Method for analyzing and detecting chemical composition of oplopanax elatus nakai petroleum ether extract and application thereof

Abstract

The invention belongs to the technical field of chemical analysis, and particularly relates to a method for analyzing and detecting chemical composition of oplopanax elatus petroleum ether extract.The method comprises the following steps that a test solution is treated, specifically, oplopanax elatus is subjected to enzymolysis treatment, enzymatic hydrolysate is removed, and medicine residues obtained after enzymolysis are collected; by taking petroleum ether as a solvent, extracting the medicine residue to obtain the petroleum ether extract of oplopanax elatus nakai; mixing the oplopanax elatus nakai petroleum ether extract solution with the re-crystallized cyclodextrin aqueous solution, stirring, standing, collecting the precipitate, purifying, adding an internal standard, uniformly mixing, centrifuging, sucking the supernate, and filtering to obtain the oplopanax elatus nakai petroleum ether extract. And carrying out full spectrum identification on the test solution by adopting an ultra-high performance liquid chromatography combined quadrupole series time-of-flight mass spectrometry and combining a Metlin network database. According to the method provided by the invention, the composition of the monomeric compound of the petroleum ether extraction part of oplopanax elatus nakai can be rapidly and qualitatively analyzed, and the monomeric compound corresponding to the pharmacological activity of the oplopanax elatus nakai can be found in a short time.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical analysis, and particularly relates to a method for analyzing and detecting the chemical composition of the petroleum ether extract of Oplopanax elatus Nakai and its use. Background Art

[0002] Due to the complex components in the roots, rhizomes and aerial stems of Oplopanax elatus Nakai, it is difficult to extract, separate and purify monomeric compounds. So far, almost no monomeric compounds corresponding to its pharmacological activities have been found, which brings great difficulties to the screening of effective components and pharmacological research of Oplopanax elatus Nakai. The components of the medicinal parts of Oplopanax elatus Nakai are complex, and it is difficult to extract, separate and purify them. Moreover, the effective components of the drug are unstable and are easily separated and degraded during the extraction, separation and identification processes.

[0003] Electrospray ionization mass spectrometry is a new type of soft ionization mass spectrometry analysis technology that has emerged in recent years. Its principle is that the sample is dissolved and diluted in a volatile solvent (such as methanol, acetone, acetonitrile, etc.), and is introduced into the mass spectrometer through an injection pump or a liquid chromatograph. Under the synergistic action of the sheath gas flow and the high electric field, the diluted solution enters the strong electric field through the nebulizing gas sleeve. According to the different properties of the substances to be measured, positively charged or negatively charged droplets are formed. Under the action of the electrostatic field, they enter the mass spectrometer. After being blown by a reverse hot nitrogen gas, the volatile solvent quickly evaporates, the volume of the droplets decreases, resulting in a continuous increase in the density of the surface charge, and the ratio of the surface charge to the surface area becomes larger. When the Rayleigh stability limit is reached, the charge repulsion force is sufficient to overcome the surface tension and cause the small droplets to explode, forming smaller charged droplets. This process repeats in the mass spectrometer. The larger small droplets form a smaller beam of small droplets through continuous splitting, and finally charged ions are obtained. During the electrospray ionization process, the sample directly enters the gas phase in the form of ions without passing through high-energy excitation, and the structure will not be fragmented. Usually, quasi-molecular ion peaks appear in the spectrum. Different from the previous hard ionization mass spectrometry analysis technologies, the structure of the sample generally does not fragment in the mass spectrometer, and the molecular ion of the substance to be measured is directly obtained. According to the different properties of the substances, the sample can form two charged modes: positive ions or negative ions.

[0004] The molecular ions obtained by electrospray ionization mass spectrometry are pushed into a radio frequency electric field. The radio frequency electric field is used to increase the kinetic energy of the precursor ions, causing the ions to collide with an inert gas (helium or argon). The bonds with weaker bond energies in the parent ions are preferentially broken, generating fragment ions. The fragment ions enter a quadrupole time-of-flight mass spectrometry detector (QTOF-MS). In the QTOF-MS detector, since ions with different m / z values have different velocities, the individual ions are distinguished and recorded according to the different arrival times of ions with different masses at the detector, forming a first-order mass spectrum. Then, the quadrupole undertakes the task of selecting ions. The selected parent ions are accelerated and then enter a collision cell to collide with an inert gas (CID). The substance structure undergoes fragmentation to produce fragment ions. The fragment ions then enter the TOF analyzer and are separated according to the mass-to-charge ratio, completing the qualitative and quantitative analysis of the molecular ions to be measured.

