CN114200059A - Method for rapidly screening toxic alkaloids in Chinese herbal medicine based on UPLC-QTOF-MS technology combined with UNIFI - Google Patents
Method for rapidly screening toxic alkaloids in Chinese herbal medicine based on UPLC-QTOF-MS technology combined with UNIFI Download PDFInfo
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- 238000012216 screening Methods 0.000 title claims abstract description 13
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- RXVGBQCEAQZMLW-UHFFFAOYSA-N alpha-solanine Natural products CC1CCC2C(C)C3C(CC4C5CC=C6CC(CCC6(C)C5CCC34C)OC7OC(CO)C(O)C(OC8OC(CO)C(O)C(O)C8O)C7OC9OC(CO)C(O)C(O)C9O)N2C1 RXVGBQCEAQZMLW-UHFFFAOYSA-N 0.000 description 2
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- ADRDEXBBJTUCND-UHFFFAOYSA-N pyrrolizidine Chemical class C1CCN2CCCC21 ADRDEXBBJTUCND-UHFFFAOYSA-N 0.000 description 2
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- 229940031352 solanine Drugs 0.000 description 2
- ZGVSETXHNHBTRK-OTYSSXIJSA-N solanine Chemical compound O([C@H]1[C@@H](O)[C@@H](CO)O[C@H]([C@@H]1O[C@@H]1[C@@H]([C@H](O)[C@@H](O)[C@H](C)O1)O)O[C@@H]1CC2=CC[C@H]3[C@@H]4C[C@@H]5N6C[C@@H](C)CC[C@@H]6[C@H]([C@@H]5[C@@]4(C)CC[C@@H]3[C@@]2(C)CC1)C)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O ZGVSETXHNHBTRK-OTYSSXIJSA-N 0.000 description 2
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- YSUIQYOGTINQIN-UZFYAQMZSA-N 2-amino-9-[(1S,6R,8R,9S,10R,15R,17R,18R)-8-(6-aminopurin-9-yl)-9,18-difluoro-3,12-dihydroxy-3,12-bis(sulfanylidene)-2,4,7,11,13,16-hexaoxa-3lambda5,12lambda5-diphosphatricyclo[13.2.1.06,10]octadecan-17-yl]-1H-purin-6-one Chemical compound NC1=NC2=C(N=CN2[C@@H]2O[C@@H]3COP(S)(=O)O[C@@H]4[C@@H](COP(S)(=O)O[C@@H]2[C@@H]3F)O[C@H]([C@H]4F)N2C=NC3=C2N=CN=C3N)C(=O)N1 YSUIQYOGTINQIN-UZFYAQMZSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 241000227129 Aconitum Species 0.000 description 1
- 241000758795 Aristolochiaceae Species 0.000 description 1
- 108700004714 Gelonium multiflorum GEL Proteins 0.000 description 1
- 241000934806 Krameria Species 0.000 description 1
- 108010022337 Leucine Enkephalin Proteins 0.000 description 1
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- 244000061457 Solanum nigrum Species 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 1
- 150000002952 aconitine derivatives Chemical class 0.000 description 1
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- URLZCHNOLZSCCA-UHFFFAOYSA-N leu-enkephalin Chemical compound C=1C=C(O)C=CC=1CC(N)C(=O)NCC(=O)NCC(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=CC=C1 URLZCHNOLZSCCA-UHFFFAOYSA-N 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- General Health & Medical Sciences (AREA)
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Abstract
In view of the characteristics of complex structure, low content in biological samples, high toxicity, non-specific clinical manifestation of poisoning and the like of toxic alkaloids, the invention develops a high-energy, high-throughput and high-efficiency accurate screening method for toxic alkaloids in Chinese herbal medicines based on the UPLC-QTOF-MS technology combined with UNIFI software. The method comprises the steps of extracting a Chinese herbal medicine sample by methanol, filtering by an organic filter membrane, detecting by UPLC-QTOF-MS, collecting data of the sample to be screened by adopting an MSE detection mode, and matching and screening information such as mass-to-charge ratio, molecular ion peak, fragment ion and the like on the original data by virtue of a natural product information base in UNIFI software. Compared with the traditional analysis and detection method, the method has the advantages of simple operation, quick analysis and reliable qualitative result, provides a good analysis and detection means for quick screening of toxic alkaloids in the Chinese herbal medicine, and has important significance in the field of clinical quality control and application of the Chinese herbal medicine.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a method for rapidly screening toxic alkaloids in Chinese herbal medicines based on UPLC-QTOF-MS technology combined with UNIFI.
