Method for simultaneously determining seven mass markers (Q-Marker) in serum of traditional Chinese medicine Jingtongling granules

Abstract

The invention discloses a method for simultaneously determining seven mass markers (Q-Marker) in serum of traditional Chinese medicine particles for treating cervical pain, which is characterized in that the seven mass markers are magnoflorine, puerarin, tetrahydropalmatine, paeoniflorin, notoginsenoside R1, ligustilide and decuricin; the method comprises the following steps: adding a serum sample into acetonitrile and acetone, carrying out vortex and centrifugation, taking a supernatant, loading the supernatant into a Hybrid SPEcolumn, washing with water to remove salt and polar interferents, eluting a target analyte with acetonitrile, filtering the eluent through a microfiltration membrane, carrying out sample introduction determination, and carrying out quantitative analysis by adopting a high performance liquid chromatography-mass spectrometry method and an electrospray ion source. And quantitative analysis is carried out in a positive / negative ion multi-reaction monitoring detection mode. The method is efficient, sensitive and high in selectivity, and solves the problems of serious matrix interference, indefinite target components, difficulty in simultaneous analysis of multiple components and the like in the prior art.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for simultaneously determining seven quality markers (Q-Markers) in serum of a Chinese medicine Jingtong Granules. BACKGROUND

[0002] Jingtong Granules are recorded in Chinese Pharmacopoeia 2020 Edition. They have the functions of activating blood and resolving stasis, and promoting blood circulation to relieve pain. They are used for treating blood stasis and qi stagnation, and collateral obstruction syndrome of nerve root type cervical spondylosis. Symptoms include neck, shoulder and upper limb pain, stiffness, numbness and pain. The Chinese medicine granule preparation for treating neck pain described in the present application is composed of the following weight ratio of medicinal materials: Sanqi 250g, Chuanxiong 750g, Yansu 500g, Qianghuo 1000g, Baishao 750g, Weilingxian 1000g, and Gegen 750g. The preparation is scientifically prepared by selecting seven kinds of Chinese medicinal materials. Clinical studies have shown that it has the functions of activating blood and resolving stasis, and dredging collaterals to relieve pain. A large number of clinical practices have confirmed that Jingtong Granules have a definite curative effect on improving symptoms such as neck pain and stiffness, and are safe. They have become the preferred drug for the clinical treatment of cervical shoulder syndrome, and are highly recognized and praised by both doctors and patients.

[0003] At present, there are detection methods for Jingtong Granules, but due to the extremely complex chemical components of Chinese patent medicines (including multiple structural types of compounds such as flavonoids, alkaloids, saponins and organic acids), it is too single to evaluate the drug only by in vitro chemical components, which is not conducive to the comprehensive and in-depth control of the quality of traditional Chinese medicines. The in vivo dynamic process is also unclear. There is no detection method for the precise quantification of the blood components of Jingtong Granules, especially the quality markers. Explaining and studying the blood components of Chinese patent medicines are of great significance to clarify the material basis of drug efficacy, mechanism of action, quality control, drug interaction and safety evaluation. However, when detecting blood components, there are problems such as serious matrix interference, low content of target components and the need to detect multiple compounds with large differences in chemical properties at the same time. Moreover, the existing mass spectrometry method has insufficient sensitivity / selectivity, and there is no specific detection method for multiple quality markers of Jingtong Granules, which leaves a certain gap in this field.

[0004] Therefore, it is urgent to establish a detection method for the quality markers of Jingtong Granules. SUMMARY

[0005] To solve the above technical problems, the present application establishes a method for simultaneously determining seven quality markers (Q-Markers) in serum of Chinese medicine Jingtong Granules. The method is efficient, sensitive and highly selective, and solves the problems of serious matrix interference, unclear target components and difficulty in simultaneous analysis of multiple components in the prior art.