[0005] The advantages of quadrupole-time-of-flight mass spectrometry are as follows: ① It can achieve high resolution within a wide mass range, obtaining the accurate molecular weight of substances, including small molecule compounds, proteins, polypeptides and other substances; ② According to the accurate molecular weight of the compound and the true isotope peak shape distribution, the elemental composition of the compound can be obtained; ③ The high-sensitivity second-order mass spectrometry function can achieve the simultaneous determination of the elemental composition of parent ions and fragment ions; ④ It has a fast detection speed and a high detection throughput, and can complete the qualitative and quantitative analysis of a large number of compounds to be measured in a short time; ⑤ It can directly analyze the components of a mixture; ⑥ For cases where chromatographic separation is incomplete, effective analysis can also be carried out.

[0006] At present, there is no report on the detection and analysis of the active components of Oplopanax elatus Nakai using ultra-high performance liquid chromatography coupled with quadrupole tandem time-of-flight mass spectrometry. Summary of the Invention

[0007] In view of the above technical problems, the present invention uses ultra-high performance liquid chromatography coupled with quadrupole tandem time-of-flight mass spectrometry in combination with the Metlin network database, which can quickly carry out qualitative analysis on the composition of monomer compounds in the petroleum ether extraction part of Oplopanax elatus Nakai, and find the monomer compounds corresponding to the pharmacological activities of Oplopanax elatus Nakai in a short time, laying a material foundation for future pharmacological research on Oplopanax elatus Nakai.

[0008] The present invention provides a method for analyzing and detecting the chemical composition of the petroleum ether extract of Oplopanax elatus Nakai, comprising the following steps:

[0009] Treatment of the test solution: Oplopanax elatus Nakai is enzymatically hydrolyzed, the enzymatic hydrolysate is removed, and the medicinal residue after enzymatic hydrolysis is collected; using petroleum ether as a solvent, the medicinal residue is extracted to obtain the petroleum ether extract of Oplopanax elatus Nakai; the petroleum ether extract solution of Oplopanax elatus Nakai is mixed with the recrystallized aqueous solution of cyclodextrin, stirred, allowed to stand, the precipitate is collected and purified, and then an internal standard is added, mixed well and centrifuged, and the supernatant is aspirated and filtered to obtain the solution.

[0010] The test solution was subjected to full-spectrum identification by ultra-high performance liquid chromatography coupled with quadrupole tandem time-of-flight mass spectrometry and combined with the Metlin network database;

[0011] Among them, the separation conditions of ultra-high performance liquid chromatography were as follows: mobile phase A was water containing 0.1-0.12% formic acid by volume, and mobile phase B was acetonitrile containing 0.08-0.1% formic acid by volume; gradient elution program: 0-2 min, 95% A-95% A; 2-12 min, 95% A-5% A; 12-15 min, 5% A-5% A; 15-17 min, 5% A-95% A; 17-20 min, 95% A-95% A;

[0012] The conditions of quadrupole tandem time-of-flight mass spectrometry were as follows: ESI source, ESI+ and ESI- scanning modes; capillary voltage: 1.4 kV and 1.3 kV; cone voltage: 40 V and 23 V; ion source temperature: 115-120 °C, desolvation gas temperature: 325-350 °C, cone gas flow rate: 45-50 L / h, desolvation gas flow rate: 600-620 L / h; collision energy: 10-40 V; ion energy: 1 V, scanning time 0.03 s; scanning interval 0.02 s; mass scanning range: 50-1500 m / z.

[0013] Preferably, the specific operation process of the enzymatic hydrolysis treatment was as follows: Oplopanax elatus and cellulase were mixed at a mass ratio of 2:0.1-0.2, added with water equivalent to 20-30 times the mass of Oplopanax elatus, and stirred at 40-55 °C, pH 4-5, and a rotation speed of 150-250 r / min for 65-80 h.

