CN112924583B

CN112924583B - Method for determining pyrrolizidine alkaloid in eupatorium

Info

Publication number: CN112924583B
Application number: CN202110110743.4A
Authority: CN
Inventors: 昝珂; 左甜甜; 金红宇; 马双成; 王莹; 刘丽娜; 李耀磊; 王丹丹
Original assignee: National Institutes for Food and Drug Control
Current assignee: National Institutes for Food and Drug Control
Priority date: 2021-01-27
Filing date: 2021-01-27
Publication date: 2022-12-06
Anticipated expiration: 2041-01-27
Also published as: CN112924583A

Abstract

The method for measuring pyrrolizidine alkaloid in eupatorium comprises the following steps: (1) Preparing a mixed reference solution containing penamedine, dan Ansong, lindellodine, N-oxidized penamedine and N-oxidized Dan Ansong; (2) Putting the eupatorium powder into a conical flask with a plug, and adding 0.05 mol.L ^–1 Ultrasonic extracting sulfuric acid solution, filtering, and purifying the filtrate; (3) determination, chromatographic column: waters acquisition UPLC HSS T3 chromatography column; column temperature: 40 ℃; mobile phase: 0.05% formic acid and 2.5 mmol. Multidot.L ^–1 Aqueous ammonium formate solution (A) and a mixture containing 0.05% formic acid and 2.5 mmol. Multidot.L ^–1 Acetonitrile (B) of ammonium formate, gradient elution; flow rate: 0.3 mL/min ^–1 (ii) a Sample injection amount: 1 mu L of the solution; and (4) detecting by mass spectrometry. The method provides scientific basis for safe administration of herba Eupatorii.

Description

Method for determining pyrrolizidine alkaloid in eupatorium

Technical Field

The invention relates to the field of quality detection of traditional Chinese medicinal materials, and in particular relates to a method for determining pyrrolizidine alkaloid in eupatorium.

Background

Eupatorium fortunei is a dry aerial part of Eupatorium fortunei Turcz of Eupatorium plant of Eupatorium of Compositae, is a collected variety in the 2020 edition of Chinese pharmacopoeia, has the effects of aromatizing, eliminating dampness, activating spleen, stimulating appetite, relieving exterior syndrome and relieving summer heat, and is used for treating diseases such as damp stagnation in middle energizer, wrist fullness, nausea, vomiting, sweet and greasy taste in mouth, halitosis, excessive salivation, summer-heat and damp exterior syndrome, early stage of damp warmth, fever, lassitude, chest discomfort and the like. The eupatorium is produced in Shandong, jiangsu, zhejiang, jiangxi, hubei, hunan, yunnan, sichuan, guizhou, guangxi, guangdong and Shanxi, is rare in wild and has many cultivators. Wilderness grow around the road and beside the mountain ditches. The whole plant and flower have fragrance like lavender. Eupatorium fortunei is an ancient common aromatic dampness-resolving medicine, is worn on a body as a sachet at first, and is listed as a superior product in Shen nong Ben Cao Jing as a medicine with the name of 'lan Cao'. The eupatorium fortunei is recorded in a list of articles which are published by the national Wei Jian committee and can be used for health food due to the special aroma and certain curative effect of the eupatorium fortunei, and the eupatorium fortunei is used as a raw material in health food formulas for clearing heat from throat, regulating immunity and the like at present. The chemical components of the eupatorium are reported more, and the eupatorium mainly contains various compounds such as volatile oil, monoterpene, phenylpropanoid, pyrrolizidine alkaloids and the like.

