A method for simultaneously detecting chiral and achiral components of Notopterygium coumarin and its application

Abstract

The invention discloses a method for simultaneously detecting chiral-achiral components of notopterygium coumarin and an application thereof, and belongs to the field of drug detection and analysis. The detection method adopts high performance liquid chromatography-tandem mass spectrometry for detection, wherein the chromatographic column adopted by the high performance liquid chromatography is Chiralpak IG, and the mobile phase is methanol-acetonitrile; the chiral components are oxidized peucedanum, hydrated oxidized peucedanum and notopterygium alcohol, and the achiral components are imperatorin, isoimperatorin, bergamot lactone and purpurogenol; the application is used for content determination and pharmacokinetic analysis of medicinal materials of notopterygium; the invention can simultaneously detect the configurations of oxidized peucedanum, hydrated oxidized peucedanum and notopterygium alcohol of notopterygium and imperatorin, isoimperatorin, bergamot lactone and purpurogenol in notopterygium extract and blood samples of rats after notopterygium gavage, and the peaks can be obtained within 25 minutes, the detection time is greatly shortened, and the separation of chiral components can be realized, the detection method is stable, and the detection result is reliable.

Description

Technical Field

[0001] The invention relates to the field of drug detection and analysis, and in particular to a method for simultaneously detecting chiral and achiral components of notopterygium coumarin and an application thereof. Background Art

[0002] Notopterygium wilfordii is the dried root and rhizome of Notopterygium wilfordii or Notopterygium wilfordii of the Umbelliferae family. It has the effects of dispelling cold, expelling wind and dampness, and relieving pain. It mainly contains coumarins, polyene acetylenes, sesquiterpenes, phenolic acids, steroids, flavonoids and other ingredients. As the characteristic ingredients of Umbelliferae plants, coumarin components are the earliest discovered and most studied components in Notopterygium wilfordii. They have anti-tumor, antibacterial, antiviral and other biological activities (China Journal of Traditional Chinese Medicine, 46(05): 1179-1190, 2021). They include bergamot lactone, notopterygium alcohol, imperatorin and purpurogenol. Among them, notopterygium alcohol and isoimperatorin are the quality control indicators of Notopterygium wilfordii in the 2020 edition of the Chinese Pharmacopoeia. A large number of literatures have reported on its pharmacological activities.

[0003] At present, the chemical composition detection of Notopterygium wilfordii slices and their compound preparations is mainly aimed at racemic compounds, or they are considered to be a single configuration for research. Chinese patent CN 108593833 B discloses an HPLC detection method for broad-leaved Notopterygium wilfordii, and simultaneously quantitatively detects five components in broad-leaved Notopterygium wilfordii, including purpurogenol, bergamot lactone, notopterygium wilfordii alcohol, and isoimperatorin. Octadecylsilane bonded silica gel is used as the stationary phase, wherein the mobile phase is 0.2-0.4% acetic acid aqueous solution (A), acetonitrile (B); the detection wavelength is 310-320nm, and the gradient elution is within 50min. Chinese patent CN 112881570 A uses HPLC to use acetonitrile-water as the mobile phase to gradiently elute notopterygium wilfordii alcohol and isoimperatorin in the compound preparation of Notopterygium wilfordii, with a detection wavelength of 305-315nm and a detection time of 55min. The document "Evaluation of the Effect of Different Drying Temperatures on the Quality of Notopterygium Wilfordii Residues Based on UPLC Fingerprint and Content Detection Method" reported a method for detecting three coumarin components in Notopterygium Wilfordii by UPLC using acetonitrile-0.1% phosphoric acid as the mobile phase for gradient elution.

[0004] The above existing reports generally have the problems of only being able to detect a small number of components and being unable to detect enantiomers, having a long retention time and low detection efficiency, and even if there is a method for detecting enantiomers, the retention time is also long.

[0005] In addition, there are few studies on the blood-entering components of coumarins in Notopterygium wilfordii. The closest existing technology is "Simultaneous determination of seven coumarins by UPLC-MS / MS: Application to a comparative pharmacokinetic study in normal and arthritic rats after oral administration of Huo Luo Xiao Ling Dan or single-herb extract" (J ChromatogrB Analyt Technol Biomed Life Sci, 2015; 991: 108-117), which discloses the liquid mass detection of three coumarin compounds, imperatorin, isoimperatorin and notopterygium wilfordii extract. The three coumarin compounds in Notopterygium wilfordii were simultaneously quantified by UPLC-MS / MS and applied to the pharmacokinetic study of rats after oral gavage. The chromatographic conditions are: in Shim-pack On an XR-ODS column (75 mm × 3.0 mm, 2.2 μm particles), a mobile phase consisting of methanol (A) and 0.05% formic acid water (B) was used for gradient elution at a flow rate of 0.4 mL / min. However, no relevant research was conducted on its enantiomers, and only a few coumarins were studied.