[0014] Preferably, the specific operation process of extracting the medicinal residues was as follows: The dried medicinal residues were mixed with petroleum ether, and extracted by Soxhlet extraction at 80-95 °C for 6-10 h, and the petroleum ether was recovered to obtain the petroleum ether extract of Oplopanax elatus.

[0015] Preferably, the cyclodextrin aqueous solution after recrystallization treatment was prepared according to the following steps:

[0016] Water equivalent to 3-5 times the mass of cyclodextrin was added to cyclodextrin, stirred at a rotation speed of 350-550 r / min for 0.5-2 h, allowed to stand overnight, and the supernatant was discarded; the above operation was repeated 5-8 times to obtain rock sugar-shaped cyclodextrin recrystallization, and water equivalent to 3-5 times the mass of the cyclodextrin recrystallization was added thereto and stirred until a solution was obtained.

[0017] Preferably, the stirring was carried out at a rotation speed of 350-600 r / min for 0.5-1.5 h.

[0018] Preferably, the specific operation process of the purification treatment is as follows: after drying the precipitate, add methanol and wash it by ultrasonic wave, filter, recover methanol from the filtrate, and obtain a white paste.

[0019] More preferably, the internal standard is 2-chlorophenylalanine, and the mass ratio of the internal standard to the white paste is 0.014∶1.

[0020] Preferably, in ultra-high performance liquid chromatography, mobile phase A is water containing 0.1% (v / v) formic acid, mobile phase B is acetonitrile containing 0.1% (v / v) formic acid, the column temperature is 40 °C, the flow rate is 0.3 mL / min, and the injection volume is 6 μL;

[0021] In quadrupole tandem time-of-flight mass spectrometry, the ion source temperature is 120 °C, the desolvation gas temperature is 350 °C, the cone gas flow rate is 50 L / h, and the desolvation gas flow rate is 600 L / h.

[0022] The present invention also provides a use of the method, and the use includes any one of (a) to (d):

[0023] (a) The use of the method in the analysis, separation or detection of carnitine;

[0024] (b) The use of the method in the analysis, separation or detection of nervonic acid;

[0025] (c) The use of the method in the analysis, separation or detection of adrenic acid;

[0026] (d) The use of the method in the analysis, separation or detection of glycerophosphoethanolamine.

[0027] Preferably, the carnitine is palmitoyl carnitine, stearoyl carnitine, tetradecanoyl carnitine or decanoyl carnitine, etc.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] 1. The present invention uses ultra-high performance liquid chromatography coupled with a quadrupole tandem time-of-flight mass spectrometer, with water (V / V) (containing 0.1% formic acid)-acetonitrile (V / V) (containing 0.1% formic acid) as the mobile phase for gradient elution, collects data under ESI positive and negative ion modes, analyzes the data with Masslynx4.1 software, and performs full spectrum identification in combination with the Metlin network database. The ACQUITYTM UPLC-QTOF-MS system is used in combination with the network database Metlin to qualitatively identify the types and specific names of the compounds contained in the petroleum ether extract of Panax ginseng after removing interfering substances such as volatile oils and higher fatty acids. The method provided by the present invention has a short analysis process and a huge analysis sample, avoids the destruction of unstable compounds in the analysis process, and can quickly obtain a large number of compounds and their structures and names in a short time, laying a material foundation for further pharmacological research on Panax ginseng.

[0030] 2. Through the method provided by the present invention, neuraminic acid, adrenic acid, glycerophosphoethanolamine, and palmitoylcarnitine, stearoylcarnitine, tetradecanoylcarnitine, decanoylcarnitine and other conjugated carnitines combined with aliphatic compounds were detected from the spiny ginseng for the first time, providing a chemical basis for the research on the anti-inflammatory and antibacterial, sedative, memory-enhancing, sleep-improving, anti-aging and anti-fatigue effects of the spiny ginseng.

