% to 1.78%. The methodological investigation proves that the method has good repeatability and stability, is accurate, sensitive, specific and has other characteristics, and can be used for the quality control of Yangxin Dingji Capsules.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical quality control technology, specifically to a method for simultaneously determining multiple important components in Yangxin Dingji capsules based on LC-MS / MS. Background Technology

[0002] The Yangxin Dingji Capsules are derived from the classic formula "Zhi Gan Cao Tang" (Prepared Licorice Decoction), which nourishes blood and qi, restores pulse, and calms palpitations. It is used to treat arrhythmia, palpitations, and qi and blood deficiency. Clinically, it is widely used to treat various types of arrhythmias and their complications, effectively improving symptoms and cardiac function, and is characterized by few adverse reactions and high safety. The Yangxin Dingji Capsules consist of prepared licorice root, rehmannia root, red ginseng, cinnamon twig, ophiopogon root, ginger, donkey-hide gelatin, jujube, and black sesame. Prepared licorice root is the principal herb, playing a major therapeutic role, invigorating qi and restoring pulse, and has a significant clinical effect in treating arrhythmias. Rehmannia root is used in the largest quantity, nourishing heart yin and heart blood, strengthening the heart, and increasing myocardial blood flow. Red ginseng, also an assistant herb, greatly replenishes vital energy and benefits heart qi, protecting the cardiovascular system, improving myocardial structure, and preventing myocardial ischemia. Therefore, prepared licorice root, rehmannia root, and red ginseng are the core medicinal components of the Yangxin Dingji Capsules.

[0003] Studies have shown that glycyrrhizin in roasted licorice has a certain effect on right ventricular remodeling in PAH rats; glycyrrhizin has antioxidant, anti-inflammatory, and myocardial damage-improving effects, and has a significant protective effect on important organs such as the heart and brain in humans. It can also reduce stress overload-induced myocardial hypertrophy and Ang II-induced cardiomyocyte hypertrophy in mice. At the same time, glycyrrhizin and glycyrrhizin can synergistically inhibit myocardial fibrosis. Rehmannia glutinosa contains rehmannia glycoside D, which nourishes yin and replenishes blood, and can significantly improve blood indices in yin-deficient and blood-deficient model mice. 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 damage in HL-1 cardiomyocytes; both can increase the expression of tight junction proteins and inhibit the mRNA expression of inflammation and apoptosis-related genes. In summary, the pharmacological activities of the five components mentioned above—glycyrrhizin, glycyrrhizin, rehmannia glycoside D, ginsenoside Re, and ginsenoside Rg1—are closely linked to the clinical application of Yangxin Dingji Capsules. Their content is a key element in evaluating the quality of the components of prepared licorice, raw rehmannia, and red ginseng, and is also an important factor in evaluating the efficacy of Yangxin Dingji Capsules.

[0004] Currently, research on Yangxin Dingji Capsules mainly focuses on clinical studies, lacking quantitative analysis and quality control of its components. Existing technologies also lack methods for detecting the chemical components of traditional Chinese medicine compound preparations with similar formulations to Yangxin Dingji Capsules. Furthermore, even compound preparations with similar formulations can exhibit differences in the content of active ingredients due to variations in the combination of medicinal materials, adjustments in the proportion of medicinal materials, and changes in the preparation process. Therefore, methods for detecting specific chemical components in compound preparations with similar formulations are not applicable to Yangxin Dingji Capsules; and the detection of components solely from medicinal materials is even less suitable for complex compound preparations.

[0005] LC-MS / MS, or Liquid Chromatography-Tandem Mass Spectrometry, is a tool in analytical chemistry used to separate, quantify, 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 resolve individual compounds. This invention aims to establish an LC-MS / MS method for the simultaneous determination of glycyrrhizin, glycyrrhizin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in Yangxin Dingji Capsules, thereby effectively assessing and controlling the quality of Yangxin Dingji Capsules. Summary of the Invention

[0006] (a) Technical problems to be solved In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a method for simultaneous determination of multiple important components in Yangxin Dingji capsules based on LC-MS / MS. This method can simultaneously and quantitatively determine the content of five important chemical components in Yangxin Dingji capsules, namely glycyrrhizin, glycyrrhizin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1. The mobile phase used in this method can obtain chromatographic peaks with good peak shape and high resolution. The detection method is accurate, sensitive, and specific, and can be used for the quality control of Yangxin Dingji capsules.