Pyrrolizidine Alkaloids (PAs) are widely present in a variety of plants, and statistically, about 3% of the flowering plants in the world, i.e., more than 6000 plants, contain PAs. PAs have been detected in plants of 13 families, most of which belong to the family Boraginaceae, the family Compositae, the families Senecio (Sterculia, selaginella, ligularia and Eupatorium), and the family Leguminosae. Western botanicals researchers refer to plants containing PAs as PA plants. PAs with a double bond at 1,2 of the pyrrolidine ring have hepatotoxicity and are known as Hepatotoxic Pyrrolizidine Alkaloids (HPAs), with over 600 HPAs reported to date. HPAs can cause irreversible hepatocyte damage and lead to adverse consequences such as Hepatic Sinus Obstruction Syndrome (HSOS), hepatic giant cell disease, hepatic fibrosis and cirrhosis. HSOS cases caused by taking traditional Chinese medicines containing HPAs, such as gynura segetum (also known as madder), senecio scandens and the like, are reported every year in China, and in recent years, the HSOS cases caused by taking traditional Chinese medicines are collected by the national knowledge network (CNKI) in thousands of cases. Besides hepatotoxicity, PAs also have toxicity to lung, heart, kidney, pancreas, brain, etc., and obvious carcinogenic, mutagenic and teratogenic effects; if the medicine is taken for a long time, the medicine has a great safety risk. As early as 1992, liu et al isolated and identified 3 pyrrolizidine alkaloid components from Eupatorium, such as Linderan (Rinderine), 7-O-Acetyl Linderan (7-O-Acetyl Linderan), and supine mustard (Suplinine), and Avula et al established UHPLC-HRMS method to identify and measure 2 pyrrolizidine alkaloid contents from Eupatorium, such as penamedine and Dan Ansong. The eupatorium fortunei can cause chronic poisoning of cattle and sheep and cause diabetes by invading liver and kidney; the alcohol extract of fresh or dry leaves contains toxic components, has acute toxicity, and can be used for anesthetizing rabbit after administration, even inhibiting respiration, slowing heart rate, reducing body temperature, and treating diabetes due to hyperglycemia. Therefore, the toxic components of Eupatorium have been regarded as important.

Disclosure of Invention

The invention aims to provide a method for measuring pyrrolizidine alkaloids in eupatorium, which adopts UPLC-MS/MS method to measure contents of nimedidine, dan Ansong, linderan, N-oxidized memantine and N-oxidized Dan Ansong pyrrolizidine alkaloids in eupatorium, wherein the compounds of the Tradescandine, N-oxidized Linderan, N-oxidized Tradescandine, N-oxidized Nemadeitine and N-oxidized triamcinolone acetonide are firstly found from the eupatorium and the contents thereof are measured.

In order to realize the purpose, the invention is realized by adopting the following technical scheme:

the method for measuring pyrrolizidine alkaloid in eupatorium comprises the following steps:

step 1, mixing a reference substance solution

Accurately weighing appropriate reference substances of the penamedine, dan Ansong, linderesine, N-oxidized penamedine and N-oxidized Dan Ansong, respectively preparing reference substance stock solutions with a certain concentration by 50% of methanol water, respectively accurately weighing appropriate reference substance stock solutions, and preparing a mixed reference substance solution with a certain concentration by 50% of methanol water;

step 2, sample solution

Taking proper amount of eupatorium herb, crushing and sieving by a No. 4 sieve, placing proper amount of eupatorium herb powder into a conical bottle with a plug, and adding 0.05 mol.L ^–1 Ultrasonic extracting sulfuric acid solution, filtering, collecting subsequent filtrate, enriching and purifying SPE-PCX, collecting eluate, concentrating, diluting with 50% methanol water, diluting to a certain volume in a measuring flask, and filtering with microporous membrane;

step 3, measurement

Precisely absorbing 1 mu l of each of the mixed reference solution and the test solution, injecting the solutions into an ultra-high performance liquid chromatography-mass spectrometer, measuring, recording a 30min chromatogram, and calculating the contents of the penamedine, dan Ansong, lindelphine, N-oxidized Linderesine, N-oxidized penamedine and N-oxidized Dan Ansong according to an external standard method;

wherein, the chromatography-mass spectrometry conditions-chromatographic column: a Waters ACQUITY UPLC HSS T3 chromatography column; column temperature: 40 ℃; mobile phase: 0.05% formic acid and 2.5 mmol. Multidot.L ^–1 Aqueous ammonium formate solution (A) and a mixture containing 0.05% formic acid and 2.5 mmol. Multidot.L ^–1 Acetonitrile (B) of ammonium formate, gradient elution conditions: 0 to 1695in, 0% by volume B → 4%B; 16-25min, 4-B → 20-B; flow rate: 0.3 mL/min ^–1 (ii) a Sample injection amount: 1 mu L of the solution; the mass spectrum ion source is an electrospray ionization source, positive ions and a multi-reaction monitoring mode, the capillary voltage is 3.0kV, the ion source temperature is 150 ℃, and the solvent removal temperature is 500 ℃.

