Method for simultaneously detecting various components in callicarpa nudiflora medicinal material

Abstract

The invention provides a method for simultaneously detecting multiple components in a callicarpa nudiflora medicinal material. Specifically, the invention provides the detection method for the callicarpa nudiflora medicinal material based on an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer, the target components detected by the method comprise geniposidic acid, decaffeoyl verbascoside, caffeic acid, cistanche glycoside F, protocatechuic aldehyde, ferulic acid, luteoloside, verbascoside, isoverbascoside, 2-acetyl eugenol glycoside and luteolin. The method disclosed by the invention can be used for accurately, quickly and comprehensively detecting and evaluating the quality of the callicarpa nudiflora medicinal material.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical analysis, and particularly to a method for simultaneously detecting multiple components in Callicarpa nudiflora Hook.ex Arn. Background Art

[0002] Callicarpa nudiflora Hook.ex Arn. is a traditional Li ethnic medicine. It is the dried leaf of Callicarpa nudiflora Hook.ex Arn., a plant of Verbenaceae family, and has the effects of anti-inflammatory, detoxifying, astringing, and hemostasis. As early as in the 1977 edition of the Chinese Pharmacopoeia, Callicarpa nudiflora Hook.ex Arn. was included, and its anti-inflammatory and hemostatic activities have been widely recognized.

[0003] The chemical constituents in Callicarpa nudiflora Hook.ex Arn. are complex, mainly including flavonoids, phenylpropanoids, saponins, monosaccharides and polysaccharides. At present, the quality control of Callicarpa nudiflora Hook.ex Arn. in the Chinese Pharmacopoeia mainly focuses on the content control of acteoside and verbascoside. However, with the in-depth research, the number of active ingredients in Callicarpa nudiflora Hook.ex Arn. that have attracted attention has increased. Therefore, there is a need in the art to provide a method that is more suitable for further research on Callicarpa nudiflora Hook.ex Arn. and its quality control. Summary of the Invention

[0004] The purpose of the present invention is to provide an accurate, rapid and comprehensive detection method that can simultaneously determine 11 chemical components in Callicarpa nudiflora Hook.ex Arn.

[0005] In the first aspect of the present invention, a detection method for Callicarpa nudiflora Hook.ex Arn. is provided, wherein the target components detected by the method include: geniposidic acid, decaffeoylacteoside, caffeic acid, cistanoside F, protocatechuic aldehyde, ferulic acid, luteoloside, verbascoside, isoverbascoside, 2-acetylacteoside and luteolin;

[0006] The method includes the steps:

[0007] (i) Sample preparation:

[0008] (a1) Preparation of the sample to be tested: Weigh precisely the powder of Callicarpa nudiflora Hook.ex Arn., use 50 - 75% v / v alcohol-water as the extraction solvent for ultrasonic or reflux extraction, make up the volume, and obtain the sample to be tested after passing through a microporous filter membrane;

[0009] (a2) Preparation of the reference substance solution: Weigh precisely the reference substances of each target component, dilute with a solvent and make up the volume to obtain a mixed reference substance solution;

[0010] (ii) Inject the sample to be tested and the reference substance solution into an ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometer for detection respectively,

[0011] wherein the chromatographic conditions include:

[0012] (b1) The chromatographic column is an octadecylsilyl bonded silica column;

[0013] (b2) The mobile phase is (0.1 ± 0.02)% formic acid in water (A) - acetonitrile (B); and

[0014] (b3) Gradient elution, and the elution program is as follows: 0 - 15 min, 10 - 20% B; 15 - 16.5 min, 20 - 90% B; 16.5 - 18.5 min, 90% B; 18.5 - 20 min, 90 - 10% B;

[0015] Among them, the mass spectrometry conditions include:

[0016] Ionization mode: ESI - mode, spray voltage 4500 ± 100 V; Ionization temperature (TEM): 550 ± 20 °C; Nebulizing gas (Gas 1): 55 ± 5 kPa; Auxiliary gas (Gas 2): 55 ± 5 kPa; Curtain gas (CUR): 30 ± 3 kPa; Collision gas (CAD) Medium; The scanning mode is multiple reaction monitoring (MRM);

[0017] The mass spectrometry parameter values of each ion pair are: The mass spectrometry parameter values of each ion pair are:

[0018]

[0019] In another preferred example, the method further includes: (iii) According to the chromatogram obtained in step (ii), quantify each target component based on the peak area, so as to obtain the content of each target component in the Callicarpa nudiflora Hook. et Arn. medicinal material.

[0020] In another preferred example, in step (a1), the Callicarpa nudiflora Hook. et Arn. medicinal material powder passes through a No. 4 sieve.

[0021] In another preferred example, in step (a1), the alcohol is methanol or ethanol.

[0022] In another preferred example, in step (a1), the extraction solvent is 50 - 70% v / v methanol - water, preferably 60 - 70% v / v methanol - water.

[0023] In another preferred example, in step (a1), the dosage ratio of the Callicarpa nudiflora Hook. et Arn. medicinal material powder to the extraction solvent is 1 g : (40 - 100) mL, preferably 1 g : 45 - 50 mL.

[0024] In another preferred example, in step (a1), volume fixation means fixing the volume of the solution after extraction to 40 - 100 mL per 1 g of medicinal material with the extraction solvent, preferably 45 - 50 mL per 1 g of medicinal material.

[0025] In another preferred example, in step (a1), the extraction method is ultrasonic or reflux for 25 - 60 minutes, preferably 30 - 45 min.

[0026] In another preferred example, the extraction method is by ultrasound for 25 - 60 minutes, preferably 30 - 45 minutes.