[0006] However, there are some differences between the research on non-single enantiomer compounds and enantiomeric compounds of Notopterygium wilfordii, mainly reflected in the fact that different enantiomers may have different pharmacological and toxicological effects. Taking notopterygii alcohol as an example, "Separation and quantification of notopterol enantiomers in notopterygii rhizoma et radix using solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry" (JPharm Biomed Anal, 186: 113255, 2021) uses high performance liquid chromatography-tandem mass spectrometry on a chiral column to separate the enantiomers of notopterygii alcohol and detect the content. The chromatographic conditions are: on a Chiralpak IA column, the mobile phase consists of acetonitrile-water (50:50, v / v), isocratic elution, and a flow rate of 0.6 mL / min for 50 min. The study found that different configurations of notopterygii alcohol enantiomers in notopterygii have different contents, among which (+)-notopterygii alcohol has a higher content in notopterygii, which is 1.98-2.48 times that of (-)-notopterygii alcohol. Detecting the content of other enantiomers in notopterygii is of great significance for the study of its pharmacokinetics.

[0007] The matrix in biological samples is complex and the drug concentration is low. HPLC technology has problems such as low sensitivity and long analysis time for the detection of Notopterygium wilfordii content in plasma, and it is difficult to completely separate isomers or enantiomers with similar polarity. UPLC-MS / MS has not yet risen to the configuration level in the research of Notopterygium wilfordii components. For the enantiomers of chiral components, only the racemic compounds are detected, or they are considered to be studied as a single configuration, while the simultaneous detection of many enantiomers in the active ingredients and other components with the same parent core has not been studied, which makes the chiral-achiral research of Notopterygium wilfordii still in a blank stage. Summary of the invention

[0008] One of the purposes of the present invention is to provide a method for simultaneously detecting chiral and achiral components of Notopterygium coumarin in order to solve the above-mentioned problem.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows: a method for simultaneously detecting chiral and achiral components of Notopterygium coumarin, using high performance liquid chromatography-tandem mass spectrometry for detection, wherein:

[0010] The chromatographic column used in the high performance liquid chromatography is Chiralpak IG, and the mobile phase is methanol-acetonitrile;

[0011] The chiral components are oxidized peucedanum, hydrated oxidized peucedanum and chasteberry alcohol, and the achiral components are imperatorin, isoimperatorin, bergamot lactone and purslane glycoside.

[0012] As a preferred technical solution, the volume ratio of the mobile phase is methanol: acetonitrile = 75: 25. The chromatographic condition of this ratio obtained through a large number of experiments is conducive to the complete separation of the chiral components in Notopterygium wilfordii with short elution time and high efficiency.

[0013] As a preferred technical solution, carbamazepine is used as an internal standard during detection. The use of the internal standard method for content determination can eliminate the interference caused by the matrix on the one hand, and can eliminate the system error on the other hand. The present invention screens internal standards through a large number of experiments, and examines acetaminophen, puerarin, diphenhydramine and carbamazepine as internal standards respectively, and comprehensively examines their chromatographic behavior, mass spectrometry response and recovery rate as indicators, and finally preferably selects carbamazepine as the internal standard.

[0014] As a preferred technical solution, during detection, an HPLC-CD high-performance chromatograph is used to determine the configuration of the chiral component. An HPLC-CD high-performance chromatograph is used to determine the configuration of each chiral compound on Chiralpak IG. Through UV and CD chromatograms, it is determined that under methanol-acetonitrile conditions, the first elution peak of hydrated oxidized praeruptorin is (+) configuration, and the second peak is (-) configuration; the first elution peak of oxidized praeruptorin is (-) configuration, and the second peak is (+) configuration; the first peak of notopterygium alcohol is (+) configuration, and the second peak is (-) configuration.

[0015] The second object of the present invention is to provide an application of the above method, and the technical scheme adopted is to be used for the determination of the medicinal material content and pharmacokinetic analysis of Notopterygium incisum. A fully complete pharmacokinetic study of Notopterygium incisum can reveal the mechanism of action of Notopterygium incisum, thereby providing guidance for the rational use of drugs, more accurately predicting the efficacy and toxicity, and optimizing the drug treatment plan.