As a preferred method of the invention, the enrichment and purification process of SPE-PCX comprises the following steps: methanol-activated SPE-PCX small column, 0.05 mol. L ^–1 Balancing the small column with sulfuric acid solution, loading the subsequent filtrate into the column, eluting with methanol to remove impurities, eluting with 8% ammonia water-methanol solution, collecting eluate, and concentrating.

As preferred for the present invention, the mass spectrometric conditions of penmedidine, dan Ansong, linderan, N-oxide Nemedidine and N-oxide Dan Ansong are as follows:

the invention has the advantages and positive effects that:

(1) The method adopts a UPLC-MS/MS method to determine contents of the indidine, dan Ansong, linderan, spine, N-oxidized Linderan, N-oxidized spine, N-oxidized indidine and N-oxidized Dan Ansong pyrrolizidine alkaloids in the eupatorium, wherein the contents of the indidine, the N-oxidized spine and the N-oxidized triamcinolone acetonide are firstly found from the eupatorium and determined, and the method can provide scientific basis for quality evaluation and safe medication of the eupatorium medicinal materials.

(2) The determination method provided by the invention is simple, convenient and accurate, has good repeatability, high detection sensitivity and accurate detection result; through detection by the method, the eupatorium fortunei is found to be a traditional Chinese medicinal material which can be used for health-care food and contains high-content PAs, so that a larger medication risk is possibly existed, the curative effect risk ratio of the medicinal material is further researched, the limit regulation of pyrrolizidine alkaloid is increased in the quality standard, the risk prompt is increased in the use instruction, the feasibility of the eupatorium fortunei as the health-care food is re-evaluated, and the medication safety of people is guaranteed.

Drawings

FIG. 1 is a graph of the standard curve for penmedidine;

FIG. 2 is a graph of the Dan Ansong standard;

FIG. 3 is a graph of a Linderan standard curve;

FIG. 4 is a graph of standard spikenard base curves;

FIG. 5 is a graph of a standard curve of Lindelbrune N-oxide;

FIG. 6 is a graph of the standard curve of the N-oxide antipyrine base;

FIG. 7 is a graph of the N-oxide Nemetidine standard;

FIG. 8 is a graph of the N-oxide Dan Ansong standard;

FIG. 9 is an MRM chromatogram of a control mixed solution;

FIG. 10 is a MRM chromatogram of a sample solution (S1).

Detailed Description

The present invention is further described in the following with reference to the drawings and the detailed description so that the technical solutions of the present invention will be more understood by those skilled in the art, but the present invention is not limited thereto.

Apparatus and materials

Waters Acquity ^TM UPLC chromatograph (Waters corporation); waters Xevo TQ-S triple quadrupole tandem mass spectrometry detectors (Waters Corp.); KQ-500DE ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); XS105DU one tenth ten thousand electronic analytical balance (METTLER TOLEDO Corp.); mili-Q ultrapure water system (Millipore Corp.).

Controls were either signedine (lot 13420), dan Ansong (lot 12183), linalol (lot 13984), N-oxidized linalol (lot 15599), N-oxidized linalol (lot 13880), N-oxidized signedine (lot 13418) and N-oxidized triamcinolone acetonide (lot 13484) from phytola GmbH, linalol (lot ZZS 19092908) from shanghai screening biotechnology limited, all with a purity of greater than 98%; acetonitrile, methanol, formic acid and ammonium formate were all in mass spectral order (Fisher corporation) with other reagents being analytical grade.

15 Eupatorium samples (numbered as S1-S15) were purchased from Chinese medicinal material market, and were identified as dry aerial parts of Eupatorium fortunei DC by the investigator of the secondary study of Keda Ke, a Chinese institute of Hospital medicine.

Wherein the chemical structural formulas of the penmedine 1, the Dan Ansong, the Lingdaline 3, the Lingdaline 4, the N-oxidized Lingdaline 5, the N-oxidized Lingdaline 6, the N-oxidized penmedine 7 and the N-oxidized Dan Ansong are as follows:

example 1

step 1, mixing a reference substance solution

Accurately weighing appropriate reference substances of the penmedine, dan Ansong, the Lingdaline, the N-oxidized penmedine and the N-oxidized Dan Ansong, and respectively preparing reference substance stock solutions with the mass concentrations of 1, 5, 1, 10, 1 and 1 mug.mL < -1 > by using 50% methanol water; accurately weighing appropriate amount of each reference stock solution, and preparing mixed reference solutions with mass concentrations of 20, 100, 20, 200, 20 and 20 ng.mL < -1 > respectively with 50% methanol-water.