[0027] In another preferred example, in step (a2), the solvent is methanol or ethanol, preferably methanol; and / or

[0028] In another preferred example, in step (a2), a series of concentration gradient mixed reference substance solutions (which can be used to draw an external standard curve) are prepared, preferably containing 5, 6 or 7 concentration gradients.

[0029] In another preferred example, the column length of the chromatographic column is 15 cm, the column diameter is 2.1 mm, and the filler particle size is 1.7 μm.

[0030] In another preferred example, the chromatographic column is Waters ACQUITY UPLC BEH C18.

[0031] In another preferred example, the mass spectrometry conditions include:

[0032] Ionization mode: ESI - mode, spray voltage 4500 ± 50 V; ionization temperature (TEM): 550 ± 10 °C; nebulizing gas (Gas 1): 55 ± 3 kPa; auxiliary gas (Gas 2): 55 ± 3 kPa; curtain gas (CUR): 30 ± 1 kPa; collision gas (CAD) Medium; the scanning mode is multiple reaction monitoring (MRM).

[0033] In another preferred example, the column temperature of the chromatographic column is 10 - 35 °C, more preferably 20 - 30 °C.

[0034] In another preferred example, the injection volume of the sample is 0.5 - 5 μL, more preferably 1 - 2 μL.

[0035] In another preferred example, the total flow rate of the elution mobile phase is 0.25 - 0.4 mL / min, preferably 0.3 - 0.35 mL / min.

[0036] In another preferred example, the elution program further includes: 20 - 25 min, 10% B (in order to balance the chromatographic column before the next injection).

[0037] In another preferred example, the conditions in the method of the present invention can optionally and independently be the corresponding conditions used in the examples of the present invention.

[0038] In the second aspect of the present invention, a method for evaluating Callicarpa nudiflora Hook. et Arn. medicinal materials is provided, including the steps:

[0039] The Callicarpa nudiflora Hook. et Arn. medicinal material sample is detected by using the detection method described in claim 1, so as to obtain the contents of the respective target components in the Callicarpa nudiflora Hook. et Arn. medicinal material sample;

[0040] The content results of the target components are compared with their content indexes. If they meet the said indexes, they are judged as qualified; otherwise, they are judged as unqualified.

[0041] In another preferred example, the content indexes of the said target components are as follows:

[0042] Geniposidic acid ≥ 0.028 w / w%;

[0043] De-caffeoylacteoloside ≥ 0.515 w / w%;

[0044] Caffeic acid ≥ 0.735 w / w%;

[0045] Cistanoside F ≥ 0.349 w / w%;

[0046] Protocatechuic aldehyde ≥ 0.018 w / w%;

[0047] Ferulic acid ≥ 0.015 w / w%;

[0048] Luteoloside ≥ 0.259 w / w%;

[0049] Acteoside ≥ 3.238 w / w%;

[0050] Isoacteoside ≥ 1.452 w / w%;

[0051] 2-Acetylvilloside ≥ 0.009 w / w%;

[0052] And luteolin ≥ 0.050 w / w%.

[0053] It should be understood that within the scope of the present invention, the above-mentioned respective technical features of the present invention and the respective technical features specifically described hereinafter (such as in the examples) can be combined with each other, so as to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0054] Figure 1 It is the base peak intensity of the Callicarpa nudiflora Hook. et Arn. administered serum in the positive (A top) / negative (B top) ion mode and the control group serum in the negative (A bottom) / positive (B bottom) ion mode.

[0055] Figure 2MRM chromatograms of the extracted ions of 11 components in Callicarpa nudiflora Hook. et Arn. medicinal materials under different mobile phases; A. Mobile phase: acetonitrile - water; B. Mobile phase: acetonitrile - 0.1% formic acid in water; 1. Geniposidic acid; 2. Decaffeoylacteoloside; 3. Caffeic acid; 4. Cistanoside F; 5. Protocatechuic aldehyde; 6. Ferulic acid; 7. Luteoloside; 8. Acteoloside; 9. Isoacteoloside; 10. 2 - Acetylsyringaresinol diglucoside; 11. Luteolin.

[0056] Figure 3 MRM chromatograms of the extracted ions of 11 components in Callicarpa nudiflora Hook. et Arn. medicinal materials; A. Reference substance; B. Sample; 1. Geniposidic acid; 2. Decaffeoylacteoloside; 3. Caffeic acid; 4. Cistanoside F; 5. Protocatechuic aldehyde; 6. Ferulic acid; 7. Luteoloside; 8. Acteoloside; 9. Isoacteoloside; 10. 2 - Acetylsyringaresinol diglucoside; 11. Luteolin. Detailed implementation manners

[0057] Through extensive and in - depth research, and through a large number of screenings and tests, the present inventors have provided a method for detecting multiple components in Callicarpa nudiflora Hook. et Arn. medicinal materials. In particular, through research and identification, the present inventors have found that there are 11 components from Callicarpa nudiflora Hook. et Arn. in the blood of subjects after taking Callicarpa nudiflora Hook. et Arn., which indicates that these components are potential active components of Callicarpa nudiflora Hook. et Arn.; based on this, the present invention further provides a detection method for these 11 specific components, and the method can simultaneously and accurately detect all these components, so as to comprehensively control the quality of Callicarpa nudiflora Hook. et Arn. medicinal materials. The present invention has been completed on this basis.

[0058] Terms

[0059] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0060] As used herein, the term "comprising" or "including" can be open - ended, semi - closed and closed. In other words, the term also includes "consisting essentially of...", or "consisting of...".