[0016] As a preferred technical solution, the method is used to detect drug-containing plasma in rats after intragastric administration of Notopterygium wilfordii.

[0017] As a further preferred technical solution, blood sample pretreatment is performed before detection.

[0018] Compared with the prior art, the advantages of the present invention are:

[0019] (1) The present invention adopts a UPLC-MS / MS method with specific detection conditions, which can simultaneously detect oxidized peucedanum, hydrated oxidized peucedanum, peucedanum alcohol, imperatorin, isoimperatorin, bergamot lactone, and purpurogenol in the extract of Notopterygium wilfordii and the blood samples of rats after intragastric administration of Notopterygium wilfordii, and the peaks can be detected within 25 minutes, which greatly shortens the detection time and can achieve the separation of chiral components;

[0020] (2) The present invention selects the chiral stationary phase method and uses the chiral column Chiralpak IG to perform enantiomeric separation on the chiral components in Notopterygium wilfordii. The enantiomeric chromatographic peak separation R>1.5. The chromatograms of the 10 coumarin components detected have a relatively stable baseline and a good peak shape. The quantitative calculation can be performed directly through the retention time and peak area ratio of the chromatogram combined with the standard curve, which is conducive to batch detection.

[0021] (3) The UPLC-MS / MS method adopted in the present invention uses blood sample pretreatment, mobile phase methanol-acetonitrile, and an isocratic elution process, which can effectively reduce the detection effect of plasma substrate components and secondary metabolites of the original drug on coumarin components, and has fewer impurity peaks, providing conditions for quantitative detection. The results of methodological studies have determined that the detection method is stable and the detection results are reliable. It can also be used for the pharmacokinetic study of chiral-achiral components of Notopterygium incisum, but is not limited to this. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the chromatogram of three chiral components under the condition of methanol-acetonitrile = 70:30 in Example 1;

[0023] Figure 2 This is the chromatogram of three chiral components under the condition of methanol-acetonitrile=80:20 in Example 1;

[0024] Figure 3 This is the chromatogram of chiral-achiral components under the condition of methanol-acetonitrile=70:30 in Example 1;

[0025] Figure 4 This is the chromatogram under the condition of acetonitrile-water=50:50 in Example 1;

[0026] Figure 5 This is the chromatogram of chiral-achiral components under the condition of acetonitrile-water=50:50 in Example 1;

[0027] Figure 6 UV and CD chromatograms of three chiral compounds in Example 2;

[0028] Figure 7 This is the chiral-achiral component-specific chromatogram of blank plasma in Example 3;

[0029] Figure 8 This is the chiral-achiral component-specific chromatogram of blank plasma to which chiral-achiral components are added in Example 3;

[0030] Fig. 9 This is a chromatogram exclusive for chiral-achiral components in drug-containing plasma in Example 3;

[0031] Fig.10This is a time-blood concentration curve of the chiral-achiral components in Notopterygium wilfordii of Example 4;

[0032] Fig.11 This is the TIC diagram of each component to be tested in Example 3 under the condition of methanol-acetonitrile = 75:25. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the accompanying drawings.

[0034] Example 1

[0035] Screening of test conditions:

[0036] 1.1 Selection of chromatographic column and chromatographic conditions:

[0037] Using Chiralpak IA, Chiralpak IC, and Chiralpak IG chiral columns under normal phase, reverse phase, and polar organic phase conditions, the three chiral components of oxidized desperadoxin, hydrated oxidized desperadoxin, and notopterygium wilfordii alcohol were separated by changing the type of stationary phase, the type of mobile phase, and the ratio of mobile phase. It was found that the Chiralpak IG column has a better chiral recognition effect on these three enantiomers.

[0038] Under normal phase conditions, the mobile phase is usually alkane-alcohol modifiers, where alkane is the non-polar part of the mobile phase and has little effect on chiral separation. Normally, n-hexane is selected, and conventional isopropanol and anhydrous ethanol are selected as alcohol modifiers. Isocratic elution is performed at v = 1.0 mL / min. As shown in the table below, under n-hexane-isopropanol mobile phase conditions, Chiralpak IA can only achieve complete separation of the three chiral components under the condition of 90:10, but the analysis time of hydrated oxidized pursedrin is relatively long, about 45 minutes. Under normal phase conditions, Chiralpak IC can only achieve chiral separation of hydrated oxidized pursedrin, and the other two chiral components are partially separated, and the analysis time is long. Chiralpak IG has good separation of the three chiral components under the condition of 70:30, but qianghuo alcohol still cannot be completely separated within 30 minutes.