Step 2, sample solution

Taking 0.8g of eupatorium powder (sieved by a No. 4 sieve) at different parts, placing the eupatorium powder in a conical bottle with a plug, and adding 0.05 mol.L ^–1 40mL of sulfuric acid solution, ultrasonic (300W, 40kHz) extraction for 30min, filtration, 10mL of subsequent filtrate for enrichment and purification of SPE-PCX, and the enrichment and purification specific process of SPE-PCX: methanol 5mL activated SPE-PCX small column, 0.05 mol. L ^–1 5mL of sulfuric acid solution is used for balancing a small column, 10mL of the subsequent filtrate is loaded on the column, 10mL of methanol is used for leaching and removing impurities, 10mL of 8% ammonia water-methanol solution is used for eluting, eluent is collected and concentrated to about 1mL, 50% methanol water is used for diluting and fixing the volume to a 100mL measuring flask, a 0.22 mu m microporous filter membrane is used for filtering, and the subsequent filtrate is taken, so that the compound microbial inoculum is obtained.

Step 3, precisely absorbing 1 mu l of each of the mixed reference solution and the test solution, injecting the solution into an ultra-high performance liquid chromatography-mass spectrometer, measuring, recording a chromatogram for 30min, and calculating the contents of the penamedine, dan Ansong, the Linderesine, the N-oxidized penamedine and the N-oxidized Dan Ansong according to an external standard method, wherein the result is shown in Table 2;

wherein, the chromatography-mass spectrometry conditions-chromatographic column: waters ACQUITY UPLC HSS T3 column (2.1 mm. Times.150mm, 1.8 μm); column temperature: 40 ℃; mobile phase: 0.05% formic acid and 2.5 mmol. Multidot.L ^–1 Aqueous solution of ammonium formate (A) and a mixture of 0.05% formic acid and 2.5 mmol. Multidot.L ^–1 Acetonitrile (B) of ammonium formate, gradient elution (0-1695in, 0% B → 4% B16-25min, 4% B → 20% B); flow rate: 0.3 mL/min ^–1 (ii) a Sample introduction amount: 1 mu L of the solution; the mass spectrum ion source is electrospray ionization source (ESI), positive ions, multiple reaction monitoring mode (MRM), capillary voltage is 3.0kV, ion source temperature is 150 ℃, desolvation temperature is 500 ℃, mass spectrum conditions of 8 pyrrolizidine alkaloids are shown in Table 1, and reference substances are obtainedAnd MRM chromatogram of sample S1 is shown in FIG. 9 and FIG. 10, wherein 1 is illustrated as dimedine; 2 is Dan Ansong; 3 is Lindelphine; 4. is the matrine; 5 is N-oxidized Lindellidine; 6 is N-oxidized antipyrine; 7 is N-oxidized trimethoprim; 8 is N-oxide Dan Ansong.

TABLE 1 Mass spectrometric detection parameters for target components

TABLE 2 measurement of sample content (. Mu.g. G) ^-1 ,n＝2)

The nimesulide, dan Ansong, the Linderan, the Tradescanline, the N-oxidized Linderan, the N-oxidized Tradescanline, the N-oxidized nimesulide, the N-oxidized Dan Ansong and the like are the most main pyrrolizidine alkaloid components in the Eupatorium and are trace or ultra-trace components, and the detection of a high-sensitivity UPLC-MS/MS method is the preferred method. The invention adopts UPLC-MS/MS method to measure 8 components simultaneously, positive ion and MRM ion pair, the quantitative ion peak shape is good, other substances are not interference, and the substance to be measured can be measured accurately.

In addition, the invention also considers the influence of acetonitrile-water with different mixture ratios and acid-containing solution containing formic acid, ammonium formate and the like as the separation capability of the mobile phase relative to the pyrrolizidine alkaloid. However, since compounds 1 to 4 and 5 to 8 are all diastereoisomers, the molecular ion peak and mass spectrum cracking behavior are basically consistent, and the compounds need to be separated by a chromatographic column, the 2 groups of diastereoisomers reach baseline separation within 25min by adjusting the gradient proportion, and finally the mobile phase is determined to contain 0.05% of formic acid and 2.5 mmol.L ^–1 Aqueous solution of ammonium formate and a mixture containing 0.05% formic acid and 2.5 mmol. Multidot.L ^–1 Acetonitrile in ammonium formate, and gradient elution; under the system, 8 components are better retained and separated.