[0007] (II) Technical Solution A method for simultaneous determination of multiple important components in Yangxin Dingji capsules based on LC-MS / MS includes the following steps: S1. Working solution for preparing the reference standard Weigh appropriate amounts of rehmannia glycoside D, ginsenoside Re, ginsenoside Rg1, glycyrrhizin, and glycyrrhizin reference standards, accurately determine their amounts, dissolve them in pure methanol, vortex and mix well to prepare five stock solutions. Take each stock solution and dilute it stepwise with a methanol solution of 0.1% formic acid to obtain working solutions of rehmannia glycoside D, ginsenoside Re, ginsenoside Rg1, glycyrrhizin, and glycyrrhizin with a series of gradient concentrations. S2. The working solutions of rehmannia glycoside D, ginsenoside Re, ginsenoside Rg1, glycyrrhizin, and glycyrrhizin with a series of gradient concentrations are detected by the chromatographic and mass spectrometric conditions described below. The peak area is calculated based on the mass spectrometric intensity of the target component within the retention time of the target component, and the working curve of reference concentration versus peak area is obtained. Chromatographic conditions: Column: C18 column; Column temperature: 40℃; Mobile phase: A is 0.1% formic acid-1mM ammonium acetate aqueous solution, B is acetonitrile; Gradient elution program includes positive ion gradient elution mode and negative ion gradient elution mode, switching to negative ion gradient elution mode after the positive ion gradient elution mode is completed; In positive ion elution mode, the injection volume is 5μL and the flow rate is 0.3mL / min; In negative ion elution mode, the injection volume is 2μL and the flow rate is 0.4mL / min; The positive ion gradient elution modes are: 0-0.1 min, 5% B; 0.1-2.0 min, 30% B; 2.0-4.0 min, 65% B; 4.0-6.0 min, 65% B; 6.0-6.01 min, 5% B; 6.01-8.0 min, 5% B; and the negative ion gradient elution modes are: 0-0.1 min, 15% B; 0.1-3.0 min, 30% B; 3.0-4.0 min, 65% B; 4.0-5.0 min, 65% B; 5.0-5.01 min, 15% B; and 5.01-9.0 min, 15% B. Mass spectrometry conditions: Electrospray ionization source (ESI), multiple reaction monitoring (MRM) scanning mode, electrospray voltage 5500V for positive ion mode and -4500V for negative ion mode; dry nitrogen gas was used. S3. Take a sample of Yangxin Dingji Capsules, weigh it accurately, dissolve it in 50% methanol, vortex to mix, centrifuge, take the supernatant, and prepare a sample solution with a mass concentration of 20-100 mg / mL. After filtering through a membrane, obtain the test sample. Detect the test sample according to the detection conditions in S2, obtain the peak area of ​​the test sample, and obtain the content of glycyrrhizin, glycyrrhizin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in the test sample according to the working curve in S2.

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

[0009] According to a preferred embodiment of the present invention, in S2, the chromatographic retention times of the test components glycyrrhizin, glycyrrhizin, rehmannia glycoside D, ginsenoside Re, and ginsenoside Rg1 are as follows: glycyrrhizin 4.242 min, glycyrrhizin 2.489 min, rehmannia glycoside D 1.405 min, ginsenoside Re 4.087 min, and ginsenoside Rg1 4.094 min.

[0010] According to a preferred embodiment of the present invention, in S2, the mass spectrometry parameters of the five analytes—glycyrrhizin, glycyrrhizin, 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 solution in S1 includes: Take the stock solution of rehmannia glycoside D and dilute it stepwise with 0.1% formic acid in methanol to prepare working solutions with rehmannia glycoside D 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; Stock solutions of ginsenoside Re, ginsenoside Rg1, glycyrrhizin, and glycyrrhizin were taken separately and serially diluted 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, the sample of Yangxin Dingji Capsules is dissolved in 50% methanol, vortexed and mixed, centrifuged, and the supernatant is collected to obtain 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 glycyrrhizin 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] - ; Ginsenoside Rg1 [M+HCOO-C2H6O-2(C6H 10 O5)] - .