[0039] Under reverse phase conditions, water was used as the base, and methanol and acetonitrile were used as organic modifiers to separate the three coumarin chiral components. When the mobile phase was methanol-water, the column pressure of the three chiral columns was high. At the maximum column pressure, the flow rate v=0.3mL / min was maintained, resulting in slow peak elution. Therefore, the mobile phase composed of acetonitrile-water was mainly studied, and isocratic elution was performed under v=0.5mL / min. As shown in the table below, on the Chiralpak IA column, the three chiral components can be completely separated under the conditions of acetonitrile-water=50:50 (v / v), but the analysis time has not been well improved; with acetonitrile-water as the mobile phase, on the Chiralpak IC column, hydrated oxidized praeruptosin and angelica alcohol can only be partially separated; on the Chiralpak IG column, the three chiral components can be completely separated under this mobile phase composition, but the analysis time is still long and has not been optimized.

[0040] Under polar organic phase conditions, methanol-acetonitrile was used as the mobile phase and isocratic elution was performed at v = 0.5 mL / min. As shown in the table below, on Chiralpak IA and Chiralpak IC columns, only oxidized praeruptosin could achieve complete chiral separation, while on Chiralpak IG column, all three chiral components could achieve enantiomeric separation with good separation effect. Considering the peak elution time and separation degree, methanol-acetonitrile (75:25) was selected to achieve the separation of chiral compounds.

[0041] Table A Enantiomer separation of three chiral components using Chiralpak IA column in different mobile phases

[0042]

[0043] Table B Enantiomer separation of three chiral components using Chiralpak IC column in different mobile phases

[0044]

[0045] Table C Enantiomer separation of three chiral components using Chiralpak IG column in different mobile phases

[0046]

[0047] In Tables A to C, “-” indicates a single peak and no separation was observed within 60 minutes; “*” indicates no peak within 60 minutes.

[0048] 1.2 Selection of internal standard

[0049] The inventors, through a large number of experiments, selected components that do not have a chiral center and are not present in Notopterygium as internal standards, and finally determined quercetin, diphenhydramine, acetaminophen, puerarin and carbamazepine as five components, and the present invention investigated these five components respectively to determine their mass spectrometry conditions. It was found that the responses of diphenhydramine and carbamazepine were high, and the mass spectrum peaks were relatively clean, but by observing the chromatogram, it was found that diphenhydramine had two chromatographic peaks, indicating that impurities may be present, so carbamazepine was finally selected as the internal standard.

[0050] Table D Mass spectrometry conditions for various internal standards

[0051]

[0052] 1.3 Different mobile phase ratios:

[0053] The separation of the three chiral components was investigated using a Chiralpak IG column with methanol-acetonitrile = 70:30 (e.g. Figure 1 ), methanol-acetonitrile = 80:20, the separation of the three chiral components (such as Figure 2 ), separation of chiral-achiral components under the condition of methanol-acetonitrile = 70:30 (such as Figure 3 );

[0054] As can be seen from the figure, the three chiral components can be completely separated under methanol-acetonitrile conditions.

[0055] It was also investigated that the separation of three chiral components (e.g. Figure 4 ), and the separation of some non-chiral components (such as Figure 5 ), this method can be further applied to liquid quality.

[0056] Embodiment 2:

[0057] The detection method includes the following steps:

[0058] (1) Preparation of standard stock solution: Take 5 mg each of oxidized peucedanum, hydrated oxidized peucedanum, notopterygium alcohol, imperatorin, isoimperatorin, bergamot lactone, and purpurogenol, accurately weigh, place in a 5 ml volumetric flask, add methanol to dissolve and dilute to the mark, shake well, and obtain a 1.00 mg / mL standard stock solution;

[0059] (2) Preparation of internal standard working solution

[0060] Take 5 mg of carbamazepine, weigh it accurately, put it in a 5 mL volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain a 1.00 mg / mL internal standard stock solution; accurately pipette an appropriate amount of the internal standard stock solution, dilute it with methanol, and prepare an internal standard solution with a concentration of 0.1 μg / mL;

[0061] (3) Plasma test sample processing: plasma from rats gavaged with Notopterygium wilfordii was taken, the internal standard was added and mixed, ethyl acetate was added, vortexed, and centrifuged; the supernatant was taken, dried with nitrogen gas, re-dissolved with methanol, vortexed, centrifuged, and the supernatant was taken and filtered through a membrane to obtain a plasma test sample;