Methodology examination

(1) Linear relationship investigation and detection lower limit, quantitative lower limit determination and sensitivity investigation

Precisely sucking 0.05 mL, 0.1 mL, 0.2 mL, 0.5 mL, 1.0mL and 2.0mL of each reference substance stock solution, respectively placing in 6 50mL measuring bottles, adding 50% methanol to constant volume to scale, shaking up to obtain reference substance mixed solution with each series of concentrations, filtering with 0.22 μm microporous membrane, collecting filtrate, and injecting sample. The measurement was carried out according to the chromatographic conditions described above. Taking the peak area Y of the reference substance as the ordinate and the corresponding concentration X as the abscissa, and performing linear regression. The lowest concentration corresponding to the chromatographic peak signal-to-noise ratio S/N of the quantitative ions is greater than or equal to 3 and is used as the lower limit of detection (LOD), the lowest concentration corresponding to the chromatographic peak signal-to-noise ratio of the quantitative ions is greater than or equal to 10 and is used as the lower limit of quantification (LOQ), and the results are shown in Table 3 and fig. 1 to 8.

TABLE 3 Linear equation, linear Range, lower detection limit and lower quantification limit

(2) Precision test

Precisely sucking 1 mu L of the mixed reference substance solution in the step 1, continuously injecting the sample for 6 times, and recording the peak area. Results RSDs (N = 6) for peak areas of 2.06%, 2.87%, 2.65%, 2.56%, 2.92%, 2.26%, 1.89%, and 2.61% for nimedine, dan Ansong, linderesine, tradescanine, N-oxidized Linderesine, N-oxidized Tradescanine, N-oxidized nimedine, and N-oxidized Dan Ansong, respectively (table 4), indicate good precision of the instrument.

TABLE 4 results of precision test

(3) Stability test

The same sample solution (sample S1) is taken and injected for measurement for 0, 2, 4, 8, 12 and 24 hours respectively. Results RSDs (N = 6) for the peak areas of vadex, dan Ansong, linderopine, tradescanine, N-oxidized Linderopine, N-oxidized Tradescanine, N-oxidized vadex and N-oxidized Dan Ansong were 1.35%, 2.07%, 1.97%, 1.15%, 1.33%, 1.65%, 0.82% and 1.56%, respectively (table 5), indicating that the test solutions were stable within 24 h.

TABLE 5 test results of solution stability of test article

Time	Pymedine (Pymetrix)	Dan Ansong	Lingderne alkali	Lingderne alkali	N-oxidized Lindelbrusine	N-oxidized antipyrinthine	N-oxide trimethoprim	N-oxide Dan Ansong
									0h	514434	139592	554434	1694769	129354	4243434	744257	1384035
2h	523514	134924	546245	1674735	129590	4341283	735585	1330702
									4h	519531	137552	564967	1703134	132910	4362130	731831	1342930
6h	514095	132086	570258	1658426	133676	4210202	738835	1339297
									8h	509231	132941	540591	1647701	129911	4397384	731428	1330484
12h	506497	133193	566393	1674368	130273	4301507	746241	1317658
									24h	504386	137030	558834	1682252	129840	4239092	732970	1343339
Average peak area	513098	135331	557389	1676484	130793	4299290	737307	1341206
									RSD(％)	1.35	2.07	1.97	1.15	1.33	1.65	0.82	1.56

(4) Repeatability test

6 parts of sample solution of the sample S1 are prepared according to the method of the step 2 and are respectively injected for measurement. As a result, the average contents of penmedine, dan Ansong, indirubine, linagliptin N-oxide, indidine N-oxide and triamcinolone N-oxide were 7.99, 4.36, 31.63, 135.35, 21.35, 363.75, 45.34 and 88.12. Mu.g.g.g.g. ^-1 RSD (n = 6) were 1.71%, 2.70%, 2.49%, 2.64%, 2.30%, 1.94%, 2.81%, and 2.61%, respectively (table 6), indicating that the reproducibility of the method was good.