[0014] (III) Beneficial Effects This invention utilizes liquid chromatography-tandem mass spectrometry (LC-MS / MS) to simultaneously determine the contents of glycyrrhizin, glycyrrhizin, rehmannia glycoside D, ginsenoside Re, and ginsenoside Rg1 in Yangxin Dingji capsules. These five components exhibited good linearity within their respective ranges (r ≥ 0.9992), with average recoveries ranging from 98.91% to 100.55% and RSDs from 1.37% to 1.78%. Methodological studies have demonstrated that the quantitative method of this invention exhibits good repeatability and stability, and is accurate, sensitive, and specific, making it suitable for the quality control of Yangxin Dingji capsules.

[0015] This invention eliminates the need for pretreatment techniques such as solid-phase extraction (SPE) and liquid-liquid extraction (LLE) to purify the target components of the sample. The procedure is simple and the detection is rapid, with the determination completed within 5 minutes, resulting in a short analysis time. Attached Figure Description

[0016] Figure 1 The LC-MS / MS chromatograms of glycyrrhizin, glycyrrhizin, rehmannia glycoside D, ginsenoside Re, and ginsenoside Rg1 in the reference standard and the test sample are shown. Detailed Implementation

[0017] To better explain and facilitate understanding of the present invention, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the composition of the mobile phase in the following embodiments and comparative examples refers to volume percentage; the composition of the mixed solution refers to volume percentage, such as 50% methanol meaning a methanol-to-water volume ratio of 1:1; and a 0.1% formic acid methanol solution meaning a formic acid-to-methanol volume ratio of 0.1:99.9.

[0018] The sources of instruments or materials, reagents, and drugs in the following examples 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 Saiwell Biotechnology Co., Ltd.).

[0019] Reagents and drugs: Yangxin Dingji Capsules (Hebei Yongfeng Pharmaceutical Co., Ltd., batch numbers 06724030141, 06724030241, 06724030341); glycyrrhizin, glycyrrhizin, rehmannia glutinosa D, ginsenoside Re, ginsenoside Rg1 (batch numbers AB0563-0020, AB0619-0020, AB0199-0020, AB0700-0020, AB1275-0020, all with a purity of 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 describes a method for the simultaneous determination of multiple important components in Yangxin Dingji capsules based on LC-MS / MS. The content of glycyrrhizin, glycyrrhizin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in Yangxin Dingji capsules is determined simultaneously by liquid chromatography-tandem mass spectrometry. The specific detection steps are as follows: (1) Preparation of reference standard working solution Accurately weigh an appropriate amount of rehmannia glycoside D reference standard, dissolve it in pure methanol, vortex to mix, and prepare a stock solution with a mass concentration of 2 mg / mL. Store at 4℃ for later use. Accurately weigh appropriate amounts of ginsenoside Re, ginsenoside Rg1, glycyrrhizin, and glycyrrhizin reference standards, dissolve them in pure methanol, vortex to mix, and prepare a stock solution with a mass concentration of 1 mg / mL. Store at 4℃ for later use.

[0021] Take an appropriate amount of stock solution of rehmannia glycoside D and serially dilute it with 0.1% formic acid in methanol to prepare working solutions with rehmannia glycoside D 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. Take appropriate amounts of stock solutions of ginsenoside Re, ginsenoside Rg1, glycyrrhizin, and glycyrrhizin, respectively, and serially dilute them with 0.1% formic acid in methanol to obtain working solutions with gradient 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.

[0022] (2) Preparation of test sample 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 Yangxin Dingji Capsule sample). After filtering through a membrane, the test sample is obtained.

[0023] (3) The working solutions of rehmannia glycoside D, ginsenoside Re, ginsenoside Rg1, glycyrrhizin, and glycyrrhizin with a series of gradient concentrations were analyzed by chromatography and mass spectrometry under the following conditions. The analysis conditions are as follows: ①Chromatographic conditions: Column: C18 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 refers to an aqueous solution with 0.1% formic acid and 1mM ammonium acetate added.

[0024] Since the target components are ginsenoside Re and ginsenoside Rg1, which belong to the saponin class, and glycyrrhizin and glycyrrhizin, which belong to the flavonoid class, both of which are compounds with low polarity, a C18 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 switches to the negative ion gradient elution mode. In the positive ion elution mode, the injection volume is 5 μL and the flow rate is 0.3 mL / min; in the negative ion elution mode, the injection volume is 2 μL and the flow rate is 0.4 mL / min.

[0026] Table 1: Gradient elution procedure .

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

[0028] Table 2: Retention times and mass spectrometry parameters of each component in chromatography

[0029] Note: In the above table, # indicates quantitative ions, and # indicates qualitative ions.