[0062] (4) Preparation of standard curve: adding oxidized praeruptorin, hydrated oxidized praeruptorin, notopterygium alcohol, imperatorin, isoimperatorin, bergamot lactone, and purpurogenol to blank rat plasma to prepare a series of standard curve samples; using the method of step (3), replacing the plasma of rats gavaged with notopterygium wilfordii with the standard curve samples to prepare standard curve plasma samples; analyzing the standard curve plasma samples by UPLC-Qtrap-MS / MS; using the ratio of the peak area of ​​the analyte to the internal standard (Y) as the ordinate and the concentration of the analyte (X) as the abscissa, performing regression calculation using the weighted least squares method to obtain each standard curve, with the calculation weight being 1 / X 2 ;

[0063] (5) Liquid phase conditions were as follows: chromatographic column, Chiralpak IG (250×4.6 mm id, 5 μm); mobile phase, methanol-acetonitrile (75:25, v / v); flow rate, 0.5 mL / min; column temperature, 25°C; injection volume, 5 μL; detection time, 25 min;

[0064] (6) Mass spectrometry conditions were: ion source, ESI; positive and negative ion mode detection; temperature, 550°C; spray voltage, +5500V / -4500V; curtain gas (CUR), 20Psi; nebulizer gas pressure (GAS1), 55Psi; auxiliary heating gas (GAS2), 55Psi; entrance voltage (EP), +10V / -10V; collision chamber exit voltage (CXP), +i3V / -13V. The mass spectrometry conditions for each component are shown in Table 1.

[0065] Table 1 Mass spectrometry conditions monitoring scanning mode

[0066]

[0067] (7) Determination of configuration: HPLC-CD (JASCO) was used to determine the elution order of each chiral compound under the condition of methanol-acetonitrile. It was determined that the first elution peak of hydrated oxidized despermatin was (+) configuration and the second peak was (-) configuration; the first elution peak of oxidized despermatin was (-) configuration and the second peak was (+) configuration; the first peak of notopterygium alcohol was (+) configuration and the second peak was (-) configuration. The results are shown in Figure 6 .

[0068] Embodiment 3:

[0069] Establishment of detection method and determination of multiple components in Notopterygium wilfordii

[0070] 1. Materials

[0071] 1.1 Drugs: oxidized purpurogenol, hydrated oxidized purpurogenol (provided by Chengdu Desite Co., Ltd.), notopterygium alcohol, imperatorin, purpurogenol (provided by Chengdu Mingrui Technology Co., Ltd.), isoimperatorin, bergamotolactone, carbamazepine (provided by Weiqiqi Biological Co., Ltd.)

[0072] 1.2 Reagents: ethyl acetate (provided by Concord Technology Co., Ltd.), methanol, acetonitrile (provided by ThermoScientific Co., Ltd.)

[0073] 1.3 Animals: SPF grade SD rats, weighing 200g-250g, male, all provided by Beijing Huafukang Biotechnology Co., Ltd.

[0074] 1.4 Instrument: Aglient 1290 ultra-high performance liquid chromatograph, equipped with AB 6500 quadrupole linear ion trap mass spectrometer (Qtrap-MS / MS)

[0075] Chromatographic conditions: chromatographic column, Chiralpak IG (250×4.6 mm id, 5 μm); mobile phase, methanol-acetonitrile (75:25, V / V); flow rate, 0.5 mL / min; column temperature, 25°C, injection volume, 5 μL.

[0076] Mass spectrometry conditions: ion source, ESI; positive and negative ion mode detection; temperature, 550°C; spray voltage, +5500V / -4500V; curtain gas (CUR), 20Psi; nebulizer gas pressure (GAS1), 55Psi; auxiliary heating gas (GAS2), 55Psi; entrance voltage (EP), +10V / -10V; collision chamber exit voltage (CXP), +13V / -13V.

[0077] 2. Methods and Results

[0078] 2.1 Plasma sample collection

[0079] Five SD rats in the blank group were selected for blood collection from the abdominal aorta. The rats were fasted for 12 hours before blood collection and were allowed to drink water freely. The collected whole blood was placed in a test tube containing sodium heparin, centrifuged at 4°C, 3500rpm for 10min, and the supernatant was aspirated and frozen in a -80°C refrigerator.

