A method for simultaneously detecting multiple main components in shengmaiyin (dangshen recipe) in plasma by LC-MS / MS

Abstract

This invention provides an LC-MS / MS method for the simultaneous detection of multiple major components of Shengmai Yin (Codonopsis pilosula formula) in plasma. The analytes include syringin, schisandrol A, gomisin D, schisandrol B, gomisin G, schisandrin A, schisandrin A, schisandrin B, codonopsis glycoside I, codonopsis alkynyl glycoside, methyl ophiopogon flavanone A, and methyl ophiopogon flavanone B. The method includes the following steps: preparation of a series of standard curve working solutions; preparation of standard curve samples; preparation of quality control working solutions; preparation of quality control samples; preparation of a mixed internal standard working solution; pretreatment of the standard curve, quality control, and analyte plasma samples using protein precipitation; sample detection; and quantitative determination based on the peak area ratio of the analyte to the internal standard to obtain the standard curve. The methodological investigation results of this invention show that the established method meets the requirements for in vivo biological sample determination, the pretreatment process is simple, and the method has good sensitivity, strong specificity, and high specificity, making it suitable for the detection of compounds with low concentrations.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to an analytical method for simultaneously determining multiple major components of Shengmai Yin (Codonopsis pilosula formula) in plasma and its application in pharmacokinetics. Background Technology

[0002] Shengmai Yin (Codonopsis Formula) (hereinafter referred to as SMY) is a derivative formula of Shengmai San, a ginseng formula in "Medical Origins". It is composed of three Chinese herbs: Codonopsis, Ophiopogon japonicus and Schisandra chinensis. It has the effects of invigorating qi and restoring pulse, nourishing yin and promoting body fluid. Modern research shows that SMY has the effects of improving the cardiovascular system, central nervous system and endocrine system. It has been widely used in modern clinical practice for the treatment of diseases such as chronic heart failure. Although the clinical application of SMY is widely recognized, existing literature mainly focuses on the qualitative and quantitative analysis of SMY's in vitro chemical components, fingerprinting, and in vivo pharmacokinetic studies of indicator components in single Chinese herbal medicines. Research on the blood entry and in vivo processes of SMY compound chemical components is relatively weak. For example, Chinese patent CN 103822975 B, application number 201310342928.3 discloses a method for detecting four indicator components in a Shengmai preparation of Codonopsis pilosula formula. Chinese patent CN 113391003 B, application number 202110797813.8 discloses a method for simultaneously detecting seven components in Shengmai decoction. Although this research model of tracking one or several components in the compound has played a certain role in improving the quality control level of SMY, it has not been confirmed whether these indicator components are the effective components in SMY. Only compounds that are absorbed into the blood circulation and can maintain an effective blood drug concentration are likely to be the main pharmacodynamic substances that exert its pharmacological effect. In pharmacokinetic studies, although there are relatively many in vivo pharmacokinetic studies on the index components of single Chinese herbal medicines, their pharmacokinetic properties are obviously not equivalent to those of compound preparations. SMY is composed of three Chinese herbal medicines: Codonopsis pilosula, Ophiopogon japonicus, and Schisandra chinensis. Polyacetylenes, simple phenylpropanoids, flavanones, and lignans are all active ingredients in SMY. Codonopsis pilosula contains codonopsis pilosula glycosides, which have cardioprotective effects; syringin has anti-inflammatory, antioxidant, and anti-apoptotic effects; flavonoids in Ophiopogon japonicus have promising applications in cardiovascular and cerebrovascular diseases; and lignans in Schisandra chinensis can effectively inhibit inflammatory responses. These components are not only the material basis of SMY but also commonly used indicators for its quality control. However, there are currently no reports on the simultaneous determination of multiple components in SMY in vivo, which greatly limits the elucidation of the mechanism of action of SMY and its safety in clinical use. Therefore, establishing a rapid, efficient, and accurate HPLC-MS / MS method for the simultaneous determination of the in vivo drug concentrations of multiple index components in SMY is of great significance. Summary of the Invention

[0003] The purpose of this invention is to provide a method for the simultaneous detection of the main components of Shengmai Yin (Codonopsis pilosula formula) in plasma samples using liquid chromatography-tandem mass spectrometry (LC-MS / MS), including syringin, schisandrol A, gomisin D, schisandrol B, gomisin G, schisandrin A, schisandrin A, schisandrin B, codonopsis glycoside I, codonopsis alkynyl glycoside, methyl ophiopogon flavanone A, and methyl ophiopogon flavanone B. This method features a simple pretreatment process, high sensitivity, strong specificity, and high specificity, making it suitable for the detection of compounds with low concentrations.

[0004] The method includes the following steps:

[0005] (1) Preparation of standard curve working solution

[0006] Accurately weigh appropriate amounts of the reference standards for each analyte, dissolve and dilute them separately in methanol to prepare mixed standard curve working solutions; specifically: accurately weigh approximately 10 mg of each reference standard into a brown vial, dissolve in methanol, mix well, and obtain reference standard stock solutions with a concentration of 5 mg / mL; then accurately measure appropriate amounts of each reference standard stock solution, dilute with methanol to obtain the following concentrations: syringin (20.0-20000 ng / mL), schisandrol A (4.00-4000 ng / mL), gomisin D (10.0-10000 ng / mL), and schisandrol B (10.0-1000 ng / mL). A mixed standard curve working solution with concentrations ranging from 0 ng / mL, Gomicin G (0.400-400 ng / mL), Schisandrin A (0.400-400 ng / mL), Schisandrin A (0.200-200 ng / mL), Schisandrin B (0.200-200 ng / mL), Codonopsis pilosula glycoside I (4.00-4000 ng / mL), Codonopsis pilosula glycoside (10.0-10000 ng / mL), Methyl Ophiopogonin A (0.400-400 ng / mL), and Methyl Ophiopogonin B (0.200-200 ng / mL);