[0006] To solve the above problems, the following technical solutions are provided: A method for simultaneously determining 7 quality markers in Jingtong granules serum, the 7 quality markers are magnoflorine, puerarin, tetrahydropalmatine, paeoniflorin, ginsenoside R1, ligustrazine and decursin; serum samples are added with acetonitrile and acetone, vortexed, centrifuged, and the supernatant is taken and loaded into a HybridSPE column, first washed with water to remove salt and polar interferents, and then eluted with acetonitrile, the eluent is filtered through a microporous filter, and then determination is carried out, high performance liquid chromatography-mass spectrometry is used, an electrospray ion source is used, and quantitative analysis is carried out by positive / negative ion multiple reaction monitoring detection mode.

[0007] Further, the serum sample is specifically processed as follows: 800-1000 muL of serum sample is added with 2000-2500 muL of acetonitrile and acetone solution, the volume ratio of acetonitrile and acetone is 9:1, vortexed for 10 min at 2-8 DEG C and 10000-12000 r / min -1 , centrifuged for 10-15 min, and the supernatant is taken.

[0008] Further, the chromatographic conditions are as follows: the mobile phase is acetonitrile (A) and 0.1% formic acid (B); the gradient elution conditions are 0-1 min, 5% A; 1-3 min, 5-10% A; 3-20 min, 10-25% A; 20-45 min, 25-98% A; the flow rate is 0.3 mL / min; the column temperature is 30 DEG C; and the injection amount is 1 muL.

[0009] Further, the mass spectrometry conditions are as follows: the mode is set as a mass detector, an electrospray ion source, positive / negative ion multiple reaction monitoring, the spray voltage is 5500V / -4500V; the atomizer temperature is 550 DEG C; the atomization gas pressure is 50 psi; the auxiliary gas pressure is 50 psi; and the curtain gas pressure is 30 psi.

[0010] Further, the mass spectrometry detection conditions of each quality marker are as follows:

[0012] A method for simultaneously determining 7 quality markers in Jingtong granules serum in the application of drug evaluation of Jingtong granules.

[0013] The target efficacy components selected by the application are derived from the paper "Prediction and analysis of quality markers of Jingtong granules based on UHPLC-Q-Exactive-Orbitrap MS technology and network pharmacology", in our previous research, and the Q-Marker for treating nerve root type cervical spondylosis of Jingtong granules is determined. The application is based on this to carry out quantitative research on efficacy substances.

[0014] This invention establishes a method for serum pretreatment using an acetonitrile-acetone mixed solution. This method can significantly reduce matrix effects, effectively remove interference from in vivo components such as phospholipids and proteins, and improve the exposure of active ingredients.

[0015] This invention utilizes an AB SCIEX Triple Quad 6500+ ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) system to detect quality markers in serum. The chromatographic method references the chromatographic conditions described in the literature "Predictive Analysis of Quality Markers for Neck Pain Granules Based on UHPLC-Q-Exactive-Orbitrap MS Technology and Network Pharmacology," and further specifies the parent ion, daughter ion, and pyrolysis voltage of the analytes, enabling more precise quantification of each analyte in the sample.

[0016] The key innovation of this invention lies in the design of the pretreatment method and the specific combination and optimization of mass spectrometry detection conditions. The method is simple, rapid, and reproducible, providing a reliable analytical tool for elucidating the pharmacodynamic material basis of this traditional Chinese medicine. Attached Figure Description

[0017] Figure 1 The TIC chromatogram of the drug-treated serum (A) and a magnified view (B) are shown. The chromatographic peaks are 1: puerarin, 2: paeoniflorin, 3: magnoflorine, 4: corydaline, 5: ligustilide, and 6: notoginsenoside R1. Figure 2 This is a comparison chart of reference standard, medicinal material, and finished drug fragments at level 2. Figure 3 The standard curve linear relationship graph; where the vertical axis is the peak area and the horizontal axis is the concentration (ng / ml). Figure 4 TIC graphs were obtained from multiple serum component measurements using this method. Detailed Implementation To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below with reference to embodiments. The described embodiments are only some embodiments of this application, not all of them, and the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to the conditions of specific manufacturers, and the implementation conditions not specified are generally the conditions in conventional experiments.