[0080] 2.2 Plasma sample processing method

[0081] The frozen plasma was thawed at room temperature, 100 μL was taken and placed in a 1.5 mL EP tube, 10 μL carbamazepine (0.1 ug / mL) was added, and then 1 mL ethyl acetate was added, vortexed and mixed for 5 min, and high-speed centrifuged at 4°C and 12000 r / min for 10 min. The supernatant was transferred to a clean test tube and blown dry with nitrogen at room temperature. The residue was vortexed and dissolved with 200 μL mobile phase, and high-speed centrifuged at 4°C and 12000 r / min for 10 min, and the supernatant was taken for analysis.

[0082] 2.3 Preparation of standard and quality control samples

[0083] 10 μL of a mixed standard working solution containing 10 components was added to 90 μL of blank plasma, so that the concentration of each component was: 0.25-100 ng / ml of oxidized peucedanum, 0.025-100 ng / mL of isoimperatorin and hydrated oxidized peucedanum, 12.5-2500 ng / mL of notopterygium wilfordii alcohol, 0.1-100 ng / mL of imperatorin, 0.025-50 ng / mL of bergamot lactone, and 1.25-200 ng / mL of purslane glycosides. At least 5 concentrations (including the lowest and highest concentrations) were set within this range. After adding the internal standard and performing sample pretreatment, a series of standard samples with different concentrations were obtained.

[0084] 10 μL of mixed standard working solution containing 10 components was added to 90 μL of blank plasma, so that the concentrations of each component were as follows: 0.75, 5, and 80 ng / mL of oxidized desperadoxin (0.375, 2.5, and 40 ng / mL of single enantiomer), 0.075, 5, and 80 ng / mL of hydrated oxidized desperadoxin (0.045, 3, and 48 ng / mL (+)-hydrated oxidized desperadoxin, 0.03, 2, and 32 ng / mL (-)-hydrated oxidized desperadoxin), and 3.5 μL of quinoline alcohol. 7.5, 250, 2000 ng / mL (18.75, 125, 1000 ng / mL single enantiomer), bergamot lactone 0.075, 2.5, 40 ng / mL, imperatorin 0.3, 5, 80 ng / ml, isoimperatorin 0.075, 5, 80 ng / mL and purpurogenol 3.75, 25, 160 ng / mL, and quality control samples with low, medium and high concentrations were obtained after adding internal standards and performing sample pretreatment.

[0085] 2.4 Methodological validation

[0086] 2.4.1 Exclusivity

[0087] The specificity of the method was evaluated by comparing the extracted ion patterns of each component in blank plasma of rats, blank plasma containing 10 components and 1 internal standard, and drug-containing plasma collected after intragastric administration of Notopterygium wilfordii.

[0088] 2.4.2 Linear range and minimum limit of quantification

[0089] Linearity was determined by standardizing at least five different concentrations of each component. The weighted least squares method (1 / x 2 ) Linear regression was performed on the ratio of the peak area of ​​the analyte to the internal standard under the same ion mode and the corresponding concentration to fit the standard curve. The linear range refers to the range of the lowest and highest concentrations that constitute the standard curve, the lowest limit of quantification (LLOQ) refers to the lowest concentration level that constitutes the calibration curve, and the correlation coefficient (R) was used to evaluate the linear correlation of the data within the respective concentration range of each component.

[0090] 2.4.3 Accuracy and precision

[0091] QC samples of three concentrations, low, medium and high, were prepared, and five samples were prepared for each concentration. Three consecutive batches of QC samples were measured within and between days. The measured concentrations of QC samples were calculated according to the accompanying standard curve, and the intra-day and inter-day accuracy and precision of the method were evaluated. The measured enantiomer concentrations were compared with the standard addition values, and the intra-day and inter-day precision (RSD) of the method was investigated based on the QC sample measurement results. The accuracy results were expressed as RE values.

[0092] 2.4.4 Extraction recovery and matrix effect

[0093] The extraction recovery was calculated by comparing the peak areas of the analytes in blank plasma before and after extraction. Before extraction refers to the plasma sample pretreatment after adding the mixed standard and internal standard to the blank plasma, and after extraction refers to the plasma sample pretreatment before adding the mixed standard and internal standard to the blank plasma. The matrix effect was calculated by comparing the peak area of ​​the analyte in blank plasma after extraction with the peak area of ​​the analyte dissolved in methanol at the same concentration. The extraction recovery and matrix effect of the 10 components were evaluated at three QC concentration levels: low, medium, and high, with 5 samples at each concentration.