[0007] (2) Preparation of standard curve samples

[0008] The mixed standard curve working solution obtained in (1) was added to blank plasma, and the volume ratio of the mixed standard curve working solution to blank plasma was controlled at 1:19. This yielded a solution containing syringin (1.00-1000 ng / mL), schisandrol A (0.200-200 ng / mL), gomisin D (0.500-500 ng / mL), schisandrol B (0.500-500 ng / mL), gomisin G (0.020-20 ng / mL), and schisandrol D. A series of standard curve samples of schisandrin A (0.020-20 ng / mL), schisandrin A (0.010-10 ng / mL), schisandrin B (0.010-10 ng / mL), codonopsis glycoside I (0.200-200 ng / mL), codonopsis glycoside (0.500-500 ng / mL), methyl ophiopogon flavanone A (0.020-20 ng / mL) and methyl ophiopogon flavanone B (0.010-10 ng / mL).

[0009] (3) Preparation of quality control working solution

[0010] The stock solutions of each analyte were diluted with methanol to prepare the following concentrations: syringin (20.0, 60.0, 1500, and 15000 ng / mL), schisandrol A (4.00, 12.0, 300, and 3000 ng / mL), gomisin D (10.0, 30.0, 750, and 7500 ng / mL), schisandrol B (10.0, 30.0, 750, and 7500 ng / mL), gomisin G (0.400, 1.20, 30.0, and 300 ng / mL), and schisandrin A (0.400, 1.20, 30.0, and 300 ng / mL). A mixed quality control working solution containing schisandrin A (0.200, 0.600, 15.0 and 150 ng / mL), schisandrin B (0.200, 0.600, 15.0 and 150 ng / mL), codonopsis glycoside I (4.00, 12.0, 300 and 3000 ng / mL), codonopsis glycoside (10.0, 30.0, 750 and 7500 ng / mL), methyl ophiopogon flavanone A (0.400, 1.20, 30.0 and 300 ng / mL), and methyl ophiopogon flavanone B (0.200, 0.600, 15.0 and 150 ng / mL);

[0011] (4) Preparation of quality control samples

[0012] Add the mixed quality control working solution obtained in (3) to blank plasma, and control the volume ratio of the mixed quality control working solution to blank plasma to be 1:19. The concentrations of each analyte (according to LLOQ QC, LQC, MQC and HQC) are as follows: syringin (1.00, 3.00, 75.0 and 750 ng / mL), schisandrol A (0.200, 0.600, 15.0 and 150 ng / mL), gomisin D (0.500, 1.50, 37.5 and 375 ng / mL), schisandrol B (0.500, 1.50, 37.5 and 375 ng / mL), gomisin G (0.020, 0.060, 1.50 and 15.0 ng / mL), schisandrin A (0.020, 0.060, 1.50 and 15.0 ng / mL), and schisandrin B (0.020, 0.060, 1.50 and 15.0 ng / mL). Mixed quality control samples of schisandrin A (0.010, 0.030, 0.750 and 7.50 ng / mL), schisandrin B (0.010, 0.030, 0.750 and 7.50 ng / mL), codonopsis glycoside I (0.200, 0.600, 15.0 and 150 ng / mL), codonopsis glycoside (0.500, 1.50, 37.5 and 375 ng / mL), methyl ophiopogon flavanone A (0.020, 0.060, 1.50 and 15.0 ng / mL), and methyl ophiopogon flavanone B (0.010, 0.030, 0.750 and 7.50 ng / mL);

[0013] (5) Preparation of internal standard working solution

[0014] Accurately weigh appropriate amounts of carbamazepine and nimodipine reference standards, dissolve and dilute them in methanol to prepare mixed internal standard working solutions with concentrations of carbamazepine (50 ng / mL) and nimodipine (200 ng / mL);

[0015] (6) Sample preparation

[0016] Add the mixed internal standard working solution to the plasma sample to be tested and mix well; the internal standard in the mixed internal standard working solution is carbamazepine and nimodipine, wherein carbamazepine is used as the internal standard in positive ion mode and nimodipine is used as the internal standard in negative ion mode, and vortex oscillate; after adding the precipitant to precipitate the protein, vortex oscillate, centrifuge, collect the supernatant, blow dry with nitrogen, add the reconstitution solvent, vortex oscillate, centrifuge, and collect the supernatant to obtain the sample to be tested;

[0017] (7) Sample testing

[0018] The test samples were analyzed by LC-MS / MS. A standard curve was obtained based on the peak area ratio of the analyte to the internal standard. Based on the standard curves established for each analyte reference standard, the plasma concentrations of these main components in rats after administration of Shengmai Yin (Codonopsis pilosula formula) aqueous extract were quantitatively determined.

[0019] Preferably, the chromatographic conditions are as follows:

[0020] chromatographic column is HSS T3, 3.5μm, 2.1×100mm column;

[0021] The mobile phase consists of 0.05% formic acid acetonitrile (A) and 0.05% formic acid aqueous solution (B) by volume.

[0022] The flow rate was 0.4 mL / min;

[0023] The column temperature is 40℃;

[0024] The injection volume was 5 μL;

[0025] The syringe washing solution is methanol;

[0026] The elution method is gradient elution;

[0027] Preferably, the gradient elution procedure is as follows:

[0028]

[0029] Preferably, the mass spectrometry conditions are as follows: the ion source is an electrospray ionization source (ESI source), with simultaneous scanning of positive and negative ions; the source temperature is 600℃; the ion spray voltage is ±4500V; Gas 1, Gas 2 and Curtain gas are 50psi, 50psi and 30psi respectively; and the scanning mode is multiple reaction monitoring (MRM).

