A method for detecting the content of active substances in waste residues from traditional Chinese medicine extraction

Abstract

The present invention discloses a method for detecting the content of active substances in waste residues extracted from traditional Chinese medicine. The active substances are chlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, lonicerae sempervirens saponin A and lonicerae sempervirens saponin B. Ultra-high pressure liquid chromatography-time-of-flight mass spectrometer is used for detection, and the steps are as follows: (1) preparing a test solution; (2) preparing a reference solution; (3) chromatographic conditions and mass spectrometry conditions; (4) determination method: respectively accurately aspirating the test solution and the reference solution, injecting them into the ultra-high pressure liquid chromatography-time-of-flight mass spectrometer, and determining them according to the above-mentioned chromatographic conditions and mass spectrometry conditions. The detection method adopted by the present invention can realize the separation and analysis of complex components in the residues extracted from traditional Chinese medicine. After methodological investigations such as precision, stability, repeatability, and recovery rate experiments, the method is simple and fast, and the quantitative analysis results are reliable and stable, which provides a basis for the analysis and resource recycling of components in waste or residues extracted from traditional Chinese medicine.

Description

Technical Field

[0001] The invention relates to a detection method, in particular to a method for detecting the content of active substances in waste residues from extraction of traditional Chinese medicine. Background Art

[0002] Mailuoning is a Chinese herbal compound intravenous injection, made from ingredients such as Dendrobium, Scrophularia, Achyranthes, Honeysuckle, and Lonicera japonica. It has the effects of clearing away heat and nourishing yin, promoting blood circulation and removing blood stasis. It plays a positive role in the prevention and treatment of acute ischemic stroke, vasculitis, arteriosclerosis, angina pectoris, blood circulation disorders and other cardiovascular and other related diseases. It has the advantages of high efficacy, quick effect, low price and high safety.

[0003] The ester extraction waste residue of Mailuoning injection was analyzed and found to contain active chemical substances chlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, honeysuckle saponin A and honeysuckle saponin B. Chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C are all phenolic acid compounds, which are the main active ingredients of many medicinal materials and Chinese patent medicines for antibacterial, detoxification, anti-inflammatory and choleretic effects. Studies have shown that chlorogenic acid reduces diet-induced inflammation and cardiovascular, liver and metabolic changes; isochlorogenic acid A can significantly reduce the phosphorylation of AMP-activated kinase and acetyl-CoA carboxylation, as well as the level of fatty acid synthase protein, and reduce fatty acid anabolism in 3T3-L1 adipocytes; isochlorogenic acid C exerts anti-inflammatory effects by inhibiting the activation of nuclear factor κB and MAPK pathways in vitro and reducing carrageenan-induced edema in vivo. Both Lonicera saponin A and Lonicera saponin B are saponin compounds. Among them, Lonicera saponin A has anti-inflammatory effects and has been shown to protect liver cells damaged by drugs or toxins; Lonicera saponin B can enhance the level of oxidative stress and reactive oxygen in cells, thereby leading to cell apoptosis, and can inhibit tumor cells such as colorectal cancer. The physiological functions, medical and economic values ​​of these compounds have attracted attention.

[0004] At present, the above five active substances are mainly determined by HPLC-ELSD (high performance liquid chromatography-evaporative light scattering) or UPLC (ultra-high performance liquid chromatography) and other methods in the literature and patents. The above detection methods are mainly aimed at single Chinese medicinal materials with a small number of chemical components. In the ester extraction waste residue of Mailuoning Injection, because the drug is a compound Chinese medicine, a large number of medicinal materials are used, and the drug has undergone multiple steps of process treatment during the extraction process, the chemical structure of some components has changed. Therefore, there are many chemical components in the waste residue, which are difficult to separate by ordinary high performance liquid chromatography. Summary of the invention

[0005] Purpose of the invention: In order to solve the technical problems existing in the prior art, the present invention aims to provide a method for detecting the content of active substances in waste residues of traditional Chinese medicine extraction with high detection efficiency and high detection accuracy and suitable for use with waste residues of traditional Chinese medicine extraction with complex components.