Background
The Chinese herbal medicine refers to a substance which is used for preventing, treating and diagnosing diseases and has the functions of rehabilitation and health care under the guidance of the theory of the traditional Chinese medicine, and is also an important mark for distinguishing the traditional Chinese medicine from other medicines. The Chinese herbal medicine is mainly from natural medicine and processed product thereof, including plant medicine, animal medicine, mineral medicine and partial chemical and biological product medicine. Because the Chinese herbal medicines are mostly plant medicines, there is a saying that the medicines are based on herbs. At present, a lot of people think that most of Chinese herbal medicines come from natural animals, plants and pure Chinese herbal medicine preparations, have mild and safe property compared with chemical medicines, and do not generate toxic and side effects of the medicines. However, just as ancient human clouds "being three drugs toxic", toxic side effects will also occur if the ancient human clouds are arbitrarily abused and the medicine stones are disorganized. The toxicity of Chinese herbal medicine is caused by toxic reaction caused by toxic components contained in the medicine, and most of the toxic components are toxic alkaloid.
Alkaloids are nitrogen-containing basic organic compounds that exist in nature and have base-like properties, and therefore have been referred to as pseudobases. Some of these alkaloids are toxic and easily cause poisoning. The alkaloid has complex structure, low content in biological samples, small toxic dose, high toxicity and no specific clinical manifestation of poisoning, which undoubtedly increases the difficulty of screening and analyzing toxic alkaloids, so that the establishment of a method for simply, conveniently and rapidly screening various toxic alkaloids has important significance for the application of clinical Chinese herbal medicines.
According to domestic and foreign literature reports, methods for analyzing and detecting toxic alkaloids comprise liquid chromatography, liquid chromatography tandem mass spectrometry, gas chromatography tandem mass spectrometry, thin-layer chromatography, molecular imprinting technology, high-efficiency capillary electrophoresis and the like. However, most methods are complicated in steps, long in time consumption, limited in detection objects and low in accuracy.
In conclusion, the inventor considers and develops a high-energy, high-flux and high-efficiency accurate screening method for toxic alkaloids in common Chinese herbal medicines based on a UPLC-QTOF-MS technology combined with UNIFI, so that the detection efficiency and accuracy of the toxic alkaloids in the Chinese herbal medicines are greatly improved.
Disclosure of Invention
The invention aims to provide a method for rapidly screening toxic alkaloids in Chinese herbal medicines based on UPLC-QTOF-MS technology combined with UNIFI, and the method has the advantages of rapidness, sensitivity and accuracy.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for rapidly screening toxic alkaloids in Chinese herbal medicines based on UPLC-QTOF-MS technology combined with UNIFI, which comprises the following steps:
step a), crushing and grinding dry Chinese herbal medicines into powder, and sieving the powder; mixing the Chinese herbal medicine powder with a solvent, performing ultrasonic extraction at room temperature, centrifuging, filtering supernate with an organic filter membrane to obtain a sample, and storing the sample in a sample bottle for sample injection;
and b) separating and detecting the sample prepared in the step a) by using an ultra-high performance liquid chromatography tandem mass spectrometer to obtain an alkaloid component substance peak, and qualitatively analyzing the substance peak by using UNIFI software.
Further, in step a), the mesh number of the screen is 60 meshes.
Further, in the step a), the solvent is methanol, and the ratio of the Chinese herbal medicine powder to the solvent is 1:100 m/v.
Further, in the step a), the ultrasonic extraction time is 30min, the ultrasonic power is 200W, and the ultrasonic frequency is 40 kHz.
Further, in the step a), the centrifugation time is 10 min, and the centrifugation rotation speed is 10000 rpm.
Further, in the step a), the supernatant is firstly filtered by an organic filter membrane with the aperture of 0.45 mu m, and then filtered by an organic filter membrane with the aperture of 0.22 mu m.