[0031] 3. The present invention uses cellulase and cyclodextrin for the extraction and purification of the petroleum ether extract of Panax ginseng. Cyclodextrin can remove the pigment in the petroleum ether extract of Panax ginseng to prevent the pigment from interfering with the subsequent qualitative detection of Panax ginseng. Cellulase only cuts 1,4-β-D-glycosidic bonds and a few 1,6-β-D-glycosidic bonds, but does not cut any other chemical bonds, so it will not have a destructive effect on the molecular parent nucleus with active ingredients in Panax ginseng, and has strong specificity in the extraction process of the effective ingredients of Panax ginseng. Therefore, the combined use of cellulase and cyclodextrin in the present invention can improve the yield of the compound, remove interfering substances such as volatile oils and higher fatty acids, reduce the burden on the chromatographic column, and facilitate a more convenient investigation of the types and quantities of low molecular weight compounds in the petroleum ether extract of Panax ginseng.

[0032] 4. The present invention uses cellulase to extract the effective ingredients of the spiny ginseng, which can disintegrate the block plant tissue of about one cubic centimeter of the spiny ginseng into a muddy state within 72 hours, greatly increasing the extraction area of the spiny ginseng and the extraction amount of the effective ingredients of the spiny ginseng. At the same time, in view of the rarity of the spiny ginseng, the present invention uses cellulase as the extraction raw material, so that the plant tissue of the spiny ginseng raw material is rapidly disintegrated under the action of cellulase, and the contact area between the subsequent solvent and the plant tissue is increased, thereby improving the extraction effect, and contributing to the protection of this precious plant in the future research and development process.

[0033] 5. The present invention uses 2-chlorophenylalanine as an internal standard. 2-chlorophenylalanine has extremely high polarity and generally elutes first in the chromatographic peak. The retention time of other compounds that elute later minus the retention time of 2-chlorophenylalanine is the actual elution time of the compound. Using this elution time, combined with the fragment information of the compound in the quadrupole time-of-flight mass spectrometry at this retention time, referring to the Metlin network database, the molecular weight and molecular formula of the compound are determined to conduct qualitative research on the compound. Using 2-chlorophenylalanine as an internal standard in the present invention can correct the actual elution time of the compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Total ion current chromatogram (ESI+) of the petroleum ether extract of Oplopanax elatus Nakai;

[0035] Figure 2 Total ion current chromatogram (ESI-) of the petroleum ether extract of Oplopanax elatus Nakai. DETAILED DESCRIPTION OF THE INVENTION

[0036] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0037] The cyclodextrin used in the present invention is one or several of α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin.

[0038] 1 Experimental Materials

[0039] 1.1 Instruments and Reagents

[0040] ACQUITYTM UPLC-QTOF system, Xevo G2-XS QTOF quadrupole time-of-flight mass spectrometer, ACQUITY UPLC binary pump and sample manager (Waters uCo., Milford, MA, USA), Masslynx TM V4.1 workstation, Scientific informatics system V1.7 (Waters), NA35 nitrogen generator (Shanghai Jinlang Technology Co., Ltd.), XFB-small crusher (Jishou Zhongcheng Pharmaceutical Machinery Factory), RCT-3200 ultrapure water instrument (Changchun Bolepat Technology Development Co., Ltd.), DCY-12S nitrogen blowing instrument (Qingdao Haike Instrument Co., Ltd.), EYELA-N-1100 rotary evaporator (Tokyo Rika Kikai Co., Ltd., Japan), DHG-9055 forced air drying oven (Shanghai Daohan Industry Co., Ltd.), Tomos 3-18R low temperature centrifuge (Tomos Co., USA), Waters ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm); UPLC-MS grade acetonitrile was purchased from Fisher Scientific (Fair Lawn, New Jersey, USA), 2-chlorophenylalanine (Sigma Co., USA), cellulase (Aladdin Co., USA), β-cyclodextrin (Shanghai Yuanye Bio-Technology Co., Ltd.), petroleum ether (boiling range 60-90 °C, Tianjin Fuyu Fine Chemical Co., Ltd.).

[0041] 1.2 Medicinal materials

[0042] The experimental Oplopanax elatus Nakai was collected from Linjiang City, Jilin Province in May 2017. It was identified by Professor Du Xiaowei of the Pharmacognosy Teaching and Research Section of Heilongjiang University of Chinese Medicine as the fresh roots, rhizomes and aerial stems of Oplopanax elatus Nakai, a plant of the genus Oplopanax in the Araliaceae family. The collected roots, rhizomes and aerial stems of Oplopanax elatus Nakai were rinsed clean with tap water, the surface moisture was wiped off, dried in an oven at 70 °C, and cut into small sections about 6 cm long for standby.