[0030] Preferably, the analyte and internal standard ion pair and the main mass spectrometry parameters are as follows:

[0031]

[0032] Preferably, the precipitant is acetonitrile, and the volume ratio of acetonitrile to the plasma sample is 2-4:1, specifically 3:1;

[0033] Preferably, the resolvent is methanol, and the volume ratio of methanol to the plasma sample is 0.5-1.5:1, specifically 1:1;

[0034] Preferably, the parameters of the vortex are: oscillation at 1425 rpm for 3 minutes; the parameters of the centrifugation are: centrifugation at 4°C (14000 rpm) for 10 minutes;

[0035] Preferably, the nitrogen drying temperature is 37°C.

[0036] The application of the above method in the pharmacokinetics of Shengmai Yin (Codonopsis pilosula formula) also falls within the scope of protection of this invention.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] This invention establishes a method for detecting the concentration of the main components of Shengmai Yin (Codonopsis pilosula formula) in plasma using LC-MS / MS. The sample pretreatment method has been rationally optimized. Carbamazepine and nimodipine are selected as internal standards and added to the plasma sample. Proteins are then precipitated with acetonitrile, centrifuged, dried under nitrogen, and reconstituted with methanol before the supernatant is collected for analysis. The sample pretreatment process is simple. During the detection process, the chromatographic peak shape is excellent, and no peaks of the target compounds are found in blank plasma. The quantifiable concentrations of each analyte are low. Therefore, this method has high sensitivity, strong specificity, high accuracy, and high precision.

[0039] Existing technologies mostly focus on the qualitative and quantitative analysis of the in vitro chemical components of Shengmai Yin (Codonopsis pilosula formula). However, it has not been confirmed whether these indicator components are the effective components of Shengmai Yin (Codonopsis pilosula formula). Only compounds that are absorbed into the bloodstream and can maintain an effective blood drug concentration are likely to be the main pharmacodynamic substances that exert their effects. Based on the theory that "absorption into the blood is the basis for exerting pharmacodynamic effects", this invention establishes an LC-MS / MS method to simultaneously determine twelve major components of Shengmai Yin (Codonopsis pilosula formula) after it enters the bloodstream, laying the foundation for subsequent pharmacokinetic studies of Shengmai Yin (Codonopsis pilosula formula). Attached Figure Description

[0040] Figure 1 This is a scan of the analytes and internal standards obtained in this invention. 1. Syringin; 2. Codonopsis pilosula glycoside I; 3. Codonopsis pilosula glycoside; 4. Schisandrol A; 5. Gomisin D; 6. Schisandrol B; 7. Methyl Ophiopogonin A; 8. Gomisin G; 9. Methyl Ophiopogonin B; 10. Schisandrin A; 11. Schisandrin A; 12. Schisandrin B; 13. Carbamazepine (internal standard in positive ion mode); 14. Nimodipine (internal standard in negative ion mode).

[0041] Figure 2 The following are MRM chromatograms of 12 analytes and internal standards in rat plasma from Example 1. 1. Syringin; 2. Codonopsis pilosula I; 3. Codonopsis pilosula glycoside; 4. Schisandrol A; 5. Gomisin D; 6. Schisandrol B; 7. Methyl Ophiopogonin A; 8. Gomisin G; 9. Methyl Ophiopogonin B; 10. Schisandrin A; 11. Schisandrin A; 12. Schisandrin B; 13. Carbamazepine (internal standard in positive ion mode); 14. Nimodipine (internal standard in negative ion mode); A: Blank plasma; B: Blank plasma + standard solution (quantitatively lower limit plasma sample); C: Drug-containing plasma.

[0042] Figure 3The image shows the drug concentration-time curves of 12 analytes in the plasma of one rat after gavage administration of Shengmai Yin (Codonopsis pilosula formula) water extract in Example 2. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0045] Example 1

[0046] I. Test Item

[0047] Syringin, Schisandrol A, Gomisin D, Schisandrol B, Gomisin G, Schisandrin A, Schisandrin A, Schisandrin B (positive ion mode)

[0048] Codonopsis glycoside I, Codonopsis alkynyl glycoside, Methyl ophiopogon flavanone A, Methyl ophiopogon flavanone B (negative ion mode)

[0049] Carbamazepine (internal standard in positive ion mode), Nimodipine (internal standard in negative ion mode)

[0050] II. Materials

[0051] See Table 1 for details of the reference standard.

[0052] Table 1

[0053]

[0054] Reagents are shown in Table 2

[0055] Table 2

[0056]

[0057] medicinal materials

[0058] The medicinal materials Codonopsis pilosula (produced in Longxi, Gansu), Ophiopogon japonicus (produced in Mianyang, Sichuan) and Schisandra chinensis (produced in Antu, Jilin) were identified as the dried roots of Codonopsis pilosula (Franch.) Nannf. of the Campanulaceae family, the dried tuberous roots of Ophiopogon japonicus (L.f) Ker-Gawl. of the Liliaceae family, and the dried ripe fruits of Schisandra chinensis (Turcz.) Baill. of the Magnoliaceae family.

[0059] Experimental animals

[0060] The experimental animals were SPF-grade male SD rats, 6 - 7 weeks old, with a body weight of (200 - 220) g. They were purchased from Spf (Beijing) Biotechnology Co., Ltd., and the animal license number was SCXK (Beijing) 2019 - 0010. They were housed in an environment with a temperature of (22 ± 2) °C, a humidity of (40 ± 10) %, and a natural light-dark cycle, and were allowed to eat and drink freely. The ethical approval number was: SXWJYLL2022 - 002.

[0061] III. The main instruments are shown in Table 3

[0062] Table 3

[0063]

[0064] IV. Analysis conditions

[0065] Chromatographic conditions

[0066] Organic phase (A): 0.05% formic acid acetonitrile

[0067] Aqueous phase (B): 0.05% formic acid water

[0068] Chromatographic column: HSS T3, 3.5 μm, 2.1×100 mm, P / N: 186006465, L / N: 0173340591 Flow rate: 0.4 mL / min

[0069] Column temperature: 40 °C

[0070] Injection volume: 5 μL

[0071] Wash needle solution: methanol

[0072] The gradient elution program is shown in Table 4 below.