[0061] Combination of active components

[0062] The present invention provides a combination of active components from Callicarpa nudiflora Hook. et Arn. medicinal materials, which comprises the following 11 components: Geniposidic acid; Decaffeoylacteoloside; Caffeic acid; Cistanoside F; Protocatechuic aldehyde; Ferulic acid; Luteoloside; Acteoloside; Isoacteoloside; 2 - Acetylsyringaresinol diglucoside; and Luteolin.

[0063] The present invention has for the first time discovered that after a subject takes Callicarpa nudiflora, the components of Callicarpa nudiflora that can enter the blood include the above 11 kinds, which indicates that these components are the potential active components of Callicarpa nudiflora. Therefore, it is necessary to detect the contents of these 11 components in Callicarpa nudiflora medicinal materials, so as to conduct more comprehensive quality control of the medicinal materials.

[0064] Detection method / Quality evaluation method of Callicarpa nudiflora medicinal materials

[0065] Callicarpa nudiflora Hook.ex Arn. refers to the plant Callicarpa nudiflora in the Verbenaceae family and is a traditional Li medicine. The "Callicarpa nudiflora medicinal materials" described in the present invention refer to its dried leaves, which have the effects of anti-inflammatory, detoxifying, astringing, and hemostatic.

[0066] Based on the in-depth research of the active components of Callicarpa nudiflora medicinal materials by the present invention, the present invention provides a detection method for Callicarpa nudiflora medicinal materials. Among them, the target components detected by the method include: geniposidic acid, decaffeoylacteoloside, caffeic acid, cistanoside F, protocatechuic aldehyde, ferulic acid, luteoloside, acteoside, isomartynoside, 2-acetylacteoside, and luteolin;

[0067] The method includes the steps:

[0068] (i) Sample preparation:

[0069] (a1) Preparation of the sample to be tested: Weigh accurately the powder of Callicarpa nudiflora medicinal materials, use 50 - 75% v / v alcohol-water as the extraction solvent for ultrasonic or reflux extraction, make up the volume, and obtain the sample to be tested after passing through a microporous filter membrane.

[0070] (a2) Preparation of the reference substance solution: Weigh accurately each reference substance of the target components, dilute and make up the volume with a solvent to obtain a mixed reference substance solution.

[0071] (ii) Inject the sample to be tested and the reference substance solution into an ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometer (UHPLC-QqQ-MSMS) for detection,

[0072] Among them, the chromatographic conditions include:

[0073] (b1) The chromatographic column is an octadecylsilane-bonded silica column;

[0074] (b2) The mobile phase is (0.1 ± 0.02)% formic acid water (A) - acetonitrile (B); and

[0075] (b3)Gradient elution, and the elution program is as follows: 0 - 15 min, 10 - 20% B; 15 - 16.5 min, 20 - 90% B; 16.5 - 18.5 min, 90% B; 18.5 - 20 min, 90 - 10% B.

[0076] Among them, the mass spectrometry conditions include:

[0077] Ionization mode: ESI - mode, spray voltage 4500 ± 100 V; Ionization temperature (TEM): 550 ± 20 °C; Nebulizing gas (Gas 1): 55 ± 5 kPa; Auxiliary gas (Gas 2): 55 ± 5 kPa; Curtain gas (CUR): 30 ± 3 kPa; Collision gas (CAD) Medium; The scanning mode is multiple reaction monitoring (MRM).

[0078] The mass spectrometry parameter values of each ion pair are: The mass spectrometry parameter values of each ion pair are:

[0079]

[0080] In another preferred example, the method further includes: (iii) According to the chromatogram obtained in step (ii), quantifying each target component based on the peak area, so as to obtain the content of each target component in the Callicarpa nudiflora Hook. et Arn. medicinal material. The present invention uses a triple quadrupole mass spectrometer as the detector, and the chromatogram is usually the corresponding ion current diagram.

[0081] In another preferred example, in step (a1), the Callicarpa nudiflora Hook. et Arn. medicinal material powder passes through a No. 4 sieve.

[0082] In another preferred example, in step (a1), the alcohol is methanol or ethanol.

[0083] In another preferred example, in step (a1), the extraction solvent is 50 - 70% v / v methanol - water, preferably 60 - 70% v / v methanol - water.

[0084] In another preferred example, in step (a1), the dosage ratio of the Callicarpa nudiflora Hook. et Arn. medicinal material powder to the extraction solvent is 1 g : (40 - 100) mL, preferably 1 g : 45 - 50 mL.

[0085] In another preferred example, in step (a1), volume fixation means fixing the volume of the extracted solution to 40 - 100 mL per 1 g of medicinal material with the extraction solvent, preferably 45 - 50 mL per 1 g of medicinal material.

[0086] In another preferred example, in step (a1), the extraction method is ultrasonic or reflux for 25 - 60 minutes, preferably 30 - 45 min.

[0087] In another preferred example, the extraction is carried out by ultrasound for 25 - 60 minutes, preferably 30 - 45 minutes.

[0088] In another preferred example, in step (a2), the solvent is methanol or ethanol, preferably methanol; and / or

[0089] In another preferred example, in step (a2), a series of concentration gradient mixed reference substance solutions (which can be used to draw an external standard curve) are prepared, preferably including 5, 6 or 7 concentration gradients.

[0090] In another preferred example, the column length of the chromatographic column is 15 cm, the column diameter is 2.1 mm, and the filler particle size is 1.7 μm.

[0091] In another preferred example, the chromatographic column is Waters ACQUITY UPLC BEH C18.