[0006] Technical solution: The method for detecting the content of active substances in the waste residue of traditional Chinese medicine extraction of the present invention, wherein the active substances are chlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, Lonicera japonica saponin A and Lonicera japonica saponin B, uses ultra-high pressure liquid chromatography-time-of-flight mass spectrometry for content detection, and the steps are as follows:

[0007] (1) Preparation of test solution: Prepare the test solution by diluting and filtering the waste residue of traditional Chinese medicine extraction to prepare the test solution;

[0008] (2) Preparation of reference solution: accurately weigh chlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, Lonicera japonica saponin A, and Lonicera japonica saponin B, and mix them and prepare a reference solution with methanol;

[0009] (3) Chromatographic conditions: Chromatographic column: Agilent Poroshell 120 SB-AQ C18, mobile phase A: 0.1% formic acid in water, mobile phase B: methanol, elution method: gradient elution, flow rate: 0.3-0.5 mL / min, injection volume: 5 μL;

[0010] Mass spectrometry conditions: ESI ion source, ionization mode was negative ion mode: 0-45min, 135V, 45-75min, 175V, capillary voltage was 3.5kV, and drying gas temperature was 350°C;

[0011] (4) Determination method: Accurately aspirate the test solution and the reference solution respectively, inject them into an ultra-high pressure liquid chromatography-time-of-flight mass spectrometer, and perform determination according to the chromatographic conditions and mass spectrometry conditions described in step (3).

[0012] Furthermore, in step (1), the process of preparing the test solution from the ester extraction waste residue of Mailuoning injection of traditional Chinese medicine extraction waste residue is specifically as follows: taking the ester extraction waste residue of Mailuoning injection, diluting it with 10 times of water, passing it through an ADS-7 macroporous resin column, gradient eluting it with water, 10% ethanol, 90% ethanol and 100% ethanol, collecting the 90% ethanol elution part, taking 500 μL, blowing it to dryness with nitrogen, dissolving it with 1 mL of methanol, and then diluting it 10 times with methanol, filtering it through a 0.22 μm filter membrane to obtain the test solution;

[0013] Furthermore, in step (2), the mass ratio of chlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, Lonicera japonica saponin A, and Lonicera japonica saponin B is 2:1:2:1:4.

[0014] Furthermore, in step (3), the specification of the chromatographic column is 3.0 mm×100 mm, the particle size is 2.7 μm, and the column temperature of the chromatographic column changes in a gradient manner, 0-20 min, 20±5° C.; 20-75 min, 40±5° C.

[0015] Furthermore, in step (3), the elution program of the gradient elution is: 0-20 min, 80%→75% mobile phase A, 20%→25% mobile phase B; 20-25 min, 75%→65% mobile phase A, 25→35% mobile phase B; 25-40 min, 65%→50% mobile phase A, 35→50% mobile phase B; 40-75 min, 50%→30% mobile phase A, 50→70% mobile phase B.

[0016] Furthermore, in step (3), the ionization mode used in the mass spectrometry is a negative ionization mode. When performing mass spectrometry analysis, the primary mass spectrum of each active substance is subjected to secondary mass spectrometry fragmentation, and the fragmentation conditions are: fragmentation voltage is: phenolic acid, flavonoids and cyclopentadiene 90V, saponin 210V, collision energy is 195V, nebulizing gas is N2, product ion scanning range m / z is 100-1700, nebulizing gas pressure is 50psi, drying gas flow rate is 10.0L / min, and Skimmer voltage is 65V.

[0017] Furthermore, the chlorogenic acid has a frontal time of 11-13 min; the isochlorogenic acid A has a frontal time of 35-36 min; the isochlorogenic acid C has a frontal time of 38-40 min; the Lonicera japonica saponin B has a frontal time of 52-54 min; and the Lonicera japonica saponin A has a frontal time of 54-56 min.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The detection method of the present invention simultaneously determines the contents of five active ingredients in the waste residue of Chinese medicine myclobutanil ester by adopting ultra-high pressure liquid chromatography-time-of-flight mass spectrometry, and establishes a convenient, reliable and accurate quantitative method, which not only provides a basis for the content determination of active ingredients in waste residue of Chinese medicine extraction containing complex components, but also provides a reference and evaluation basis for the comprehensive utilization of Chinese medicine resources and waste recycling and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the TIC diagram of the reference solution of Example 1 of the present invention;

[0020] Figure 2 This is the TIC diagram of the test solution of Example 1 of the present invention, peak 1: chlorogenic acid, peak 2: isochlorogenic acid A, peak 3: isochlorogenic acid C, peak 4: Lonicera japonica saponin B, peak 5: Lonicera japonica saponin A. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with specific embodiments and drawings.