Further, in step b), the conditions of the liquid chromatographic separation are as follows: the chromatographic column is ACQUITY UPLC-BEH C18(2.1 mm × 100 mm, 1.7 μm, Waters Corp.); the sample size is 1 mu L, the flow rate is 0.2 mL/min, and the column temperature is 40 ℃; the mobile phase of the elution system is as follows: the mobile phase A is water (containing 10 mmol/L ammonium formate), and the mobile phase A is waterB is methanol; the procedure for gradient elution was: 0-1 min, 95% A; 1-3 min, 95% A → 70% A; 3-12 min, 75% A → 55% A; 12-20 min, 55% A → 1% A; 20-25min, 1% A; 25-25.1min, 1% A-95% A; 25.1-30min, 95% A.
Further, in step b), the mass spectrometry detection conditions are as follows: electrospray ion source (ESI), MSE positive ion scan mode; the nitrogen is cone hole back-blowing gas, desolventizing gas and atomizing gas, and the argon is collision gas; the capillary voltage is 2.00 kV, the cone hole voltage is 40V, the ion source temperature is 150 ℃, the desolvation gas flow is 600L/h, the solvent gas temperature is 350 ℃, the cone hole back-blowing gas flow is 50L/h, the low-energy collision voltage is 6V, the high-energy collision voltage is 30-50V, the mass scanning range is 50-1200 Da, the scanning time is 0.250 s, and the scanning time interval is 0.250 s.
Further, in the step b), the model of the UNIFI software is Waters UNIFI 1.8.
The invention has the following remarkable advantages:
compared with the existing detection method, the method provided by the invention can accurately measure the relative molecular mass of the compound and accurately determine the nature of the compound, thereby avoiding false detection and missed detection of the sample, and has the advantages of simple sample treatment, multiple screened varieties, accurate and reliable qualitative result, capability of greatly improving the screening capability of the sample and great significance for the clinical quality control application of the Chinese herbal medicine.
Drawings
FIG. 1 shows the secondary mass spectrum of aristolochic acid I.
FIG. 2 is a secondary mass spectrum of aconitine.
FIG. 3 is a secondary mass spectrum of Krameria alkali.
FIG. 4 is the second-order mass spectrum of solanine.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example 1
The herbs used in this example were selected from Table 1, which are commercially available.
TABLE 1 Chinese herbal medicine information
The instruments and reagents used in this example were as follows:
the instrument comprises the following steps: a Waters Acquity UPLC H-Class ultra high performance liquid chromatograph and a Xevo G2 QTOF/MS mass spectrometer.
Reagent: acetonitrile (Mass Spectrometry pure, Merck, Germany), methanol (chromatography pure, Merck, Germany), formic acid (chromatography pure, ACS, USA), sodium hydroxide (analytical pure, Sigma-Aldrich, USA), leucine enkephalin (Mass Spectrometry pure, Waters, USA), ammonium formate (chromatography pure, Mackin).
1 sample preparation
Cutting purchased dried Chinese herbal medicines into small pieces, then putting the small pieces into a grinder for grinding into powder, sieving the powder with a 60-mesh sieve, and putting the sieved powder into a PE self-sealing bag for storage and later use. Weighing 0.3 g of Chinese herbal medicine powder, placing the Chinese herbal medicine powder into a 50 mL centrifuge tube, adding 30 mL of methanol into the centrifuge tube, performing ultrasonic extraction at room temperature for 30min, wherein the ultrasonic power is 200W, the ultrasonic frequency is 40 kHz, then centrifuging the centrifuge tube for 10 min under the condition of 10000 r/min, filtering supernatant by an organic filter membrane with the aperture of 0.45 mu m, then filtering by an organic filter membrane with the aperture of 0.22 mu m, storing filtrate in a sample bottle, and storing the filtrate to be detected at 4 ℃ in a dark place.
2 separation and detection of samples
And (3) separating and detecting the prepared sample by using an ultra-high performance liquid chromatography tandem mass spectrometer, wherein the ultra-high performance liquid chromatography tandem mass spectrometer consists of a Waters acquisition UPLC H-Class ultra-high performance liquid chromatograph and a Xevo G2 QTOF/MS mass spectrometer.
The liquid chromatography conditions were: ACQUITY UPLC-BEH C18Chromatography column (2.1 × 100 mm, 1.7 μm); sample introduction amount is 1 mu L, flow rate is 0.2 mL/min, and column temperature40 ℃; the mobile phase of the elution system is as follows: the mobile phase A is water (containing 10 mmol/L ammonium formate), and the mobile phase B is methanol; the procedure for gradient elution was: 0-1 min, 95% A; 1-3 min, 95% A → 70% A; 3-12 min, 75% A → 55% A; 12-20 min, 55% A → 1% A; 20-25min, 1% A; 25-25.1min, 1% A → 95% A; 25.1-30min, 95% A.