[0092] In another preferred example, the mass spectrometry conditions include:

[0093] Ionization mode: ESI - mode, spray voltage 4500 ± 50 V; ionization temperature (TEM): 550 ± 10 °C; nebulizing gas (Gas 1): 55 ± 3 kPa; auxiliary gas (Gas 2): 55 ± 3 kPa; curtain gas (CUR): 30 ± 1 kPa; collision gas (CAD) Medium; the scanning mode is multiple reaction monitoring (MRM).

[0094] In another preferred example, the column temperature of the chromatographic column is 10 - 35 °C, more preferably 20 - 30 °C.

[0095] In another preferred example, the injection volume of the sample is 0.5 - 5 μL, more preferably 1 - 2 μL.

[0096] In another preferred example, the total flow rate of the elution mobile phase is 0.25 - 0.4 mL / min, preferably 0.3 - 0.35 mL / min.

[0097] In another preferred example, the elution program further includes: 20 - 25 min, 10% B (in order to balance the chromatographic column before the next injection).

[0098] In another preferred example, the conditions in the method of the present invention can optionally and independently be the corresponding conditions used in the examples of the present invention.

[0099] Quality evaluation method for Callicarpa nudiflora Hook. et Arn. medicinal materials

[0100] The present invention also provides an evaluation method for Callicarpa nudiflora Hook. et Arn. medicinal materials, including the steps:

[0101] Detect the Callicarpa nudiflora Hook. et Arn. medicinal material sample using the detection method as described in the present invention, so as to obtain the contents of each target component in the Callicarpa nudiflora Hook. et Arn. medicinal material sample;

[0102] Compare the content results of the target components with their content indicators. If they meet the indicators, it is judged as qualified; otherwise, it is unqualified.

[0103] The content indicators of each target component can be obtained by using common methods in the art. For example, the average value of each component obtained by measuring the contents in multiple batches of medicinal materials, and multiplying by a certain percentage (such as 70%, 80% or 90%) as the content standard. Or some of the target components can use known content indicators, such as pharmacopoeia indicators.

[0104] In another preferred example, the content indicators of the target components are as follows:

[0105] Geniposidic acid ≥ 0.028 w / w%;

[0106] Decaffeoylacteoloside ≥ 0.515 w / w%;

[0107] Caffeic acid ≥ 0.735 w / w%;

[0108] Cistanoside F ≥ 0.349 w / w%;

[0109] Protocatechuic aldehyde ≥ 0.018 w / w%;

[0110] Ferulic acid ≥ 0.015 w / w%;

[0111] Luteoloside ≥ 0.259 w / w%;

[0112] Acteoloside ≥ 3.238 w / w%;

[0113] Isoacteoloside ≥ 1.452 w / w%;

[0114] 2-Acetylacteoside ≥ 0.009 w / w%;

[0115] And luteolin ≥ 0.050 w / w%.

[0116] The main advantages of the present invention include:

[0117] The invention provides a method for simultaneously determining the contents of 11 components in Callicarpa nudiflora Hook. et Arn. medicinal materials by liquid chromatography-triple quadrupole mass spectrometry (LC-QQQ-MS / MS), and the contents of 11 components in 6 batches of Callicarpa nudiflora Hook. et Arn. medicinal materials are determined by this method. Through methodological investigation, the specificity, linearity, repeatability, stability, and precision of the method all meet the relevant regulations. Compared with the existing method for determining the contents of various components in Callicarpa nudiflora Hook. et Arn. medicinal materials using HPLC, the pretreatment of this method is simpler, the required determination time is shorter, it can simultaneously measure components with large differences in content in Callicarpa nudiflora Hook. et Arn. medicinal materials, and it is more accurate and reliable, providing a reference for comprehensively evaluating the quality of Callicarpa nudiflora Hook. et Arn. medicinal material extracts, studying their pharmacodynamic material basis, and ensuring the safety and effectiveness of their medication.

[0118] The method of the invention makes up for the blank of simultaneously determining the contents of multiple chemical components in Callicarpa nudiflora Hook. et Arn. medicinal materials, and provides a scientific basis for the quality control research of Callicarpa nudiflora Hook. et Arn. medicinal materials and their related preparations. The method provided by the invention is simple to operate, has a short analysis time, and can complete the quantification of 11 components within only 20 minutes. Its precision, repeatability, and stability are good, and it has the characteristics of strong specificity and high sensitivity.

[0119] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0120] Instruments and reagents

[0121] Exion LC TM The high-performance liquid chromatograph (Japan, Shimadzu Corporation) includes; AB Q-TRAP4500 triple quadrupole mass spectrometer (AB Sciex Company, USA, data acquisition Analyst AB Sciex; data analysis SCIEX OS), METTLER MS105(DU) 1 / 100,000 analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.), SB-5200DTD ultrasonic cleaner, ultrasonic power 250W, 50kHz (Ningbo Xinzhi Biotechnology Co., Ltd.), ultrapure water instrument (Merck Chemical Technology (Shanghai) Co., Ltd.).

[0122] Reference substances of geniposidic acid (1) (batch number CFS202301); reference substances of decaffeoyl verbascoside (2) (batch number CFS202301); reference substances of caffeic acid (3) (batch number CFS202302); reference substances of Cistanoside F (4) (batch number CFS202301); reference substances of Procatechualdehyde (5) (batch number CFS202302); reference substances of ferulic acid (6) (batch number CFS202302), reference substances of Luteolin (7) (batch number CFS202302); reference substances of acteoside (8) (batch number CFS202302), reference substances of isoacteoside (9) (batch number CFS202301); reference substances of 2'-acetylacteoside (10) (batch number CFS202301), reference substances of Luteolin (11) (batch number CFS202301). The above reference substances were all purchased from Wuhan Zhongbiao Technology Co., Ltd., with a purity of 98%. Water is ultrapure water, acetonitrile, and formic acid are LCMS grade (Adamas Company), and the rest of the reagents are all of analytical grade. The batch numbers of 6 batches of Callicarpa nudiflora Hook. & Arn. herbs are 211107, 210608, 211217, 211106, 210803, and 211219.