[0073] Table 4

[0074]

[0075]

[0076] Mass spectrometry conditions

[0077] The ion source was an electrospray ionization (ESI) source, simultaneously scanning positive and negative ions; the source temperature was 600℃; the ion spray voltage was ±4500V; Gas 1, Gas 2, and Curtain gas were 50psi, 50psi, and 30psi, respectively; the scanning mode was multiple reaction monitoring (MRM). The retention times of the analytes and internal standards, ion pairs, and main mass spectrometry parameters are shown in Table 5. The fraction ion scan is shown in... Figure 1 .

[0078] Table 5

[0079]

[0080] V. Preparation of analytes, internal standard stock solutions, and working solutions

[0081] (1) Analyte and internal standard stock solution: Take about 10 mg of the analyte and internal standard into a brown vial, weigh accurately, dissolve in methanol, mix well to obtain a standard stock solution with a concentration of 5 mg / mL, and store in a constant temperature incubator at 4℃ for later use.

[0082] (2) Preparation of standard curve working solution

[0083] Each stock solution was serially diluted with methanol to prepare working solutions with the following concentrations: syringin (250 μg / mL), schisandrol A (250 μg / mL), gomisin D (250 μg / mL), schisandrol B (250 μg / mL), gomisin G (10 μg / mL), schisandrin A (10 μg / mL), schisandrin A (10 μg / mL), schisandrin B (10 μg / mL), codonopsis glycoside I (250 μg / mL), codonopsis glycoside (250 μg / mL), methyl ophiopogon flavanone A (10 μg / mL), and methyl ophiopogon flavanone B (10 μg / mL).

[0084] Take appropriate amounts of the working solutions of each of the above reference standards and dilute them with methanol to obtain mixed reference working solutions with concentrations of syringin (20 μg / mL), schisandrol A (4 μg / mL), gomisin D (10 μg / mL), schisandrol B (10 μg / mL), gomisin G (400 ng / mL), schisandrin A (400 ng / mL), schisandrin A (200 ng / mL), schisandrin B (200 ng / mL), codonopsis glycoside I (4 μg / mL), codonopsis glycoside (10 μg / mL), methyl ophiopogon flavanone A (400 ng / mL), and methyl ophiopogon flavanone B (200 ng / mL).

[0085] The prepared mixed reference working solution was diluted sequentially with methanol to obtain the following concentrations: syringin (20.0-20000 ng / mL), schisandrol A (4.00-4000 ng / mL), gomisin D (10.0-10000 ng / mL), schisandrol B (10.0-10000 ng / mL), gomisin G (0.400-400 ng / mL), schisandrin A (0.400-400 ng / mL), and schisandrin A (0.400-400 ng / mL). A series of working solutions with concentrations ranging from 200-200 ng / mL, schisandrin B (0.200-200 ng / mL), codonopsis glycoside I (4.00-4000 ng / mL), codonopsis glycoside (10.0-10000 ng / mL), methyl ophiopogon flavanone A (0.400-400 ng / mL), and methyl ophiopogon flavanone B (0.200-200 ng / mL) were prepared. The concentrations of the mixed working solutions for the standard curves of each analyte are shown in Table 6.

[0086] Table 6

[0087]

[0088] (3) Preparation of quality control working solution: The stock solution of the analyte is diluted stepwise with methanol to prepare mixed quality control working solutions of each analyte with concentrations of LLOQ QC, LQC, MQC and HQC. The concentrations of each analyte in the mixed quality control working solution are shown in Table 7.

[0089] Table 7

[0090]

[0091]

[0092] (4) Preparation of internal standard working solution: The internal standard stock solution was gradually diluted with methanol to prepare a mixed internal standard working solution with carbamazepine and nimodipine concentrations of 50.0 ng / mL and 200 ng / mL, respectively.

[0093] (5) Preparation of mobile phase

[0094] Aqueous phase preparation: Measure 500 mL of purified water into a 500 mL mobile phase bottle, add 500 μL of 50% (volume ratio) formic acid suspended in the mobile phase bottle, mix well, and sonicate to obtain the aqueous phase.

[0095] Organic phase preparation: Measure 500 mL of acetonitrile into a 500 mL mobile phase bottle, add 500 μL of 50% formic acid in a suspended manner into the above mobile phase bottle, mix well, and sonicate to obtain the organic phase.

[0096] (6) Sample pretreatment methods

[0097] Transfer 100 μL of plasma sample to a 1.5 mL centrifuge tube, add 10 μL of internal standard (carbamazepine and nimodipine mixed internal standard), vortex (1425 rpm) for 3 minutes, add 300 μL of acetonitrile precipitant, vortex (1425 rpm) for 3 minutes, centrifuge at 4°C (14000 rpm) for 10 minutes, transfer the supernatant to a 2 mL centrifuge tube, dry under nitrogen at 37°C, add 100 μL of methanol reconstituter, vortex (1425 rpm) for 3 minutes, centrifuge at 4°C (14000 rpm) for 10 minutes, and transfer the supernatant to a sample vial for testing.

[0098] (7) Preparation of Shengmai Yin Water Extract

[0099] Weigh 15g of Codonopsis pilosula, 10g of Ophiopogon japonicus, and 5g of Schisandra chinensis into a 500mL round-bottom flask, add 10 times (300mL) of purified water, soak for 30min, heat under reflux for 2h, filter while hot, add 8 times (240mL) of water to the residue, continue heating under reflux for 2.0h, filter and combine the two filtrates, concentrate under reduced pressure to about 0.8g / mL, slowly add 1 times the amount of ethanol while stirring, let stand overnight, centrifuge (4500r / min, 10min) to collect the supernatant, concentrate under reduced pressure at 70℃ to a certain volume, transfer to a 25mL volumetric flask, add water to make up to the mark, and obtain SMY aqueous extract.