[0022] Example 1: The method for determining the content of active substances in the waste residue of the ceramide extraction of the present invention is as follows:

[0023] 1. Instruments and reagents

[0024] 1.1 Instrument

[0025] Agilent 1260 UPLC-DAD-6530 ESI-Q-TOF-MS liquid spectrometer (Agilent, USA); KQ-400KDE digital ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); JY / YP electronic balance (Shanghai Liangping Instrument Co., Ltd.); SPING-S5i ultrapure water meter (Nanjing Miaozhiyi Electronic Technology Co., Ltd.); OSB-2200 rotary evaporator (EYELA, Japan).

[0026] 1.2 Reagents

[0027] Methanol (chromatographically pure, TEDIA, USA), anhydrous ethanol (analytical pure, Sinopharm Chemical Reagent Co., Ltd.), formic acid (chromatographically pure, ROE SCIENTIFIC INC, USA), distilled water (Watsons), ADS-7 macroporous adsorption resin (Zhengzhou Hecheng New Material Technology Co., Ltd.), and silica gel (Qingdao Ocean Chemical Plant).

[0028] Chlorogenic acid (batch number: 0753-9607), isochlorogenic acid A (batch number: 111782-201807), and isochlorogenic acid C (batch number: 111894-202104) standards were purchased from the China Food and Drug Inspection Institute; Lonicera japonica saponin A and Lonicera japonica saponin B were isolated from the experiment of the present invention, and the purity of all reference substances was greater than 95%; the ester extraction waste residue of Mailuoning injection came from Jinling Pharmaceutical Co., Ltd.

[0029] 2. Experimental steps

[0030] (1) Chromatographic conditions: Chromatographic column: Agilent Poroshell 120 SB-AQ C18 (3.0×100 mm, 2.7 μm); column temperature: 0-20 min, 20°C; 20-75 min, 40°C; mobile phase: 0.1% formic acid solution (A)-methanol (B), gradient elution program: see Table 1, flow rate: 0.3 mL / min; injection volume: 5 μL.

[0031] Table 1 Gradient elution program

[0032]

[0033] (2) Preparation of reference solution: accurately weigh chlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, Lonicera japonica saponin A, and Lonicera japonica saponin B to prepare mixed standard solutions of 33.33 μg / ml, 16.67 μg / ml, 33.33 μg / ml, 16.67 μg / ml, and 66.67 μg / ml, and refrigerate for later use;

[0034] (3) Preparation of test solution: Weigh the ester extract of Mailuoning injection, dilute it 10 times with water, pass it through an ADS-7 macroporous resin column, and gradiently elute it with water, 10% ethanol, 90% ethanol and 100% ethanol. Collect the 90% ethanol elution part, take 500 μL, blow it to dryness with nitrogen, dissolve it in 1 ml of methanol, dilute it 10 times with methanol, filter it through a 0.22 μm filter membrane to obtain the test sample, and then inject it for analysis; the yield of macroporous resin in the 90% ethanol part was 43.31-60.77%; the mass of each batch of samples was 11.5 mg, 12.0 mg, 12.1 mg, 12.4 mg, 18.5 mg, 17.9 mg, 22.4 mg, and 28.5 mg respectively;

[0035] (4) Mass spectrometry conditions: ESI ion source, ionization mode was negative ion mode: 0-45 min, 135 V, 45-75 min, 175 V, capillary voltage was 3.5 kV, drying gas temperature was 350 °C, fragmentation voltage was: phenolic acid, flavonoids and terpenoids 90 V, saponin 210 V, collision energy was 195 V, nebulizer gas was N2, product ion scanning range was m / z 100-1700, nebulizer gas pressure was 50 psi, drying gas flow rate was 10.0 L·min-1, and skimmer voltage was 65 V;

[0036] (5) Inject the reference solution and the test solution into the LC-MS / MS instrument for determination.