[0123] Example 1

[0124] Animal experiment

[0125] Male Sprague-Dawley rats (weight 200±20 g) were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., and the experimental animal use license number is SYXK (Shanghai) 2019-0027. All animals were raised in the barrier environment of the Experimental Animal Center of Shanghai Institute of Pharmaceutical Industry. The dry extract of Callicarpa nudiflora Hook. & Arn. herbs was dissolved in distilled water and ultrasonicated for 30 min to prepare a suspension.

[0126] Preparation of the dry extract of Callicarpa nudiflora Hook. & Arn. herbs: Take Callicarpa nudiflora Hook. & Arn. herbs, decoct twice with water, add 10 times the amount of water each time, 2 hours for the first time and 1 hour for the second time. Combine the decoction, filter, concentrate the filtrate, and dry it into a dry extract.

[0127] The experiment adopted a randomized grouping design, and 20 rats were divided into two groups:

[0128] (1) Administration group (n = 10): The suspension was administered by gavage at a dose of 1.08 g / kg / d (equivalent to 3 times the clinical equivalent dose);

[0129] (2) Blank control group (n = 10): gavaged with an equal volume of 0.9% normal saline.

[0130] Blood was collected from the orbital venous plexus 30 min after administration, and the serum was separated by centrifugation at 5000 r / min for 10 min and then combined for subsequent absorption component analysis.

[0131] Serum sample pretreatment

[0132] 500 μl of serum was added with 1.5 ml of methanol to precipitate proteins, vortexed for 60 s, centrifuged at 12000 r / min at 4 °C for 20 min, the supernatant was dried by nitrogen blowing, the residue was redissolved with 100 μl of methanol, vortexed for 60 s, centrifuged at 12000 r / min at 4 °C for 10 min, and the supernatant was taken for standby.

[0133] Chromatographic conditions

[0134] Using Waters Acquity TM Ultra performance liquid chromatography system, equipped with a binary pump, an autosampler and a PDA detector. Chromatographic separation was performed using an Acquity UPLC HSS T3 analytical column (2.1 mm × 150 mm, 1.8 μm; Waters, USA), the column temperature was 35 °C, and the flow rate was 0.3 mL / min. The mobile phase was 0.1% formic acid aqueous solution (A) - acetonitrile (B), and the gradient elution program was as follows:

[0135] 0 - 3 min, 10% B; 3 - 8 min, 10% → 12.5% B; 8 - 13 min, 12.5% → 20% B; 13 - 15 min, 20% → 25% B; 15 - 30 min, 25% B; 30 - 50 min, 25% → 45% B; 50 - 55 min, 45% → 70% B; 55 - 60 min, 70% → 100% B; 60 - 62 min, 100% B; 62 - 62.1 min 100% → 10% B; 62.1 - 70 min, 10% B. The injection volume was 5 μL.

[0136] Mass spectrometry conditions

[0137] An Xevo G2-XS Q-TOF high-resolution mass spectrometer (Waters, UK) was used, equipped with an electrospray ionization source (ESI), and synchronous acquisition in positive and negative ion modes was performed. The full-scan mass range was m / z 50 - 1500 Da, and the scan time was 0.3 s. The collision energy settings in MSE mode were: low collision energy 6 eV, high collision energy 25 - 60 eV. The flow rate of desolvation gas (nitrogen) was 500 L / h, and the temperature was 400 °C; the flow rate of cone gas was 100 L / h, and the ion source temperature was 120 °C. The cone voltage was 40 V, the source compensation voltage was 40 V, and the capillary voltage was 3500 V. All data acquisition and processing were carried out using Masslynx 4.1 software (Waters, USA).

[0138] Experimental results

[0139] Table A Identification results of the blood components of the extract of Callicarpa nudiflora herbs in rats

[0140]

[0141] The results are shown in Table A and Figure 1 As shown, a total of 11 blood components in the herbs of Callicarpa nudiflora were detected in the serum samples of rats after taking the extract of Callicarpa nudiflora herbs. These components can be transported throughout the body through the blood to regulate related physiological processes in the body and are potential active components for the herbs of Callicarpa nudiflora to exert their pharmacological effects. Therefore, it is of great significance to comprehensively detect and control the quality of these 11 components in the herbs of Callicarpa nudiflora.

[0142] Example 2

[0143] I. Preparation of test samples

[0144] 1.1 Detection conditions

[0145] 1.1.1 Chromatographic conditions

[0146] The chromatographic conditions were as follows: the chromatographic column was Waters ACQUITY UPLC BEH C18 (150 mm × 2.1 mm, 1.7 μm); the mobile phase was 0.1% formic acid water (A) - acetonitrile (B), and gradient elution was performed. The elution program was as follows: 0 - 15 min, 10 - 20% B; 15 - 16.5 min, 20 - 90% B; 16.5 - 18.5 min, 90% B; 18.5 - 20 min, 90 - 10% B; 20 - 25 min, 10% B; the flow rate was 0.35 mL / min; the column temperature was 30 °C; the injection volume was 1 μL.