[0100] VI. Data Processing

[0101] Data were processed and analyzed using Analyst V 1.7 software. Data results: concentration and standard deviation were retained to three significant figures; selectivity, residue, and recovery results were expressed as percentages and retained to one decimal place; %RE and %CV were both retained to one decimal place; retention time was retained to two decimal places; correlation coefficient r was retained to four significant figures.

[0102] VII. Validation of Analytical Methods

[0103] The method described herein was evaluated in accordance with the Validation Guidelines for Quantitative Analysis of Biological Samples in General Chapter 9012 of the 2020 edition of the Chinese Pharmacopoeia, and the ICH / M10 Guidelines for Validation and Sample Analysis of Bioanalytical Methods. The accuracy and precision of the method were determined using three batches of samples at four different concentration levels: lower limit of quantitation, low, medium, and high. Recovery was assessed for accuracy using six parallel samples at low, medium, and high concentrations. Matrix effects were evaluated by analyzing three replicates of low and high concentration quality control samples, each replicate prepared from blank plasma from four different rats. The precision and accuracy of the experimental results are expressed as coefficient of variation (%CV) and accuracy (%RE). The specific steps include the following:

[0104] (1) Exclusivity

[0105] 100 μL of blank rat plasma, drug-treated plasma containing 12 reference standards and internal standards, and rat plasma collected 20 min after gavage administration of Shengmai drinking water extract (13.5 mL / kg) were processed according to the method described in the "Sample Pretreatment Methods" section, injected, and chromatograms were recorded. The results showed that no endogenous substances or metabolites interfered within the retention times of the analytes. (See attached figures). Figure 2 .

[0106] (2) Selectivity

[0107] Double blank samples (excluding analytes and internal standards): 95 μL of blank plasma from 6 different rats were placed in 1.5 mL centrifuge tubes. 5 μL of methanol was added to each tube, and the mixture was vortexed for 3 minutes. 10 μL of methanol was added, and the mixture was vortexed for 3 minutes. 300 μL of acetonitrile precipitant was added, and the procedure was followed according to the "Sample Pretreatment Methods" section to obtain blank plasma samples for analysis. The interference of the blank matrix on the retention time of the analytes and internal standards was investigated.

[0108] Zero-concentration samples (samples containing internal standards but excluding analytes): 95 μL of blank plasma from 6 different rats were placed in 1.5 mL centrifuge tubes. 5 μL of methanol was added to each tube, and the mixture was vortexed for 3 minutes. 10 μL of internal standard was added, and the mixture was vortexed for 3 minutes. 300 μL of acetonitrile precipitant was added, and the procedure was followed as described in the "Sample Pretreatment Methods" section to obtain zero-concentration samples for analysis. The effect of the internal standard on each analyte was investigated.

[0109] Upper limit of quantitation (UPQ) samples (containing analyte at UPQ concentration, excluding internal standard samples): 95 μL of blank plasma from 6 different rats was placed in 1.5 mL centrifuge tubes. 10 μL of the ULOQ working solution of the analyte was added to each tube, and the mixture was vortexed for 3 minutes. 10 μL of methanol was added, and the mixture was vortexed for 3 minutes. 300 μL of acetonitrile precipitant was added, and the procedure was followed according to the "Sample Pretreatment Methods" section to obtain the UPQ sample for analysis, thus investigating the effect of the analyte on the internal standard.

[0110] The results show that within the retention time interval of each analyte, the peak area of ​​the interfering peak is less than 20.0% of the peak area of ​​the analyte in the qualified LLOQ sample of the standard curve of this batch. Within the retention time interval of the internal standard, the peak area of ​​the interfering peak is less than 5.0% of the peak area of ​​the internal standard in the qualified LLOQ sample of the standard curve of this batch. This indicates that the six matrices from different sources do not interfere with the analytes and the internal standard. The results are shown in Tables 8-9.

[0111] Table 8 Selectivity of Double Blank Samples

[0112]

[0113]

[0114] ND = Not Detectable

[0115] Table 9 Selectivity of zero-concentration samples and samples with upper limit of quantitation

[0116]

[0117] ND = Not Detectable

[0118] (3) Standard Curve

[0119] 285 μL of blank rat plasma was collected and placed in a 1.5 mL centrifuge tube. 15 μL of standard curve working solutions were added to each tube, and the mixture was vortexed to obtain the following plasma concentrations for each analyte: syringin (1.00-1000 ng / mL), schisandrin A (0.200-200 ng / mL), gomisin D (0.500-500 ng / mL), schisandrin B (0.500-500 ng / mL), gomisin G (0.0200-20.0 ng / mL), schisandrin A (0.0200-20.0 ng / mL), and schisandrin A (0.0100 ng / mL). Standard curves were generated for a series of drug-containing plasmas with concentrations ranging from -10.0 ng / mL, schisandrin B (0.0100-10.0 ng / mL), codonopsis pilosula glycoside I (0.200-200 ng / mL), codonopsis pilosula glycoside (0.500-500 ng / mL), methyl ophiopogonin A (0.0200-20.0 ng / mL), and methyl ophiopogonin B (0.0100-10.0 ng / mL). 100 μL of the above-mentioned plasmas with added reference standards were placed in 1.5 mL centrifuge tubes (two replicates per concentration), and processed according to the "Sample Pretreatment Methods" section before injection and analysis. Linear regression was performed using the least squares method with concentration x (ng / mL) as the x-axis and the ratio of the analyte peak area to the internal standard peak area (y) as the y-axis, with a weighting factor of 1 / x. 2 The correlation coefficient (r) was calculated. The results showed that the correlation coefficients (r) of the standard curves for each analyte were all greater than 0.99, indicating a good linear relationship. The linear regression equations for each analyte are shown in Table 10.