[0018] (1) Instruments, reagents and test samples Instruments: AB SCIEX Triple Quad 6500+ ultra-high performance liquid chromatography-mass spectrometry system: a product of AB SCIEX Corporation, USA, equipped with an electrospray ionization source and MultiQuant 3.0.2 data processing system; METTLER TOLEDO XPE205 electronic balance.

[0019] Reference standards: magnoflorin (purity 98.2%, batch number MUST-22020816), puerarin (purity 96.8%, batch number 110752-201816), corydaline (purity 99.9%, batch number 110726-201414), paeoniflorin (purity 96.7%, batch number 110736-201943), notoginsenoside R1 (purity 90.4%, batch number 110704-202129), ligustilide (purity 95.2%, batch number MUST-22102401), and purslane (purity 99.6%, batch number 111821-201303). Material: Hybrid SPE ® -Phosphorlipid solid phase extraction (SPE) Cartridge (Merck Group, Germany) Test sample: Neck pain granules (Shandong Mingren Furida Pharmaceutical Co., Ltd., batch number: 22101041).

[0020] (2) Chromatographic conditions Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, 0431, Mass Spectrometry).

[0021] Using Thermo Scientific TM Hypersil GOLD TM HPLC column (100 mm × 2.1 mm × 3 μm); mobile phase: A is acetonitrile, B is 0.1% formic acid; gradient elution conditions: 0-1 min, 5% A; 1-3 min, 5-10% A; 3-20 min, 10-25% A; 20-45 min, 25-98% A; flow rate: 0.3 mL / min; column temperature: 30 ℃; injection volume: 1 μL.

[0022] (3) Mass spectrometry conditions The parameters are set as follows: mode is set to mass detector, electrospray ionization source (ESI), positive / negative ion multi-reaction monitoring, spray voltage 5500V / -4500V; nebulizer temperature (TEM) 550℃; nebulizing gas (GS1) pressure 50 psi; auxiliary gas (GS2) pressure 50 psi; curtain gas pressure 30 psi.

[0023]

[0024] (4) Serum sample preparation SD rats were randomly divided into a control group and a treatment group, with 8 rats in each group. Rats in the treatment group were administered cervical pain granules via gavage, while the control group was administered the same amount of distilled water via gavage. Blood samples were collected from the inner canthus of the eyes at 15 min, 30 min, 1 h, 1.5 h, and 2 h after gavage. After 30 min, blood samples were collected at 2–8℃ and 3500–5000 rpm. -1 Centrifuge for 10-15 min and separate the supernatant serum. Mix the eight serum samples collected at each time point from both the blank control group and the drug treatment group, vortex thoroughly, and then take 800-1000 μL of serum from each group and time point. Add 2000-2500 μL of acetonitrile:acetone (9:1) solution to each sample, vortex for 10-15 min, and centrifuge (2-8℃, 10000-12000 r / min). -1 (10-15 min), collect the supernatant and transfer it to HybridSPE. ® In the column, the analyte is first washed with water (containing 0.1% formic acid) to remove salts and polar interfering substances, and then eluted with acetonitrile (containing 0.1% formic acid). The eluent is filtered through a 0.22 μm microporous membrane and then injected for analysis.

[0025] (5) Preparation of mixed reference standards Accurately weigh appropriate amounts of magnoflorin, puerarin, corydaline, paeoniflorin, notoginsenoside R1, ligustilide, and purslane reference standards, dissolve them in 70% methanol, and obtain samples with a mass concentration of 246 ng·mL. -1 573 ng·mL -1 248 ng·mL -1 167 ng·mL -1 128 ng·mL -1 376 ng·mL -1 125 ng·mL -1 Prepare a series of mixed standard stock solutions. Accurately pipette appropriate amounts of the mixed standard stock solution and dissolve in methanol to prepare a series of mixed standard solutions of varying concentrations for later use.

[0026] (6) System suitability test and negative interference test Under the above chromatographic conditions, accurately measure 1 μL each of the reference solution, drug-containing serum sample, and blank serum control sample, inject them into the chromatograph, and record the TIC chromatogram. Figure 1 By comparing with the reference standard (). Figure 2 In the chromatogram of the drug-containing serum sample, all the analytes were successfully located.