[0147] 1.1.2 Mass spectrometry conditions

[0148] The mass spectrometry conditions were as follows: ion source Turbo V, ionization mode: ESI- mode, spray voltage 4500 V; ionization temperature (TEM): 550 °C; nebulizing gas (Gas 1): 55 kPa; auxiliary gas (Gas 2): 55 kPa; curtain gas (CUR): 30 kPa; collision gas (CAD) Medium; the scanning mode was multiple reaction monitoring (MRM), and the optimized mass spectrometry parameter values for each ion pair are shown in Table 1.

[0149] Table 1 Optimized mass spectrometry condition parameters

[0150]

[0151] 1.2 Determination of extraction solvent

[0152] Precisely weigh 1.0 g of Callicarpa nudiflora Hook. et Arn. medicinal material powder (passed through No. 4 sieve) and place it in a 50 mL volumetric flask. Precisely add 45 mL of methanol, 70% methanol aqueous solution, 50% methanol aqueous solution, ethanol, 70% ethanol aqueous solution, and 50% ethanol aqueous solution respectively, weigh, tightly stopper, ultrasonically treat for 60 min, cool, make up the volume respectively, shake well, filter through a 0.22 μm microporous filter membrane, inject for analysis, and calculate the content of each component, as shown in Table 2. The results show that the extraction rates of 70% methanol and 50% methanol for these 11 components are relatively high. In this experiment, 70% methanol was selected as the extraction solvent.

[0153] Table 2 Contents of 11 components in Callicarpa nudiflora Hook. et Arn. medicinal material extracted by different solvents (%)

[0154]

[0155] 1.3 Determination of extraction method

[0156] Precisely weigh 1.0 g of Callicarpa nudiflora Hook. et Arn. medicinal material powder (passed through No. 4 sieve), in two portions, and place them in a stoppered conical flask and a round-bottom flask respectively. Precisely add 50 mL of 70% methanol to each, weigh, ultrasonically treat and heat under reflux for 60 min respectively, cool, make up the lost weight, shake well, filter through a 0.22 μm microporous filter membrane, inject for analysis, and calculate the content of each component, as shown in Table 3. The results show that the contents of 11 components in Callicarpa nudiflora Hook. et Arn. medicinal material obtained by ultrasonic extraction are slightly higher, and the operation is simple. Therefore, ultrasonic extraction method was selected in this experiment.

[0157] Table 3 Contents of 11 components in Callicarpa nudiflora Hook. et Arn. medicinal material obtained by different extraction methods (%)

[0158]

[0159]

[0160] 1.4 Determination of solid-liquid ratio

[0161] Accurately weigh 1.0 g of Callicarpa nudiflora Hook. & Arn. medicinal material powder (passed through No. 4 sieve), take 3 portions, and place them in 25 mL, 50 mL, and 100 mL volumetric flasks respectively. Add 22.5 mL, 45 mL, and 90 mL of 70% methanol respectively, ultrasonicate for 60 min, let it cool, make up the volume with 70% methanol, shake well, filter through a 0.22 μm microporous filter membrane, inject for analysis, and calculate the content of each component, as shown in Table 4. Considering using as little solvent as possible and being easy to dissolve completely, the material-liquid ratio of 1 / 50 mL is selected.

[0162] Table 4 Contents of 11 components in Callicarpa nudiflora Hook. & Arn. medicinal material obtained with different material-liquid ratios (%)

[0163] Component 1 / 25 mL 1 / 50 mL 1 / 100 mL Geniposidic acid 0.031 0.035 0.036 De - caffeoylacteoloside 0.669 0.673 0.673 Caffeic acid 0.943 0.949 0.950 Cistanoside F 0.424 0.429 0.430 Protocatechuic aldehyde 0.022 0.025 0.026 Ferulic acid 0.016 0.018 0.019 Luteoloside 0.326 0.329 0.330 Acteoloside 4.067 4.070 4.077 Isoacteoloside 2.073 2.077 2.076 2 - Acetylsyringin 0.011 0.013 0.013 Luteolin 0.064 0.066 0.067 Total 8.646 8.684 8.697

[0164] 1.5 Determination of ultrasonic time

[0165] Accurately weigh 1.0 g of Callicarpa nudiflora Hook. & Arn. medicinal material powder (passed through No. 4 sieve), take 3 portions, and place them in 50 mL volumetric flasks respectively. Add 45 mL of 70% methanol respectively, ultrasonicate for 30, 60, and 90 min respectively, let it cool to room temperature, make up the volume to the scale line with 70% ethanol, shake well, filter through a 0.22 μm microporous filter membrane, inject for analysis, and calculate the content of each component, as shown in Table 5. The results show that the ultrasonic time has little effect on the content of compounds. Considering the time cost, the ultrasonic time of 30 min is preferably selected in this experiment.

[0166] Table 5 Contents of 11 components in Callicarpa nudiflora Hook. & Arn. medicinal material obtained with different ultrasonic times (%)

[0167]

[0168]

[0169] 1.6 Determination of the preparation method of test samples

[0170] Accurately weigh 1.0 g of Callicarpa nudiflora Hook. & Arn. medicinal material powder (passed through No. 4 sieve), place it in a 50 mL volumetric flask, add 40 ml of 70% methanol, ultrasonicate for 30 min, let it cool to room temperature, add 70% methanol to the scale line, shake well, filter (0.22 μm microporous filter membrane), and determine.

[0171] II. Determination of chromatographic mobile phase

[0172] Examine different mobile phases, including acetonitrile-water and acetonitrile-0.1% formic acid water. Considering the peak shapes and resolutions of each component, the chromatogram peak shape obtained using acetonitrile-water as the mobile phase is not good, which is not conducive to the accuracy of peak area integration ( Figure 2 ). Therefore, acetonitrile-0.1% formic acid water is selected as the mobile phase.