[0120] Table 10

[0121]

[0122] (4) Precision and accuracy

[0123] Take 665 μL of rat blank plasma into a 1.5 mL centrifuge tube, add 35 μL of quality control working solution, vortex to mix, and take 100 μL into a 1.5 mL centrifuge tube (6 replicates per concentration). Prepare QC samples for each analyte at the lower limit of quantitation (LLOQ), low, medium, and high concentrations according to the method under "Sample Pretreatment Methods". The concentrations of the QC samples for each analyte are shown in the table below. Analyze three consecutive batches and calculate the intra-batch and inter-batch precision and accuracy of the QC samples. Intra-batch or inter-batch precision, except for LLOQ QC (LLOQQC%CV≤20.0%), is %CV≤15.0%, and intra-batch or inter-batch accuracy, except for LLOQ QC (LLOQ QC%RE within ±20.0%), is %RE within ±15.0%. The results show that the intra-day and inter-day precision RSDs of each analyte meet the requirements, and the results are shown in Tables 11-12.

[0124] Table 11 Precision and accuracy of samples tested within a batch

[0125]

[0126] Table 12 Precision and accuracy of inter-batch sample testing

[0127]

[0128] (5) Extraction recovery rate

[0129] The LQC, MQC, and HQC quality control concentrations were investigated. Six samples were tested in parallel at each concentration. The coefficient of variation (%CV) of the extraction recovery rates of the analyte and internal standard at each concentration level was calculated by comparing the peak area of ​​the extracted biological sample with the average peak area of ​​the sample after extraction with the same final concentration of analyte and internal standard in the blank matrix. Furthermore, the overall %CV of the extraction recovery rates of the analyte and internal standard at the three concentration levels was calculated. The results showed that the %CV of the extraction recovery rate of the analyte at each concentration level was ≤15.0%, and the overall %CV of the extraction recovery rates of the analyte and internal standard at the three concentration levels was ≤15.0%, all meeting the requirements. The results are shown in Table 13.

[0130] Table 13 Recovery results of analytes and internal standards

[0131]

[0132]

[0133] (6) Matrix effect

[0134] The matrix effect was evaluated by analyzing three replicate low- and high-concentration quality control samples, each replicate prepared from blank plasma from four different rats. The results showed that the accuracy of the blank plasma from the four different rats was within ±15% of the labeled concentration, and the precision %CV was less than 15%. The results indicate that the established method meets the requirements for the determination of various analytes, as shown in Table 14.

[0135] Table 14

[0136]

[0137] (7) Residual effect

[0138] Following the standard curve ULOQ (Upper Loop Question) sample, a blank sample was used for evaluation. The results showed that the peak areas of each analyte in the blank sample were all less than 20.0% of the peak area of ​​the analyte in the LLOQ sample, and the peak areas of the internal standard in the blank sample were all less than 5.0% of the peak area of ​​the internal standard in the LLOQ sample, indicating that this method has no residual effects on the retention times of either the analyte or the internal standard.

[0139] (8) Dilution reliability

[0140] Add an analyte concentration higher than the ULOQ to blank plasma, and dilute the sample 5-fold to HQC with blank plasma. Take 100 μL of HQC sample into a 1.5 mL centrifuge tube, and follow the sample pretreatment procedure. Perform this procedure in parallel with 6 samples, and inject for analysis. Calculate the concentration using the standard curve of the day, and multiply by the dilution factor (5-fold) to obtain the QC concentration before dilution. The results show that the detection concentration %RE of each diluted sample is within ±15.0%, and the precision %CV is less than 15.0%, all meeting the requirements. The results are shown in Table 15.

[0141] Table 15

[0142]

[0143] (9) Stability

[0144] 100 μL of blank rat plasma was collected and low- and high-concentration quality control samples were prepared according to the method under "Sample Pretreatment Methods". The samples were injected according to the chromatographic conditions under "Chromatographic and Mass Spectrometry Analysis Conditions". Each concentration was tested in triplicate. The stability of the samples was investigated under the following conditions: before treatment, storage at room temperature for 4 h; three freeze-thaw cycles (stored at -80℃, repeated freeze-thaw cycles, with each freeze cycle lasting more than 12 h); and after treatment (stored in an autosampler at 4℃ for 48 h, then at room temperature for 4 h). The results showed that the accuracy was within ±15.0% and the precision %CV was less than 15% under all four conditions. These results indicate that the stability of each analyte in the QC samples meets the requirements of biological sample analysis methods. The results are shown in Tables 16-19.

[0145] Table 16 Stability of samples after 4 hours of storage at room temperature before treatment

[0146]

[0147]

[0148] Table 17 Stability after 3 freeze-thaw cycles (-80℃)

[0149]

[0150] Table 18 shows the stability of samples after treatment in an autosampler (4°C) for 48 hours.

[0151]

[0152] Table 19 Stability of samples after treatment and storage at room temperature for 4 hours

[0153]

[0154]

[0155] Example 2: Detection of drug-treated rat samples

[0156] Twelve male SD rats were acclimatized for one week and randomly divided into a blank control group and a Shengmai Yin group, with six rats in each group. The rats were fasted for 12 hours before administration but allowed free access to water. In the Shengmai Yin group, blank blood was collected, and the rats were then administered Shengmai Yin water extract by gavage at 13.5 mL / kg. Approximately 300 μL of blood was collected from the fundus venous plexus at 0.033, 0.083, 0.167, 0.333, 0.667, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, and 36 hours after administration. The blood samples were placed in centrifuge tubes treated with heparin sodium anticoagulation and centrifuged at 14000 rpm for 10 min (4℃). The supernatant was collected and stored at -80℃ for later use.

[0157] After processing the drug-treated plasma according to the "Sample Pretreatment Methods" section, the plasma was injected for analysis. Three quality control samples (low, medium, and high concentrations) were also included. The blood drug concentration was calculated using the standard curve of the day. A plot of blood drug concentration (conc) against blood collection time (time) was generated to obtain the drug-time curves for each component. Figure 3 Drug concentration-time curves of 12 analytes in plasma were obtained after one rat was administered Shengmai Yin (Codonopsis pilosula formula) water extract by gavage.