[0030] During the detection process, the target compound was identified by qualitative ions. The peak area was calculated by the mass spectrometry signal intensity of the quantitative ions within the retention time corresponding to the target compound. The peak area corresponding to different concentrations of each reference standard was then calculated. Regression was performed with the reference standard mass concentration 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 standards are shown in Table 3.

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

[0032] As shown in Table 3, the regression equations for each component have r ≥ 0.9992. r usually refers to the Pearson correlation coefficient; the closer r is to 1, the better the positive linear relationship. Therefore, each target component exhibits a good linear relationship within its respective range.

[0033] (4) The test sample prepared in step (2) is tested on the instrument according to the injection and detection conditions of the reference working solution in step (3). During the detection process, the target compound is identified by qualitative ions, and the peak area of ​​the test sample is calculated by the mass spectrometry signal intensity of quantitative ions within the retention time corresponding to the target compound. By comparing the working curves of the mass concentration and peak area of ​​the five reference standards, the peak area of ​​the test sample is substituted to obtain the concentrations of glycyrrhizin, glycyrrhizin, rehmannia glycoside D, ginsenoside Re, and ginsenoside Rg1 in the test sample. Then, based on the concentration of the capsule contents during the preparation of the test sample (50 mg / mL in this example), the contents (μg / mg) of glycyrrhizin, glycyrrhizin, rehmannia glycoside D, ginsenoside Re, and ginsenoside Rg1 in the Yangxin Dingji capsule sample are further calculated. Figure 1 As shown, A1 and B1 correspond to the chromatographic peaks of glycyrrhizin (1 μg / mL) and 50 mg / L test sample obtained by instrumental analysis (RT=4.242 min); A2 and B2 correspond to the chromatographic peaks of glycyrrhizin (1 μg / mL) and 50 mg / L test sample obtained by instrumental analysis (RT=2.489 min); A3 and B3 correspond to the chromatographic peaks of rehmannia glutinosa D (2 μg / mL) and 50 mg / L test sample obtained by instrumental analysis (RT=1.405 min); A4 and B4 correspond to the chromatographic peaks of ginsenoside Re (1 μg / mL) and 50 mg / L test sample obtained by instrumental analysis (RT=4.087 min). (min); A5 and B5 correspond to the chromatographic peaks of ginsenoside Rg1 (RT=4.094 min) obtained after instrumental detection of the working solution of ginsenoside Rg1 (1 μg / mL) and the test sample of 50 mg / L, respectively. Figure 1 It can be seen that the chromatographic peaks of glycyrrhizin, glycyrrhizin, rehmannia glycoside D, ginsenoside Re, and ginsenoside Rg1 in the test sample have good peak shapes and the components can be well separated without interference from other components in the capsule. This indicates that the extraction conditions (i.e., the preparation methods of the reference standard and the test sample) and the injection and detection conditions (chromatographic and mass spectrometric detection conditions) designed in this invention are reasonable and have good specificity.

[0034] Example 2 Take an appropriate amount of the stock solution of rehmannia glycoside D and dilute it to 50 μg / mL with a methanol solution of 0.1% formic acid. Take appropriate amounts of the stock solutions of ginsenoside Re, ginsenoside Rg1, glycyrrhizin, and glycyrrhizin respectively and dilute them to 25 μg / mL with a methanol solution of 0.1% formic acid. Perform the instrument detection six times according to the detection conditions in step (3) of Example 1. The peak area RSDs of glycyrrhizin, glycyrrhizin, rehmannia glycoside D, ginsenoside Re, and ginsenoside Rg1 were 1.08%, 1.66%, 1.87%, 1.24%, and 1.26%, respectively, indicating that the instrument has good precision.

[0035] Example 3 Six samples of Yangxin Dingji Capsules (batch number 06724030141) were taken and prepared according to the preparation method of the test sample in Example 1. The test samples were then tested according to the detection conditions in 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 has good repeatability.

[0036] Example 4 Six portions of the contents of Yangxin Dingji Capsules (batch number 06724030141) with known content of each component were taken, and appropriate amounts of reference solutions were added according to the proportions (2.480 μg / mg for glycyrrhizin, μg / mg for glycyrrhizin, 4.015 μg / mg for ginsenoside Re, 2.252 μg / mg for ginsenoside RG1, and 5.020 μg / mg for rehmannia glutinosa D). The samples were injected and tested according to the instrument detection conditions in step (3) of Example 1, and the recovery rate was calculated. The average recovery rates (RSD) of glycyrrhizin, glycyrrhizin, 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 three samples from each batch. The samples were injected and tested according to the instrument detection conditions in step (3) of Example 1. The average content of each batch was calculated, and the results are shown in Table 4.