[0043] 2. Experimental part

[0044] 2.1. Recrystallization pretreatment of cyclodextrin:

[0045] Weigh 3000 g of β-cyclodextrin crude powder, add it to 10 L of hot distilled water at 90 °C, stir with a stirring paddle at a speed of 500 r / min and stir to dissolve for 1 h, let it cool, stand overnight, and discard the supernatant. Repeat the above operation 5 times to obtain rock sugar-like β-cyclodextrin recrystallization. Take 1000 g of β-cyclodextrin recrystallization, add it to 3 L of hot distilled water at 90 °C, stir with a stirring paddle at a speed of 500 r / min, and stir to dissolve to obtain Solution Ⅰ.

[0046] 2.2. Preparation of test solution

[0047] 10 kg of dried and cut dried roots, rhizomes and aerial stems of Radix Panax notoginseng were crushed into coarse powder, 0.75 kg of cellulase was added, stirred in 250 L of distilled water at 48°C, pH 4-5, and a stirring speed of 200 r / min for 72 hours, the upper enzymatic solution was removed by centrifugation, and the lower residue was dried at 70°C. The Radix Panax notoginseng residue was placed in a Soxhlet extractor, petroleum ether was added and repeatedly extracted at 90°C for 8 hours, and the petroleum ether was recovered to obtain the Radix Panax notoginseng petroleum ether extract.

[0048] The petroleum ether extract of Panax ginseng was fully dissolved in anhydrous ethanol to obtain solution II. 150 mL of solution II was slowly added to solution I, and the stirring paddle was turned at 500 r / min and stirred for 1 hour, then cooled and allowed to stand overnight. The precipitate was dried at 70°C, and 3 L of methanol was added for ultrasonic washing twice, and the mixture was filtered with filter paper. The filtrate was used to recover methanol to obtain a white paste. 2 mg of white paste was accurately weighed, 500 μL of dichloromethane was added, and 10 μL of internal standard (2.8 mg / mL, 2-chlorophenylalanine) was added, and the mixture was vortexed for 30 seconds, and then placed in a 4°C centrifuge, centrifuged at 12000 rpm for 15 minutes, 200 μL of supernatant was aspirated, transferred to a sample vial, and filtered through a 0.22 μm microporous filter membrane to obtain the test solution.

[0049] 2.3 UPLC-MS analysis

[0050] 2.3.1. Chromatographic separation conditions:

[0051] The column temperature was 40°C; mobile phase A was water (V / V) (containing 0.1% formic acid), and mobile phase B was acetonitrile (V / V) (containing 0.1% formic acid); gradient elution program, 0-2 min, 95% A-95% A; 2-12 min, 95% A-5% A; 12-15 min, 5% A-5% A; 15-17 min, 5% A-95% A; 17-20 min, 95% A-95% A; the column temperature was 40°C; the flow rate was 0.3 mL / min; the injection volume was 6 μL.

[0052] 2.3.2 Mass spectrometry conditions:

[0053] ESI source, scanning mode: ESI+, ESI- mode; capillary voltage: 1.4kV and 1.3kV; cone voltage: 40V and 23V; ion source temperature: 120℃, desolvation gas temperature: 350℃, cone gas flow rate: 50L / h, desolvation gas flow rate: 600L / h; collision energy: 10-40V; ion energy: 1V, scanning time 0.03s; scanning interval 0.02s; mass scanning range: 50-1500m / z.

[0054] 2.3.3 Data Analysis:

[0055] Leveraging MasslynxTM The data obtained was preprocessed using V4.1 software (Waters Corporation) and post - edited in Excel 2010 software. The final results were organized into a two - dimensional data matrix containing information such as retention time (RT), mass - to - charge ratio (m / z), and observed quantities (samples). Based on the m / z values in the Excel data table, full - spectrum identification was performed using the online database Metlin.