[0158] Example 3 Parameter Examination Experiment

[0159] (1) Selection of pretreatment method

[0160] During sample processing, since Shengmai Yin contains both highly polar components, such as syringin, and less polar components, such as schisandrin A and schisandrin B, four pretreatment methods were investigated to remove proteins from plasma samples in order to improve the recovery rate of each analyte and reduce matrix effects: ethyl acetate liquid-liquid extraction, methyl tert-butyl ether liquid-liquid extraction, methanol precipitation, and acetonitrile precipitation. The results showed that when methanol was used as the precipitant, the recovery rate of schisandrin lignans was significantly lower than that of acetonitrile. Furthermore, the reproducibility was poor. When ethyl acetate and methyl tert-butyl ether were used as extraction reagents, the recoveries of syringin, codonopsis glycoside, codonopsis glycoside I, and methyl ophiopogonin A were significantly lower than those of acetonitrile. Only schisandrin A and schisandrin A had slightly higher recoveries than acetonitrile. Therefore, acetonitrile was chosen as the precipitant. Next, 200, 300, and 400 μL of acetonitrile were investigated as precipitants. The results showed that the response was higher with 300 μL of acetonitrile as the precipitant than with 200 μL, while no significant change in response was observed with 400 μL. Therefore, in summary, the overall recovery rate of the analytes was higher when 300 μL of acetonitrile was used as the precipitant, ranging from 77.1% to 103.1%.

[0161] Regarding the selection of the resolvent, the responses of methanol, acetonitrile, 80% methanol, and 0.05% formic acid methanol as resolvents were investigated. The results showed that when acetonitrile was used as the resolvent, the response of most compounds was less than that when methanol was used as the resolvent. When 20% water or 0.05% formic acid was added to methanol, the response of compounds with lower polarity decreased significantly. Therefore, methanol was selected as the resolvent.

[0162] (2) Investigation of liquid phase conditions

[0163] Schisandra chinensis contains a variety of lignans, and Ophiopogon japonicus contains a variety of flavonoids. Among these components are many isomers and components with very similar molecular weights, which brings great difficulties to chromatographic separation. Therefore, the chromatographic conditions were optimized. Seven mobile phase systems were investigated: acetonitrile-water, methanol-water, 0.025% formic acid acetonitrile-0.025% formic acid water, 0.05% formic acid acetonitrile-0.05% formic acid water, 0.1% formic acid acetonitrile-0.1% formic acid water, acetonitrile-5 mmol / L ammonium acetate water, and 0.05% formic acid acetonitrile-0.05% formic acid + 5 mmol / L ammonium acetate water. The results showed that acetonitrile, compared to methanol, provided a higher response from Schisandra lignans. When different concentrations of formic acid were added to the acetonitrile-water mobile phase, the response of most analytes initially increased and then decreased with increasing formic acid concentration. At a formic acid concentration of 0.05%, most analytes showed a high response. Adding ammonium acetate to the mobile phase did not significantly change the response of most compounds. Therefore, 0.05% formic acid acetonitrile-0.05% formic acid water was ultimately selected as the mobile phase.

[0164] The chromatographic column was investigated HSS T3, 3.5μm, 2.1×100mm, HSS T3, 3.5μm, 2.1×50mm and Phenomenex C 18 Three chromatographic columns were used: 2.1*50mm and 2.6μm. Results showed that when using an HSS T3 100mm column, the separation between gomisin G and schisandrin A, between schisandrin B and adjacent interfering peaks, and between methyl ophiopogon flavanone A and adjacent interfering peaks was good. Regarding analysis time, due to the large number of analytes and the abundance of low-polarity compounds, the analysis time was investigated. When the analysis time was less than 12 min, the matrix effect of four analytes—schisandrin A, schisandrin B, methyl ophiopogon flavanone A, and methyl ophiopogon flavanone B—significantly increased. This may be because endogenous components in the matrix were not separated from these four analytes, leading to an increased matrix effect. Therefore, adjusting the analysis time to 13 min significantly improved the matrix effect, meeting the requirements of the analytical method.

[0165] During the chromatographic investigation, it was found that four components, schisandrin A, schisandrin B, methyl ophiopogon flavanone A, and methyl ophiopogon flavanone B, had residues due to their low polarity. Therefore, the residue ratios of methanol and different proportions of methanol-water as washing solutions were investigated. The results showed that when 2 mL of methanol was used as the washing solution after each injection, no residues of the above four components were found.

[0166] (3) Investigation of mass spectrometry conditions

[0167] Regarding the selection of ion pairs for each analyte, schisandrol A, schisandrol B, schisandrin A, and schisandrin B [M+H] are preferred. + The response was high; when the ion pair of schisandrin A changed from 433.2→415.1 to 433.2→384.0, the interference in the sample was significantly reduced, and its lower limit of quantitation also decreased from 1 ng to 0.2 ng; syringin, gomisin D, and gomisin G [M+NH4] + High response and low interference, such as schisandrin methyl [M+NH4] + When acting as a precursor ion, its residual value is significantly smaller than that of [M+H]. + Furthermore, there were no adjacent interfering peaks, and the lower limit of quantitation changed from 1 ng to 0.02 ng; Codonopsis pilosula glycoside I, methyl ophiopogonin A, and methyl ophiopogonin B [MH] - The response was high, with codonopsis glycoside [M+HCOO] -The method exhibits high response and minimal interference. When using ion pairs 419.1→168.9 and 395.1→88.9, an interfering peak appears 0.1 min after the retention time, which is difficult to separate. However, when using 441.1→142.9, the interfering peak disappears. Since the above components exhibit high responses in both positive and negative ion modes, this method uses one internal standard for correction in both positive and negative ion modes.