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

[0039] As shown in the table above, the contents of glycyrrhizin, glycyrrhizin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in the three batches of Yangxin Dingji capsules were relatively stable.

[0040] Example 6 Weigh an appropriate amount of Yangxin Dingji capsule sample, accurately weigh it, dissolve it in 50% methanol, vortex mix, centrifuge, and collect the supernatant to obtain a sample stock solution with a mass concentration of 50 mg / mL. After filtration, the test sample is obtained. Take an appropriate amount of the test sample and place it for 0, 2 h, 4 h, 8 h, 12 h, and 24 h. Then, inject the sample for detection according to the detection conditions in step (3) of Example 1. The peak areas RSD of glycyrrhizin, glycyrrhizin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in the samples placed for different times were measured to be 1.34%, 1.71%, 1.53%, 1.95%, and 1.88%, respectively, indicating that the test sample has good stability within 24 h. The method of the present invention is applicable in actual production and sample testing.

[0041] Comparative Example 1 The conditions for preparing the reference stock solution in Example 1 were modified as follows: the reference standard was dissolved in equal amounts of 50% methanol (50% water), 70% methanol (30% water), and 100% methanol, and mixed using both ultrasonic and vortex mixing methods. When preparing the working solution, a series of working solutions with varying concentrations were prepared by stepwise dilution with 50% methanol and 100% methanol solutions. When preparing the test sample, the contents of the Yangxin Dingji capsules were dissolved in equal amounts of 50% methanol (50% water), 70% methanol (30% water), and 100% methanol.

[0042] Compared with Example 1, vortex mixing was found to be more efficient than ultrasonic mixing. When preparing the reference standard with pure methanol, the number of chromatographic peaks was more stable and the peak shape was better. Compared to dilution with 100% or 50% methanol, using a stepwise dilution with a methanol solution containing 0.1% formic acid to prepare working solutions of varying concentrations resulted in more stable peak numbers and better peak shapes. Furthermore, compared to preparing the test sample with 70% or 100% methanol, using 50% methanol for dissolution and extraction resulted in more stable peak numbers and better peak shapes.

[0043] Comparative Example 2 In this comparative example, the precursor ions of ginsenosides Re and Rg1 in Table 2 of Example 1 were selected as positive ions, and initial spectrometry was performed in positive ion mode, resulting in a low mass spectrometry signal response. Therefore, the mode was adjusted to negative ion mode, i.e., from the initial [M+H]... + Change to [M+HCOO] - Afterwards (see Example 1), the mass spectrometry response value increased.

[0044] Comparative Example 3 This comparative example uses the negative ion elution mode of Example 1, but with the mobile phase acetonitrile-water (0.1% formic acid-1mM ammonium acetate) changed to methanol-water (containing 0.1% formic acid and 5mM ammonium formate). The results show that under these elution conditions, the chromatographic peaks of glycyrrhizin and glycyrrhizin are poorly separated, exhibiting tailing and low response values. Even after adjusting the plateau time, gradient organic phase ratio, and organic phase increment rate, separation remains impossible, and the peak shapes are even worse. Therefore, replacing the acetonitrile organic phase in Example 1 with methanol will not yield high resolution and good peak shapes. In contrast, in Example 1, B is acetonitrile, and A is a 0.1% formic acid-1mM ammonium acetate aqueous solution, which effectively separates the components during liquid chromatography with better peak shapes.