[0094] 2.4.5 Stability

[0095] This experiment investigated the stability of the concentrations of each component in low and high QC plasma samples after being placed at 4°C for 24 hours, at room temperature for 12 hours, at -80°C for 45 days, and after three freeze-thaw cycles.

[0096] 2.5 Quantitative determination of ingredients in Notopterygium root solution

[0097] Take Notopterygium wilfordii medicinal material, extract it with 10 times the amount of 75% ethanol under reflux twice, each time for 1 hour, filter, combine the filtrate, concentrate the medicinal solution by rotary evaporation until there is no alcohol taste, and make up the volume with physiological saline to 1g / ml (raw drug amount) of Notopterygium wilfordii extract to obtain Notopterygium wilfordii extract.

[0098] Take 100 μL of Notopterygium root extract, dilute it step by step with methanol to 2000 times, centrifuge it at 4℃12000rpm for 15min, take 10 μL of supernatant and add it to 90 μL of blank plasma, then add internal standard working solution for sample pretreatment, and UPLC-Qtrap-MS / MS quantitative analysis. The results are as follows: Fig.11 As shown, it should be noted that the TIC diagram does not need to be separated, and the overlapping components will peak in different ion channels, so as long as the two configurations of each chiral component can be separated from each other, it will be sufficient.

[0099] 2.6 Experimental Results

[0100] 2.6.1 Exclusivity

[0101] The UPLC-Qtrap-MS / MS extracted ion patterns of blank plasma, blank plasma with 10 components and internal standard, and drug-containing plasma with internal standard are shown in Figure 2. Figure 7 , Figure 8 and Fig. 9 As shown, it can be seen from the figure that there is no interference from endogenous components.

[0102] 2.6.2 Linear range and minimum limit of quantification

[0103] The standard curves of the 10 components were linear, and the correlation coefficients were all greater than 0.99. The standard curves, linear ranges, minimum quantification limits and correlation coefficients of each component are listed in Table 2. These data show that the method has good linearity and is suitable for the quantitative detection of the 10 components.

[0104] Table 2 Standard curves and quantification limits of chiral-achiral components

[0105]

[0106] 2.6.3 Accuracy and precision

[0107] The results of the determination of QC samples of chiral-achiral components at different concentrations were used to evaluate the intra-day and inter-day precision and accuracy of the method. Accuracy is expressed as relative error (RE), which should not exceed ±15%; precision is expressed as relative standard deviation (RSD), which should not exceed 15%. RE was within ±15%, and RSD was within 15%, indicating that the accuracy and precision of the method met the requirements of quantitative determination. The results are listed in Table 3. At different concentration levels, the intra-day relative error (RE) and relative standard deviation (RSD) of the determination results of chiral-achiral components were -0.38%-14.84% and 3.12%-14.77%, respectively, and the inter-day relative error (RE) and relative standard deviation (RSD) were -1.02%-14.89% and 4.53%-13.83%, respectively, all within 15%, indicating that the method has good precision and accuracy in detecting chiral-achiral components in plasma.

[0108] Table 3 Accuracy and precision of chiral-achiral components

[0109]

[0110]

[0111] 2.6.4 Extraction recovery and matrix effect

[0112] The recovery rate of each concentration of the target analyte is greater than 60%, and the matrix effect of each concentration of the target analyte is between 64% and 119%. According to the requirements, the RSD of the extraction recovery rate and matrix effect of each component at each concentration should not exceed 15%. The results are shown in Table 4. The data show that the extraction recovery rate and matrix effect meet this requirement.

[0113] Table 4 Extraction recovery and matrix effect of chiral-achiral components

[0114]

[0115]

[0116] 2.6.5 Stability

[0117] The QC samples of low and high concentrations of chiral-achiral components were subjected to short-term storage stability (4°C for 24 hours), room temperature storage stability (25°C for 12 hours), long-term storage stability (-80°C frozen for 45 days), and repeated freeze-thaw stability (-80°C frozen to room temperature 25°C thaw, repeated 3 times). Each sample was paralleled with 5 samples, and the stability analysis results under 4 different conditions are shown in Table 5. Table 5 shows that they are relatively stable under general experimental conditions, and the RSD under all conditions is within 15%, which meets the requirements of the pharmacopoeia for quantitative methods, indicating that the storage and handling of the samples under the above conditions are relatively stable and have no significant effect on the analysis.