[0056] A total of 74 compounds were identified in the positive ion mode and 45 compounds were identified in the negative ion mode. The total ion chromatograms of the petroleum ether extract of Oplopanax elatus are shown in Figure 1 and Figure 2 . The full - spectrum identification results of the petroleum ether extract of Oplopanax elatus in the positive ion mode and negative ion mode are shown in Table 1 and Table 2, respectively.

[0057] Table 1 Full - spectrum identification results of the petroleum ether extract of Oplopanax elatus in the positive ion mode

[0058]

[0059]

[0060]

[0061] Table 2 Full - spectrum identification results of the petroleum ether extract of Oplopanax elatus in the negative ion mode

[0062]

[0063]

[0064]

[0065] The 113 identified chemical components were classified and analyzed. It was found that the petroleum ether extracts of the roots, rhizomes and aerial stems of Oplopanax elatus Nakai contained 34 fatty acid compounds, 5 fatty amine compounds, 15 amino acid compounds, among which there were 6 alkaloid compounds, 9 steroid and terpene compounds, 9 fat-soluble vitamin and coenzyme compounds, 5 conjugated carnitines cross-linked with aliphatic compounds, as well as other substances such as pigments and flavors. Among these discovered compounds, filixic acid has antibiotic, antimicrobial and anthelmintic effects; magnolin has strong anti-allergic and anti-inflammatory effects; acitretin can be effectively used to treat psoriasis; harmine has antibacterial, anti-parasitic, anti-tumor and hallucinogenic effects, and hyoscyamine can relieve fatigue and improve mood. At the same time, a large amount of conjugated carnitine combined with aliphatic compounds was also found in the petroleum ether extracts of the water extracts of the roots, rhizomes and aerial stems of Oplopanax elatus Nakai. This is the unique amino acid composition that differentiates Oplopanax elatus Nakai from other plants. Carnitine can affect and promote the body's fat metabolism, promote the body's utilization of carbohydrates and amino acids, improve the body's tolerance, prevent lactic acid accumulation, delay aging and have antioxidant effects. Nervonic acid and adrenic acid were also found in the petroleum ether extracts of Oplopanax elatus Nakai. Among them, nervonic acid can repair damaged neurons, and adrenic acid is the most abundant fatty acid in the early human brain. The contents of these two substances decrease significantly in the hippocampus as mammals age. The extracts of Oplopanax elatus Nakai have obvious effects of directly improving the functions of the hippocampus and hypothalamus, thereby improving the patient's sleep and memory. This may be related to the nervonic acid, adrenic acid and other abundant unsaturated fatty acids in the extracts of Oplopanax elatus Nakai. At the same time, a certain amount of ethanolamine glycerophosphate was also found in the petroleum ether extracts of Oplopanax elatus Nakai. It is a compound formed by the condensation of one molecule of glycerol and three molecules of cephalin, and it is an intelligent drug developed successfully abroad in recent years. In vitro experiments have proved that it can inhibit the apoptosis of brain nerve stem cells by inhibiting the production of nuclear factor NF-κB, thereby playing a role in strengthening the brain and improving intelligence and delaying aging. The combined effects of the above components constitute the chemical basis for the anti-inflammatory, antibacterial, sedative, memory-enhancing, sleep-improving, anti-aging and anti-fatigue effects of Oplopanax elatus Nakai.

[0066] In addition, the present invention uses cellulase and β-cyclodextrin for the extraction and purification of the petroleum ether extracts of Oplopanax elatus Nakai, aiming to improve the compound yield, remove interfering substances such as volatile oils and higher fatty acids, reduce the burden on the chromatographic column, and facilitate the more convenient investigation of the types and quantities of low-molecular compounds in the petroleum ether extracts of Oplopanax elatus Nakai.