[0037] Example 2 Methodology Verification

[0038] 1. System suitability test

[0039] The reference substance and the test substance solution were taken separately and analyzed according to the chromatography and mass spectrometry conditions in Example 1. The separation between the chromatographic peak of the analyte and its adjacent chromatographic peak was greater than 1.5, the theoretical plate number was not less than 2000, and the tailing factors were between 0.9 and 2.5. The reference substance solution in step (2) of Example 1 was taken and sampled 6 times continuously. The RSD of the calculated peak area was less than 5%, indicating that the system has good applicability.

[0040] 2. Exclusivity

[0041] Accurately pipette an appropriate amount of the mixed reference solution and the test solution, and perform sample analysis according to the chromatographic and mass spectrometric conditions in Example 1. Baseline separation is achieved, and there are no interfering components at the peak positions of the components, indicating that the detection method of the present invention has good specificity and meets the requirements of quantitative analysis.

[0042] 3. Linear relationship, detection limit and quantification limit

[0043] Accurately pipette appropriate amount of the reference solution in Example 1 into a 5 mL stoppered brown volumetric flask, gradually dilute with methanol to prepare a series of mixed reference solutions, inject 5 μL of the solution according to the chromatographic and mass spectrometric conditions in Example 1 to determine the peak area, perform linear regression on the peak area (Y) against the mass concentration (X) of the reference substance, and calculate the regression equation and linear correlation coefficient (R) of the five analytes. 2 ), the results are shown in Table 2 below.

[0044] Table 2 Linear relationship, detection limit and quantification limit

[0045]

[0046] 4. Precision experiment

[0047] The test solution prepared in step (3) of Example 1 was injected six times continuously according to the chromatographic and mass spectrometric conditions in Example 1, and the relative standard deviations of the peak areas of the five analytes were calculated. The intra-day RSDs of the peak areas were 2.33%, 3.56%, 2.84%, 4.49% and 3.97%, respectively, indicating that the instrument had good precision.

[0048] 5. Stability test

[0049] The test solution prepared in step (3) of Example 1 was taken and sampled and analyzed at 0, 4, 8, 12, and 24 h according to the chromatographic and mass spectrometric conditions in Example 1, and the relative standard deviations of the peak areas of the five analytes were calculated. The RSDs of the peak areas were 2.13%, 4.06%, 4.60%, 0.56%, and 2.34%, respectively, indicating that the assay components had good stability within 24 h.

[0050] 6. Repeatability test

[0051] Six test sample solutions (batch number: 20210303) were prepared in parallel according to step (3) in Example 1. The peak areas were determined according to the chromatographic and mass spectrometric conditions in Example 1. The mass fractions of the five analytes were calculated to be 10.99 mg·g -1 , 3.92mg·g -1 , 8.06mg·g -1 、36.90mg·g -1 、3.87mg·g -1, the RSDs of the peak areas were 0.82%, 1.69%, 1.57%, 0.62%, and 1.02%, respectively, indicating that the method had good repeatability.

[0052] 7. Sample recovery test

[0053] Take 9 parts of the waste residue treatment liquid extracted with known content of neuregoside (batch number: 20210314), add three mixed reference solutions of low, medium and high concentrations respectively, and perform content determination according to the chromatographic and mass spectrometry conditions in Example 1, calculate the average addition recovery rate and RSD of the five components, and the average addition recovery rates of chlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, lonicerae saponin B and lonicerae saponin A are 90.18%, 91.38%, 103.06%, 95.68% and 101.04%, respectively, and the RSDs of the peak areas are 0.76%, 0.89%, 1.37%, 1.79% and 3.55%, respectively, indicating that this method has good accuracy.