[0168] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for simultaneously detecting the main components of Shengmai Yin (Dangshen Fang) in plasma samples, namely syringin, schisandrol A, gomisin D, schisandrol B, gomisin G, schisandrin A, schisandrin A, schisandrin B, codonopsis glycoside I, codonopsis glycoside, methyl ophiopogonin A, and methyl ophiopogonin B, using liquid chromatography-tandem mass spectrometry, characterized in that... The method includes the following steps: (1) Preparation of standard curve working solution Accurately weigh appropriate amounts of reference standards for each analyte, dissolve them separately in methanol to obtain stock solutions of each analyte, and then dilute with methanol to prepare a mixed standard curve working solution. (2) Preparation of standard curve samples Add the mixed standard curve working solution obtained in (1) to blank plasma, and control the volume ratio of the mixed standard curve working solution to blank plasma to be 1:19 to obtain a series of standard curve samples containing each analyte; (3) Preparation of quality control working solution Each analyte stock solution was diluted with methanol to prepare a mixed quality control working solution. (4) Preparation of quality control samples Add the mixed quality control working solution obtained in (3) to blank plasma, and control the volume ratio of the mixed quality control working solution to the blank plasma to be 1:19 to obtain a mixed quality control sample containing each analyte; (5) Preparation of internal standard working solution Accurately weigh appropriate amounts of carbamazepine and nimodipine reference standards, dissolve and dilute them in methanol to prepare mixed internal standard working solutions with concentrations of carbamazepine (50 ng / mL) and nimodipine (200 ng / mL); (6) Sample preparation Add the mixed internal standard working solution to the plasma sample to be tested and mix well; the internal standard in the mixed internal standard working solution is carbamazepine and nimodipine, wherein carbamazepine is used as the internal standard in positive ion mode and nimodipine is used as the internal standard in negative ion mode, and vortex oscillate; after adding the precipitant to precipitate the protein, vortex oscillate, centrifuge, collect the supernatant, blow dry with nitrogen, add the reconstitution solvent, vortex oscillate, centrifuge, and collect the supernatant to obtain the sample to be tested; (7) Sample testing The test samples were analyzed by LC-MS / MS. A standard curve was obtained based on the peak area ratio of the analyte to the internal standard. Based on the standard curves established for each analyte reference standard, the plasma concentrations of these main components in rats after administration of Shengmai Yin (Codonopsis pilosula formula) aqueous extract were quantitatively determined.

2. The method according to claim 1, characterized in that, The chromatographic conditions are as follows: chromatographic column is HSS T3, 3.5 μm, 2.1 × 100 mm column; The mobile phase consists of 0.05% formic acid acetonitrile (A) and 0.05% formic acid aqueous solution (B) by volume. The flow rate was 0.4 mL / min; The column temperature is 40℃; The injection volume was 5 μL; The syringe washing solution is methanol; The elution method is gradient elution; The gradient elution procedure is as follows:

3. The method according to claim 1, characterized in that, The mass spectrometry conditions were as follows: the ion source was an electrospray ionization (ESI) source, with simultaneous scanning of positive and negative ions; the source temperature was 600℃; the ion spray voltage was ±4500V; Gas 1, Gas 2, and Curtain gas were 50psi, 50psi, and 30psi, respectively; the scanning mode was multiple reaction monitoring (MRM); the analyte and internal standard ion pairs and the main mass spectrometry parameters are as follows:

4. The method according to claim 1, characterized in that, In step (6), the precipitant is acetonitrile, and the volume ratio of acetonitrile to the plasma sample is 2-4:1, specifically 3:1; The resolvent is methanol, and the volume ratio of methanol to the plasma sample is 0.5-1.5:1, specifically 1:1; The parameters of the vortex are: 1425 revolutions per minute oscillation for 3 minutes; The centrifugation parameters are: centrifugation at 4℃ (14000 rpm) for 10 minutes; The nitrogen drying temperature is 37°C.

5. The method of claim 1, wherein, The concentration ranges of each analyte in the standard curve sample mentioned in step (2) are as follows: syringin (1.00-1000 ng / mL), schisandrol A (0.200-200 ng / mL), gomisin D (0.500-500 ng / mL), schisandrol B (0.500-500 ng / mL), gomisin G (0.020-20 ng / mL), schisandrin A (0.020-20 ng / mL), schisandrin A (0.010-10 ng / mL), schisandrin B (0.010-10 ng / mL), codonopsis glycoside I (0.200-200 ng / mL), codonopsis glycoside (0.500-500 ng / mL), methyl ophiopogon flavanone A (0.020-20 ng / mL), and methyl ophiopogon flavanone B (0.010-10 ng / mL).

6. The method of claim 1, wherein, The concentrations of each analyte in the quality control sample described in step (4), according to LLOQ QC, LQC, MQC, and HQC, are as follows: syringin (1.00, 3.00, 75.0, and 750 ng / mL), schisandrol A (0.200, 0.600, 15.0, and 150 ng / mL), gomisin D (0.500, 1.50, 37.5, and 375 ng / mL), schisandrol B (0.500, 1.50, 37.5, and 375 ng / mL), gomisin G (0.020, 0.060, 1.50, and 15.0 ng / mL), and schisandrin A (0.020, 0.060, 1.50, and 15.0 ng / mL). Schisandrin A (0.010, 0.030, 0.750 and 7.50 ng / mL), Schisandrin B (0.010, 0.030, 0.750 and 7.50 ng / mL), Codonopsis pilosula I (0.200, 0.600, 15.0 and 150 ng / mL), Codonopsis pilosula glycoside (0.500, 1.50, 37.5 and 375 ng / mL), Methyl Ophiopogonin A (0.020, 0.060, 1.50 and 15.0 ng / mL), Methyl Ophiopogonin B (0.010, 0.030, 0.750 and 7.50 ng / mL).

7. A method for determining the pharmacokinetics of Shengmai Yin (Codonopsis pilosula formula), comprising the method according to any one of claims 1-6.

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