[0173] III. Preparation of reference substance solution

[0174] Weigh appropriate amounts of each reference substance accurately, add methanol to prepare a stock solution of the single-component reference substance, and shake well. Accurately measure an appropriate amount of the above-mentioned stock solution of the reference substance, add methanol to prepare a solution with a concentration of about 0.5 - 1 mg / ml, and dilute it to the required concentration in gradients with methanol to obtain a series of mixed standard solutions.

[0175] IV. Chromatographic Detection Conditions

[0176] Chromatographic Conditions

[0177] The chromatographic conditions are as follows: chromatographic column Waters ACQUITY UPLC BEH C18 (150 mm × 2.1 mm, 1.7 μm); mobile phase: 0.1% formic acid in water (A) - acetonitrile (B), gradient elution, and the elution program is as follows: 0 - 15 min, 10 - 20% B; 15 - 16.5 min, 20 - 90% B; 16.5 - 18.5 min, 90% B; 18.5 - 20 min, 90 - 10% B; 20 - 25 min, 10% B; flow rate 0.35 mL / min; column temperature 30 °C; injection volume 1 μL.

[0178] Mass Spectrometry Conditions

[0179] The mass spectrometry conditions are as follows: ion source Turbo V, ionization mode: ESI - mode, spray voltage 4500 V; ionization temperature (TEM): 550 °C; nebulizing gas (Gas 1): 55 kPa; auxiliary gas (Gas 2): 55 kPa; curtain gas (CUR): 30 kPa; collision gas (CAD) Medium; scanning mode is multiple reaction monitoring (MRM), and the optimized mass spectrometry parameter values of each ion pair are shown in Table 1.

[0180] V. Method Validation

[0181] Specificity

[0182] Under the above LC - MS / MS conditions, the MRM quantitative ion chromatograms of the mixed reference substance and 11 components of the sample are shown in Figure 1 , and the 11 components are well separated.

[0183] Linear Range, Limit of Quantitation, Limit of Detection

[0184] Precisely pipette an appropriate amount of the prepared mixed reference solution, dilute it successively to prepare a series of linear working solutions with 6 concentrations, and analyze them respectively under the conditions described in item "1.1". Using the concentration of each component as the abscissa (X) and the peak area of the component to be measured as the ordinate (Y), the regression equations, correlation coefficients and linear ranges of 11 components are obtained. Then, gradually dilute the reference solution until the signal-to-noise ratio (S / N) is approximately 10 to calculate the limit of quantitation (LOQ), and the signal-to-noise ratio (S / N) is approximately 3 to calculate the limit of detection (LOD), as shown in Table 6. Linearity: All calibration curves show good linear regression within the test range (r2≥0.999).

[0185] Table 6: Regression equation standard curves, limits of detection and limits of quantitation of 11 components

[0186]

[0187]

[0188] Precision experiment

[0189] Take a mixed reference solution with a certain concentration, inject it continuously and repeatedly 6 times, measure the area of each component respectively, calculate the RSD value, and the RSD is between 0.04% and 1.78%, indicating that the instrument precision is good.

[0190] Repeatability experiment

[0191] According to the preparation method of the test solution described in item "1.6", take the same sample and prepare 6 parallel test solutions, inject them for analysis, and the RSD of the content of each component is between 0.05% and 2.69%, indicating that the method has good repeatability.

[0192] Stability experiment

[0193] Take the same test solution in the repeatability test, inject it for analysis at 0, 2, 4, 8, 12, and 24 h respectively, and calculate the RSD of the content of each component to be between 1.98% and 6.17%. The results show that the test solution is stable within 24 h at room temperature.

[0194] Table 7 Precision, repeatability, stability (n = 6)

[0195] Component Precision RSD(%) Repeatability RSD(%) Stability RSD(%) Geniposidic acid 0.90 1.93 5.05 De - caffeoylacteoloside 0.63 2.69 4.70 Caffeic acid 1.69 1.93 3.44 Cistanoside F 0.88 1.89 4.21 Protocatechuic aldehyde 0.68 1.13 6.17 Ferulic acid 0.76 1.49 2.74 Luteoloside 0.59 1.41 3.21 Acteoloside 0.84 1.49 2.79 Isoacteoloside 0.33 0.46 3.35 2 - Acetylsyringin 0.04 2.59 6.15 Luteolin 1.74 0.05 4.96

[0196] Recovery experiment

[0197] Precisely weigh 3 portions of the same sample with known content, about 1 g, and add the mixed reference substances of geniposidic acid, decaffeoylacteoloside, caffeic acid, protocatechuic aldehyde, ferulic acid, luteoloside, acteoloside, isoacteoloside, 2-acetylsyringolide, and luteolin respectively at the 100% level of the sample content. Prepare according to the method under item "1.6", determine the content of each component and calculate the extraction recovery rate. The results are shown in Table 8. The average recovery rate of the 11 components is between 97.22% and 103.33%, and the RSD is between 2.12% and 4.81%, indicating that the recovery rate of the method is good.

[0198] Table 8 Recovery rate (%, n = 3)

[0199]

[0200]

[0201] VI. Sample determination

[0202] Take 6 batches of Callicarpa nudiflora Hook. et Arn. medicinal material powder (passed through No. 4 sieve), precisely weigh 1.0 g, prepare the test solution according to the method under item "1.6", and perform injection analysis under the conditions of item "1.1", and calculate the content of each component respectively. The results are shown in Table 9.