[0067] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for analyzing and detecting the chemical composition of the petroleum ether extract of Oplopanax elatus, characterized in that, It includes the following steps: Treatment of the test sample solution: enzymatically hydrolyze Oplopanax elatus, remove the enzymatic hydrolysate, and collect the medicinal residues after enzymatic hydrolysis; use petroleum ether as a solvent to extract the said residues to obtain the petroleum ether extract of Oplopanax elatus; mix the petroleum ether extract solution of Oplopanax elatus with the recrystallized aqueous solution of cyclodextrin, stir, let stand, collect the precipitate, conduct purification treatment, then add an internal standard, mix well and centrifuge, and aspirate the supernatant and filter to obtain the solution. Use ultra-high performance liquid chromatography coupled with quadrupole tandem time-of-flight mass spectrometry and combine with the Metlin network database to conduct full-spectrum identification of the test sample solution. Among them, the separation conditions of ultra-high performance liquid chromatography are as follows: mobile phase A is water containing 0.1-0.12% formic acid by volume, and mobile phase B is acetonitrile containing 0.08-0.1% formic acid by volume; gradient elution program: 0-2 min, 95% A-95% A; 2-12 min, 95% A-5% A; 12-15 min, 5% A-5% A; 15-17 min, 5% A-95% A; 17-20 min, 95% A-95% A. The conditions of quadrupole tandem time-of-flight mass spectrometry are as follows: ESI source, ESI+ and ESI- scanning modes; capillary voltage: 1.4 kV and 1.3 kV; cone voltage: 40 V and 23 V; ion source temperature: 115-120 °C, desolvation gas temperature: 325-350 °C, cone gas flow rate: 45-50 L / h, desolvation gas flow rate: 600-620 L / h; collision energy: 10-40 V; ion energy: 1 V, scanning time 0.03 s; scanning interval 0.02 s; mass scanning range: 50-1500 m / z.

2. The method according to claim 1, wherein The specific operation process of the said enzymatic hydrolysis treatment is as follows: mix Oplopanax elatus and cellulase at a mass ratio of 2:0.1-0.2, add water equivalent to 20-30 times the mass of Oplopanax elatus, and stir at 40-55 °C, pH 4-5, and a rotation speed of 150-250 r / min for 65-80 h.

3. The method according to claim 1, wherein The specific operation process of extracting the medicinal residues is as follows: mix the dried medicinal residues with petroleum ether, and use the Soxhlet extraction method to extract at 80-95 °C for 6-10 h, and recover the petroleum ether to obtain the petroleum ether extract of Oplopanax elatus.

4. The method according to claim 1, wherein The recrystallized aqueous solution of cyclodextrin is prepared according to the following steps: Add water at 90 °C equivalent to 3-5 times the mass of cyclodextrin to cyclodextrin, stir at a rotation speed of 350-550 r / min for 0.5-2 h, let stand overnight, and discard the supernatant; repeat the above operation 5-8 times to obtain rock sugar-shaped cyclodextrin recrystallization, and add water at 90 °C equivalent to 3-5 times the mass of the said cyclodextrin recrystallization, and stir until it becomes a solution to obtain the solution.

5. The method according to claim 1, wherein The said stirring is carried out at a rotation speed of 350-600 r / min for 0.5-1.5 h.

6. The method according to claim 1, characterized in that, The specific operation process of the said purification treatment is as follows: dry the precipitate, add methanol and wash it ultrasonically, filter, recover methanol from the filtrate to obtain a white paste.

7. The method according to claim 6, wherein The said internal standard is 2-chlorophenylalanine, and the mass ratio of the internal standard to the said white paste is 0.014:

1.

8. According to the method described in claim 1, characterized in that In ultra-high performance liquid chromatography, mobile phase A is water containing 0.1% formic acid by volume, mobile phase B is acetonitrile containing 0.1% formic acid by volume, the column temperature is 40 °C, the flow rate is 0.3 mL / min, and the injection volume is 6 μL; In quadrupole tandem time-of-flight mass spectrometry, the ion source temperature is 120 °C, the desolvation gas temperature is 350 °C, the cone gas flow rate is 50 L / h, and the desolvation gas flow rate is 600 L / h.

9. Use of the method according to any one of claims 1 to 8, characterized in that The use includes any one of (a) to (d): (a) The use of the method in the analysis, separation or detection of carnitine; (b) The use of the method in the analysis, separation or detection of nervonic acid; (c) The use of the method in the analysis, separation or detection of adrenic acid; (d) The use of the method in the analysis, separation or detection of glycerophosphoethanolamine.

10. The use according to claim 9, characterized in that, The carnitine is palmitoyl carnitine, stearoyl carnitine, tetradecanoyl carnitine or decanoyl carnitine.

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