[0027] (7) Preparation of standard curves and investigation of linearity, determination of limit of quantitation Mouse blank plasma / tissue was collected, and a series of mixed standard solutions of different concentrations were added. The concentration of the analyte was plotted on the x-axis, and the ratio of the peak areas of each analyte was plotted on the y-axis. Figure 3 ), calculate the regression equation and correlation coefficient (R²) 2 The lowest concentration point of the standard curve is taken as the lowest concentration for quantification, i.e., the lower limit of quantification (LLQQ).

[0028] Table 1. Results of Standard Curve 1-1 Results of the Magnolia Alkine Standard Curve

[0029] Regression equation: Y =1076.2 X -312.03 Correlation coefficient: R 2 =0.9999 Table 1-2 Results of Puerarin Standard Curve

[0030] Regression equation: Y =80268 X -+253387 Correlation coefficient: R 2 =0.9993 Table 1-3 Results of the Corydalis Ethyl Standard Curve

[0031] Regression equation: Y =11621 X +4876 Correlation coefficient: R 2 =0.9999 Table of 1-4 Paeoniflorin Standard Curve Results

[0032] Regression equation: Y =2073.9 X +1552.2 Correlation coefficient: R 2 =0.999 Table of Standard Curve Results for Panax notoginseng Saponins R1

[0033] Regression equation: Y =482.94 X +35.995 correlation coefficient: R 2 =0.9996 Table 1-6 Results of the Standard Curve for Ligustilide

[0034] Regression equation:Y =1707.9 X -1984 Correlation coefficient: R 2 =0.9999 Table 1-7 Results of the Standard Curve for Imperatorin

[0035] Regression equation: Y =10999 X -14577 Correlation coefficient: R 2 =0.9991 (8) Precision test Samples with intermediate concentrations under the items (7) Preparation of standard curve and investigation of linear relationship, determination of limit of quantitation were injected and measured within one day, and measured continuously for 6 times. The peak area was recorded and the relative standard deviation was calculated. The RSD of each component was less than 2%. The results are shown in Table 2, indicating that the instrument has good precision.

[0036] Precision: Relative Standard Deviation (RSD %) = (Standard Deviation SD) / (Arithmetic Mean of Calculated Results) × 100% Table 2. Precision Data Table Table 2-1 Precision data of magnoflorine

[0037] Table 2-2 Precision Data of Puerarin

[0038] Table 2-3 Precision data of Corydalis yanhusuo

[0039] Table 2-4 Precision data of paeoniflorin

[0040] Table 2-5 Precision data of Panax notoginseng saponin R1

[0041] Table 2-6 Precision data of ligustilide

[0042] Table 2-7 Precision data of purslane glycosides

[0043] (9) Stability test After the test solution was prepared, 1 μL was accurately pipetted into the mass spectrometer, and the peak area was recorded. Measurements were then taken every 2 hours for 8 hours, and the relative standard deviation of the peak area was calculated. The results (Table 3) show that the solution of each analyte in the test sample exhibited good stability within 8 hours.

[0044] Table 3. Stability Data Table Table 3-1 Stability data of magnoflorine

[0045] Table 3-2 Stability data of puerarin

[0046] Table 3-3 Stability data of corydaline

[0047] Table 3-4 Stability data of paeoniflorin

[0048] Table 3-5 Stability data of Panax notoginsenoside R1

[0049] Table 3-6 Stability data of ligustilide

[0050] Table 3-7 Stability data of purslane glycosides

[0051] (10) Investigation of matrix effect Mouse blank plasma / tissue was collected and mixed standard solution was added for detection to obtain the peak area of ​​each compound. Separately, mixed standard solutions of corresponding concentrations were prepared using the mobile phase and analyzed to obtain the corresponding peak areas. The ratio of the two sets of peak areas was calculated to examine the matrix effect. The matrix effect was calculated according to the following formula, and the results (Table 4) showed a ratio between 85% and 105% (n=6), indicating that there was no significant matrix effect.