[0203] Table 9 Contents of 11 components in 6 batches of Callicarpa nudiflora Hook. et Arn. medicinal materials measured by LC-MS (%)

[0204]

[0205] In summary, the method established in this study for simultaneously determining the contents of 11 active components in Callicarpa nudiflora Hook. et Arn. medicinal materials by LC-QQQ-MS / MS is simple, rapid, and accurate, providing a scientific basis for comprehensively evaluating the quality of Callicarpa nudiflora Hook. et Arn. medicinal materials.

[0206] All documents mentioned in this invention are cited in this application as references, just as if each document is cited separately as a reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for detecting Callicarpa nudiflora medicinal material, wherein: The target components detected by the method include: geniposide, decaffeoyl verbascoside, caffeic acid, cistancheside F, protocatechuic aldehyde, ferulic acid, luteolin, verbascoside, isovalascoside, 2-acetyleugenol glycoside and luteolin; The method comprises the steps of: (i) Sample preparation: (a1) Preparation of the sample to be tested: accurately weigh the powder of Callicarpa nudiflora, use 50-75% v / v alcohol-water as the extraction solvent for ultrasonic or reflux extraction, fix the volume, and filter through a microporous membrane to obtain the sample to be tested; (a2) Preparation of reference solution: accurately weigh the reference substance of each target component, dilute it with a solvent and make up to volume to obtain a mixed reference solution; (ii) injecting the sample solution and the reference solution into an ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometer for detection, The chromatographic conditions include: (b1) The chromatographic column is an octadecyl bonded silica gel column; (b2) the mobile phase is (0.1±0.02)% formic acid water (A)-acetonitrile (B); and (b3) gradient elution, the elution program is as follows: 0-15 min, 10-20% B; 15-16.5 min, 20-90% B; 16.5-18.5 min, 90% B; 18.5-20 min, 90-10% B; Among them, the mass spectrometry conditions include: Ionization mode: ESI-mode, spray voltage 4500±100V; ionization temperature (TEM): 550±20℃; nebulizer gas (Gas1): 55±5kPa; auxiliary gas (Gas 2): 55±5kPa; curtain gas (CUR): 30±3kPa; collision gas (CAD) Medium; scanning mode: multiple reaction monitoring (MRM); The mass spectrometry parameter values ​​for each ion pair are:

2. The detection method according to claim 1, characterized in that The method further comprises: (iii) quantifying each target component according to the peak area based on the chromatogram obtained in step (ii), thereby obtaining the content of each target component in the Callicarpa nudiflora medicinal material.

3. The detection method according to claim 1, characterized in that The method has one or more characteristics selected from the group consisting of: In step (a1), the Callicarpa nudiflora medicinal material powder is passed through a No. 4 sieve; In step (a1), the alcohol is methanol or ethanol; and / or In step (a1), the extraction solvent is 50-70% v / v methanol-water, preferably 60-70% v / v methanol-water.

4. The detection method according to claim 1, characterized in that The method has one or more characteristics selected from the group consisting of: In step (a1), the ratio of the Callicarpa nudiflora medicinal material powder to the extraction solvent is 1 g: (40-100) mL, preferably 1 g: 45-50 mL; In step (a1), the volume adjustment means that the extracted solution is adjusted to 40-100 mL per 1 gram of medicinal material, preferably 45-50 mL per 1 gram of medicinal material, using the extraction solvent; and / or In step (a1), the extraction method is ultrasound or reflux for 25-60 minutes, preferably 30-45 minutes.

5. The detection method according to claim 1, characterized in that The method has one or more characteristics selected from the group consisting of: In step (a2), the solvent is methanol or ethanol, preferably methanol; and / or In step (a2), a series of mixed reference solutions with concentration gradients are prepared (which can be used to draw an external standard curve), preferably comprising 5, 6 or 7 concentration gradients.

6. The detection method according to claim 1, characterized in that The column length of the chromatographic column is 15 cm, the column diameter is 2.1 mm, and the filler particle size is 1.7 μm.

7. The detection method according to claim 1, characterized in that The chromatographic column is Waters ACQUITY UPLC BEH C18.

8. The detection method according to claim 1, characterized in that The method has one or more characteristics selected from the group consisting of: The column temperature of the chromatographic column is 10-35°C, more preferably, 20-30°C; The injection volume of the sample is 0.5-5 μL, more preferably, 1-2 μL; The total flow rate of the mobile phase for elution is 0.25-0.4 mL / min, preferably 0.3-0.35 mL / min; and / or The elution procedure also includes: 20-25 min, 10% B.

9. A method for evaluating Callicarpa nudiflora medicinal material, characterized in that: Includes steps: Using the detection method as described in claim 1 to detect the Callicarpa nudiflora medicinal material sample, thereby obtaining the content of each target component in the Callicarpa nudiflora medicinal material sample; The content result of the target component is compared with its content index. If it meets the index, it is judged to be qualified, otherwise it is unqualified.

10. The evaluation method according to claim 9, characterized in that The content index of the target component is as follows: Geniposide acid ≥ 0.028w / w%; Decaffeoyl verbascoside ≥ 0.515w / w%; Caffeic acid ≥ 0.735w / w%; Cistancheside F ≥ 0.349w / w%; Protocatechuic aldehyde ≥ 0.018w / w%; Ferulic acid ≥ 0.015w / w%; Luteolin ≥ 0.259w / w%; Verbascoside ≥ 3.238w / w%; Isovale glycoside ≥ 1.452w / w%; 2-Acetyleugenol glycoside ≥ 0.009w / w%; and luteolin ≥ 0.050w / w%.

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