[0045] Comparative Example 4 This comparative example, in Example 1, used a positive ion gradient elution mode with a mobile phase composition of 90% A + 10% B during the 0-0.1 min phase. The result showed a double peak in the chromatographic peak of the test sample, rehmannia glycoside D. After changing B to 5% in the initial mobile phase, the test sample, rehmannia glycoside D, showed the following... Figure 1 The peak shape of A3 is shown. Therefore, the starting organic phase for the positive ion elution mode is 5%.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions 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 simultaneous determination of multiple important components in Yangxin Dingji capsules based on LC-MS / MS, wherein the contents of glycyrrhizin, glycyrrhizin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in Yangxin Dingji capsules are simultaneously determined by liquid chromatography-tandem mass spectrometry, characterized in that... Includes the following steps: S1. Working solution for preparing the reference standard Weigh appropriate amounts of rehmannia glycoside D, ginsenoside Re, ginsenoside Rg1, glycyrrhizin, and glycyrrhizin reference standards, accurately weigh them, dissolve them in pure methanol, vortex and mix them to prepare five stock solutions. Take each stock solution and dilute it stepwise with a methanol solution of 0.1% formic acid to obtain working solutions of rehmannia glycoside D, ginsenoside Re, ginsenoside Rg1, glycyrrhizin, and glycyrrhizin with a series of gradient concentrations. S2. The working solutions of rehmannia glycoside D, ginsenoside Re, ginsenoside Rg1, glycyrrhizin, and glycyrrhizin with a series of gradient concentrations are detected by the chromatographic and mass spectrometric conditions described below. The peak area is calculated based on the mass spectrometric signal intensity of the target component within the retention time of the target component, and the working curve of the reference mass concentration versus peak area is obtained. Chromatographic conditions: Column: C18 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, switching to negative ion gradient elution mode after the positive ion gradient elution mode is completed; in positive ion elution mode, the injection volume is 5μL and the volumetric flow rate is 0.3mL / min; in negative ion elution mode, the injection volume is 2μL and the volumetric flow rate is 0.4mL / min; The positive ion gradient elution mode is: 0-0.1 min, 5% B; 0.1-2.0 min, 30% B; 2.0-4.0 min, 65% B; 4.0-6.0 min, 65% B; 6.0-6.01 min, 5% B; 6.01-8.0 min, 5% B; negative ion gradient elution mode: 0-0.1 min, 15% B; 0.1-3.0 min, 30% B; 3.0-4.0 min, 65% B; 4.0-5.0 min, 65% B; 5.0-5.01 min, 15% B; 5.01-9.0 min, 15% B; Mass spectrometry conditions: Electrospray ionization source (ESI), multiple reaction monitoring (MRM) scanning mode, electrospray voltage 5500 V for positive ion mode and -4500 V for negative ion mode; dry nitrogen gas was used. The chromatographic retention times of the analytes glycyrrhizin, glycyrrhizin, rehmannia glycoside D, ginsenoside Re, and ginsenoside Rg1 were as follows: glycyrrhizin 4.242 min, glycyrrhizin 2.489 min, rehmannia glycoside D 1.405 min, ginsenoside Re 4.087 min, and ginsenoside Rg1 4.094 min. The mass spectrometry parameters for glycyrrhizin, glycyrrhizin, rehmannia glycoside D, ginsenoside Re, and ginsenoside Rg1 are as follows: ； S3. Take a sample of Yangxin Dingji Capsules, weigh it accurately, dissolve it in 50% methanol, vortex to mix, centrifuge, take the supernatant, and prepare a sample solution with a mass concentration of 20-100 mg / mL. After filtering through a membrane, obtain the test sample. Detect the test sample according to the detection conditions in S2, obtain the peak area of ​​the test sample, and obtain the content of glycyrrhizin, glycyrrhizin, rehmannia glutinosa D, ginsenoside Re, and ginsenoside Rg1 in the test sample according to the working curve in S2.

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

3. The method according to claim 1, characterized in that, In S3, the sample of Yangxin Dingji Capsules was dissolved in 50% methanol, vortexed and mixed, centrifuged, and the supernatant was collected to obtain a sample solution with a mass concentration of 50 mg / mL.

4. The method according to claim 1, characterized in that, In S1, the method for preparing the working solution includes: taking the stock solution of rehmannia glycoside D and diluting it stepwise with a methanol solution of 0.1% formic acid to obtain working solutions of rehmannia glycoside D with gradient 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; Stock solutions of ginsenoside Re, ginsenoside Rg1, glycyrrhizin, and glycyrrhizin were taken separately and serially diluted with 0.1% formic acid in methanol solution to prepare working solutions of ginsenoside Re, ginsenoside Rg1, glycyrrhizin, and glycyrrhizin 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, respectively.

5. The method according to claim 1, characterized in that, In S2, during mass spectrometry detection, the quantitative ion for glycyrrhizin is [M-CH2OH-C6H3O2]. - The quantitative ion of glycyrrhizin 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] - ; Ginsenoside Rg1 [M+HCOO-C2H6O-2(C6H 10 O5)] - .

Images