[0054] 8. Sample content determination

[0055] The reference solution prepared according to step (2) of Example 1 and 8 batches of test solution prepared according to step (3) were respectively taken for sample analysis according to the chromatographic and mass spectrometric conditions of Example 1. The content of active substance in each batch of samples was calculated by the peak area of ​​each component using the external standard method. The results are shown in Table 3.

[0056] Table 3 List of five active ingredient content determination results

[0057]

[0058] Figure 1 and Figure 2 These are the TIC diagrams of the reference solution and the test solution.

Claims

1. A method for detecting the content of active substances in waste residues from traditional Chinese medicine extraction, wherein the active substances are chlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, Lonicera saponin A and Lonicera saponin B, characterized in that: Ultra-high pressure liquid chromatography-time-of-flight mass spectrometry was used for content detection, and the steps were as follows: (1) Preparation of test solution: Take the waste residue of traditional Chinese medicine extraction, dilute and filter it to make the test solution; (2) Preparation of reference solution: accurately weigh chlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, Lonicera japonica saponin A, and Lonicera japonica saponin B, and mix them with methanol to prepare a reference solution; (3) Chromatographic conditions: Chromatographic column: Agilent Poroshell 120 SB-AQ C18, mobile phase A is 0.1% formic acid in water, mobile phase B is methanol, elution method is gradient elution, flow rate: 0.3-0.5 mL / min, injection volume 5 μL; Mass spectrometry conditions: ESI ion source, ionization mode was negative ion mode: 0-45min, 135V, 45-75min, 175V, capillary voltage was 3.5 kV, and drying gas temperature was 350°C; (4) Determination method: accurately aspirate the test solution and the reference solution respectively, inject them into the ultra-high pressure liquid chromatography-time-of-flight mass spectrometer, and determine them according to the chromatographic conditions and mass spectrometry conditions described in step (3); in step (1), the Chinese medicine extraction waste residue is the ester extraction waste residue of Mailuoning injection, and the process of preparing the test solution is specifically as follows: take the ester extraction waste residue of Mailuoning injection, dilute it with 10 times water, pass it through an ADS-7 macroporous resin column, and then pass it through water, 10% ethanol, 90% ethanol and 100% % ethanol gradient elution, collect the 90% ethanol elution portion, take 500 μL, blow nitrogen to dryness, dissolve in 1 mL methanol, dilute 10 times with methanol, and filter through a 0.22 μm filter membrane to obtain a test solution; in step (2), the mass ratio of chlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, honeysuckle saponin A, and honeysuckle saponin B is 2:1:2:1:4; in step (3), the elution program of the gradient elution is: 0-20 min, 80%→75% Mobile phase A, 20%→25% mobile phase B; 20-25min, 75%→65% mobile phase A, 25→35% mobile phase B; 25-40min, 65%→50% mobile phase A, 35→50% mobile phase B; 40-75min, 50%→30% mobile phase A, 50→70% mobile phase B; the elution time of chlorogenic acid is 11-13min; the elution time of isochlorogenic acid A is 35-36min; the elution time of isochlorogenic acid C is 38-40min; the elution time of Lonicera japonica saponin B is 52-54min; the elution time of Lonicera japonica saponin A is 54-56min; in the mass spectrometry conditions: the ionization mode is negative ionization mode.

2. The detection method according to claim 1, characterized in that: In step (3), the specification of the chromatographic column is 3.0 mm×100 mm, and the particle size is 2.7 μm.

3. The detection method according to claim 1, characterized in that: In step (3), the column temperature of the chromatographic column changes in a gradient manner: 0-20 min, 20±5°C; 20-75 min, 40±5°C.

4. The detection method according to claim 1, characterized in that: In step (3), when performing mass spectrometry analysis, the primary mass spectrum of each active substance is subjected to secondary mass spectrometry fragmentation.

5. The detection method according to claim 4, characterized in that: The pyrolysis conditions are as follows: pyrolysis voltage is 90 V for phenolic acid, flavonoids and cyclopentadiene, 210 V for saponin, collision energy is 195 V, nebulizer gas is N2, product ion scanning range m / z is 100-1700, nebulizer gas pressure is 50 psi, drying gas flow rate is 10.0 L / min, and skimmer voltage is 65 V.

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