TABLE 6 results of the repeatability tests (μ g. G) ^-1 )

Compound (I)	Nimexidine	Dan Ansong	Lingderne alkali	Lingdaline	Linderesin N-oxide	N-oxidized ciliate	N-oxide trimethoprim	N-oxide Dan Ansong
									Sample
1	7.99	4.28	31.85	137.71	21.93	362.14	45.65	89.94
									Sample 2	7.75	4.23	32.02	135.47	21.49	359.08	45.23	86.07
Sample 3	8.10	4.36	30.35	130.99	21.62	360.39	46.24	85.83
									Sample No. 4	7.99	4.29	31.24	132.30	20.47	359.90	42.86	86.87
Sample No. 5	8.13	4.54	32.68	134.84	21.33	377.80	46.25	91.59
									Sample No. 6	7.99	4.46	31.66	140.81	21.28	363.18	45.80	88.41
Average content	7.99	4.36	31.63	135.35	21.35	363.75	45.34	88.12
									RSD(％)	1.71	2.70	2.49	2.64	2.30	1.94	2.81	2.61

(5) Sample application recovery test

Precisely weighing appropriate amount of reference substances of the nimodidine, dan Ansong, the Linderan alkali, the Tradescandine alkali, the N-oxidized Linderan alkali, the N-oxidized nimodidine and the N-oxidized Dan Ansong, precisely weighing the reference substances, and respectively adding 50% methanol water to prepare the 8 components with the mass concentrations of 3.024, 1.964, 12.27, 54.65, 8.352, 75.16, 18.56 and 38.96 mu g/mL in sequence ^–1 Each control solution of (1). 6 portions of sample S1 powder with known content, each about 0.4g, were precisely weighed, placed in conical flasks with stoppers, precisely added with 1.0mL of each of the 8 reference solutions (2 mL of N-oxidorferine), prepared in parallel according to the method of step 2, injected for analysis, and the recovery was calculated (Table 7). The results were found to be average sample recoveries (N = 6) of 92.46%, 90.99%, 83.52%, 84.39%, 108.88%, 104.55%, 87.50% and 83.01% for signet, dan Ansong, indirubine, spikiderd, spikaderd, N-oxide Dan Ansong, and RSD of 2.85%, 2.36%, 2.64%, 2.71%, 2.76%, 1.97%, 2.73% and 2.67%, respectively.

TABLE 7 sample recovery test results

Claims

1. The method for measuring pyrrolizidine alkaloid in eupatorium is characterized by comprising the following steps:

step 1, mixing a reference substance solution

Accurately weighing appropriate amounts of reference substances such as penmedidine, dan Ansong, lingdaline, N-oxidized penmedidine and N-oxidized Dan Ansong, respectively preparing reference substance stock solutions with certain concentrations by 50% methanol water, respectively accurately weighing appropriate amounts of the reference substance stock solutions, and preparing a mixed reference substance solution with certain concentrations by 50% methanol water;

step 2, sample solution

step 3, measurement

Precisely absorbing 1 mu l of each of the mixed reference solution and the test solution, injecting the solution into an ultra-high performance liquid chromatography-mass spectrometer, measuring, recording a chromatogram for 30min, and calculating the contents of penamedine, dan Ansong, linderopine, N-oxidized penamedine and N-oxidized Dan Ansong according to an external standard method;

wherein, the chromatography-mass spectrometry conditions-chromatographic column: waters acquisition UPLC HSS T3 chromatography column; column temperature: 40 ℃; mobile phase: 0.05% formic acid and 2.5 mmol. Multidot.L ^–1 Aqueous ammonium formate solution (A) and a solution containing 0.05% formic acid and 2.5mmol·L ^–1 Acetonitrile (B) of ammonium formate, gradient elution conditions: 0 to 1semin, 0% B → 4%B; 16-25min, 4-B → 20-B; flow rate: 0.3 mL/min ^–1 (ii) a Sample introduction amount: 1 mu L of the solution; the mass spectrum ion source is an electrospray ionization source, positive ions and a multi-reaction monitoring mode, the capillary voltage is 3.0kV, the ion source temperature is 150 ℃, and the solvent removal temperature is 500 ℃;

mass spectrometry conditions for penmedidine, dan Ansong, indirubine, linalodine N-oxide, indidine N-oxide and Dan Ansong N-oxide are as follows:

2. the method for detecting pyrrolizidine alkaloids in eupatorium according to claim 1, wherein the enrichment and purification process of SPE-PCX is as follows: methanol-activated SPE-PCX small column, 0.05 mol. L ^–1 Balancing the small column with sulfuric acid solution, loading the subsequent filtrate into the column, eluting with methanol to remove impurities, eluting with 8% ammonia water-methanol solution, collecting eluate, and concentrating.