[0052] Matrix effect % = RA (substrate sample) / RA (methanol sample) × 100% Table 4 Results of matrix effect investigation

[0053] (11) Recovery test Six portions of the same batch of samples were accurately weighed, and appropriate amounts of each reference standard were added. This function was used to determine the content and calculate the recovery rate. The recovery rates of each analyte are shown in Table 5 below. The results show that the determination method is accurate.

[0054] Table 5. Recovery Rate Data Table

[0055] (12) Residual effect After injection at the highest concentration point of the standard curve, blank plasma samples were injected and analyzed to assess residual effects. The results showed that after systematic injection analysis, the highest concentration point sample and blank matrix sample were analyzed, repeated three times, and the response of the analyte in the blank matrix sample was measured. The residue in the blank sample did not exceed 25% of the lower limit of quantitation (Table 6) and did not affect the sample detection.

[0056] Tables 6 and 7 show the residual effects of the components.

[0057] (13) Confirmation by serum sample testing Based on the established serum detection method, seven components, including magnoflorine, puerarin, corydaline, paeoniflorin, notoginsenoside R1, ligustilide, and imperatorin, were quantitatively analyzed in eight batches of samples (2110, 2108, etc.). Results ( Figure 4 This indicates that the method can accurately determine the content of the above components in different batches of drug-containing serum samples. Although there are some fluctuations in the content of each component in different batches of samples, which may be related to factors such as individual differences in experimental animals and differences between batches of medicinal materials, statistical analysis shows that the differences in the content of each component are not significant and are generally within a controllable range.

Claims

1. A method for simultaneously determining 7 quality markers (Q-Markers) in the serum of Jingtong granules, characterized in that, The 7 quality markers are magnoflorine, puerarin, tetrahydropalmatine, paeoniflorin, notoginsenoside R1, ligusticum lactone and decursin; the serum sample is added with acetonitrile and acetone, vortexed, centrifuged, and the supernatant is taken and loaded into a HybridSPE column, first washed with water to remove salt and polar interferents, and then eluted with acetonitrile to elute the target analyte, the eluent is filtered through a microporous filter, and then determination is performed; high performance liquid chromatography-mass spectrometry is adopted, an electrospray ion source is adopted, and quantitative analysis is performed through positive / negative ion multiple reaction monitoring detection mode.

2. The method for simultaneously determining 7 quality markers (Q-Markers) in the serum of Jingtong granules according to claim 1, characterized in that, The specific processing steps of the serum sample are as follows: take 800-1000 μL of the serum sample, add 2000-2500 μL of an acetonitrile, acetone solution, the volume ratio of acetonitrile to acetone is 9:1, vortex for 10 min at 2-8 ℃ and 10000-12000 r / min -1 , centrifuge for 10-15 min, and take the supernatant.

3. The method for simultaneously determining 7 quality markers (Q-Markers) in the serum of Jingtong granules according to claim 1, characterized in that, The chromatographic conditions are as follows: mobile phase: A is acetonitrile and B is 0.1% formic acid; gradient elution conditions 0-1 min, 5% A; 1-3 min, 5-10% A; 3-20 min, 10-25% A; 20-45 min, 25-98% A; flow rate 0.3 mL / min; column temperature 30 DEG C; injection volume 1 μL.

4. The method for simultaneously determining 7 quality markers (Q-Markers) in the serum of Jingtong granules according to claim 1, characterized in that, The mass spectrometry conditions are as follows: mode setting is mass detector, electrospray ion source, positive / negative ion multiple reaction monitoring, spray voltage 5500V / -4500V; atomizer temperature 550 DEG C; atomizing gas pressure 50 psi; auxiliary gas pressure 50 psi; curtain gas pressure 30 psi.

5. The method for simultaneously determining 7 quality markers (Q-Markers) in the serum of the Chinese medicine neck pain granules according to claim 4, characterized in that, Mass spectrometry detection conditions of each quality marker: 。 6. Application of a method for simultaneously determining 7 quality markers in serum of Chinese medicine Jingtong granules in drug evaluation of Chinese medicine Jingtong granules.

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