[0118] Table 5 Stability test results of chiral-achiral components (n=5, mean ± SD)

[0119]

[0120]

[0121] 2.7 Quantitative determination of ingredients in Notopterygium root solution

[0122] The verified UPLC-Qtrap-MS / MS method was used to quantitatively analyze the extract of Notopterygium wilfordii.

[0123] Extraction method of Notopterygium incisum extract: Take Notopterygium incisum medicinal material, extract it with 10 times the amount of 75% ethanol reflux twice, each time for 1 hour, filter, combine the filtrate, concentrate the medicinal solution by rotary evaporation until there is no alcohol taste, and make up the volume with physiological saline to 1g / ml (raw drug amount) of Notopterygium incisum extract, to obtain Notopterygium incisum extract;

[0124] Take 100uL of Notopterygium root extract, dilute it step by step to 2000 times with methanol, centrifuge it at 12000rpm at 4℃ for 15min, take 10uL of supernatant and add it to 90uL of blank plasma, then add internal standard working solution for sample pretreatment and UPLC-Qtrap-MS / MS quantitative analysis.

[0125] The results are shown in Table 6. It can be seen that the contents of the 10 chiral-achiral components in the solution range from 12.01 to 5054.67 μg / mL

[0126] Table 6 Content of chiral-achiral components in Notopterygium wilfordii medicinal solution

[0127]

[0128]

[0129] Experimental Example 4: Pharmacokinetics of the main components in rat plasma after oral administration of Notopterygium wilfordii

[0130] 1. Medication and Collection of Plasma Samples

[0131] Rats were adaptively fed for one week and fasted for 12 h before the experiment. They had free access to water. Six rats were gavaged with Notopterygium wilfordii extract at a dose of 3 g / kg. 0.5 mL of blood was collected from the inner canthus of the rats at 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, and 24 h after administration and placed in a test tube containing sodium heparin. The blood was centrifuged at 4°C, 3500 rpm for 10 min to separate the plasma, which was then stored in a -80°C refrigerator for testing.

[0132] 2. Plasma Sample Pretreatment

[0133] Take 100 μL of plasma sample, place it in a 1.5 mL EP tube, add 10 μL of carbamazepine (0.1 ug / mL), then add 1 mL of ethyl acetate, vortex mix for 5 minutes, centrifuge at 4°C and 12000 r / min for 10 minutes, transfer the supernatant to a clean test tube, and blow dry with nitrogen at room temperature. Vortex and dissolve the residue in 200 μL of mobile phase, centrifuge at 4°C and 12000 r / min for 10 minutes, and take the supernatant for analysis.

[0134] 3. Pharmacokinetic studies

[0135] According to the plasma sample pretreatment method under item "2", the UPLC-Qtrap-MS / MS conditions established under item "1.4" in Example 1 were used for detection. A running standard curve was established for each analytical batch of samples, and the peak areas of each component to be tested and the internal standard were recorded. The blood drug concentrations of each component at different time points after administration to rats were calculated. The DAS2.0 software was used to perform non-compartmental model fitting on the obtained blood drug concentrations at each time point, and the statistical moment method was used to calculate the pharmacokinetic parameters. The results are shown in Table 7 and Fig.10 .

[0136] Table 7 Pharmacokinetic parameters of chiral-achiral components of Notopterygium wilfordii

[0137]

[0138]

[0139] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for simultaneously detecting chiral and achiral components of Notopterygium coumarin, characterized in that: High performance liquid chromatography-tandem mass spectrometry was used for detection, The chromatographic column used in the high performance liquid chromatography is Chiralpak IG, the mobile phase is methanol-acetonitrile, and the volume ratio of methanol to acetonitrile is (70-80):(30-20); The chiral components are oxidized peucedanum, hydrated oxidized peucedanum and chasteberry alcohol, and the achiral components are imperatorin, isoimperatorin, bergamot lactone and purslane glycoside.

2. The method according to claim 1, characterized in that The volume ratio of the mobile phase is methanol:acetonitrile=75:

25.

3. The method according to claim 1, characterized in that Carbamazepine was used as the internal standard during the detection.

4. The method according to claim 1, characterized in that: During detection, HPLC-CD high performance chromatography is used to determine the configuration of the chiral component.

5. Use of the method according to any one of claims 1 to 4, characterized in that: Used for the determination of medicinal content and pharmacokinetic analysis of Notopterygium wilfordii.

6. The use according to claim 5, characterized in that: The method was used to detect drug-containing plasma in rats after intragastric administration of Notopterygium wilfordii.

7. The use according to claim 6, characterized in that: Blood samples were pre-treated before testing.

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