The mass spectrum conditions are as follows: an electrospray ion source (ESI) and a positive ion scanning mode (ESI +), wherein nitrogen is cone hole back blowing gas, desolvation gas and atomization gas, argon is collision gas, the capillary voltage is 2.00 kV, the cone hole voltage is 40V, the ion source temperature is 150 ℃, the desolvation gas flow rate is 600L/h, the solvent gas temperature is 350 ℃, the cone hole nitrogen flow rate is 50L/h, the low-energy collision voltage is 6V, the high-energy collision voltage is 30-50V, the mass scanning range is 50-1200 Da, the scanning time is 0.250 s, and the scanning time interval is 0.250 s.
3 qualitative analysis of toxic alkaloid components in Chinese herbal medicine
And (3) introducing the alkaloid component substance peak data measured by the ultra-performance liquid chromatography tandem mass spectrometer into Waters UNIFI 1.8 software for analysis.
Before data processing, related documents of alkaloid chemical components in 43 Chinese herbal medicines reported at home and abroad (the information of the Chinese herbal medicines is shown in table 1) are consulted, English names, molecular formulas, precise molecular weights and CAS numbers of the chemical components are recorded into an Excel table, a structural formula of each chemical component is obtained from Chemmpider through the CAS number, the Excel table and the structural formula are introduced into UNIFI 1.8 software of Waters, a toxic alkaloid database is built by self, and 92 compounds are collected in the toxic alkaloid database. Subsequently, the extracted alkaloid component substances in the 43 Chinese herbal medicines are qualitatively analyzed in Waters UNIFI 1.8 software, the cracking mode of the extracted alkaloid component substances is analyzed through secondary mass spectrum fragment information of toxic alkaloids, and 47 alkaloid components including aristolochia alkaloids, aconitum alkaloids, pyrrolizidine alkaloids and other types of alkaloids are separated and identified in the 43 Chinese herbal medicines, and are specifically shown in Table 2. The references referred to are as follows:
[1] qualitative and quantitative analysis of aristolochic acid components in 9 Chinese medicinal materials containing aristolochic acid toxic substances [ J ] pharmaceutical science, 2021, 56(7):8.
[2] Based on UHPLC-Q/TOF-MS technology, the content [ J ] of 8 aristolochic acid substances in five plants of the Aristolochiaceae family is rapidly determined, and the content 2015, 024(006):364-375 is prepared in Chinese pharmacy (English edition).
[3] Yuan Yue, Lin \203429ultra high performance liquid chromatography-time of flight mass spectrometry qualitatively and quantitatively detects the content of 14 aconite alkaloids and metabolites thereof in aconite plants [ J ]. food safety and quality detection academy, 2018, 9(21):6.
[4] Suyu, Jingjing, the identification and comparison of Chinese medicine Sichuan aconite root and wild aconite root and the analysis of pharmacological activity [ J ]. the current generation medical complex 2020, 18(7):2.
[5]Wang J ,Zhang M ,Chen L , et al. Determination of Toxic Pyrrolizidine Alkaloids in Traditional Chinese Herbal Medicines by UPLC-MS/MS and Accompanying Risk Assessment for Human Health[J]. Molecules, 2021, 26(6):1648.
[6] Zhimin, LUO, Xuan, et al. Development of UPLC-Q-TOF-MS Coupled with Cation-exchange Solid-phase Extraction Method for the Determination of Ten Pyrrolizidine Alkaloids in Herbal Medicines[J]. Analytical Sciences, 2019, 35(12):1317-1325.
[7] Chemical component researches of gelonin [ J ] Chinese herbal medicines, 2017, 48(10):5.
[8] Pongyong, Chenpefeng, the chemical composition and pharmacological and toxicological research progress of traditional Chinese medicine Solanum nigrum [ J ]. Shanxi Chinese medicine, 2011, 27(1):3.
Wherein the co-separation identified 6 aristolochia alkaloids, exemplified by compound 1 (FIG. 1), and in positive ion mode, the first mass spectrum M/z of compound 1 was 359.0873, corresponding to [ M + NH ]4]+The fragment ions M/z observed in the secondary mass spectrogram are 324.04990, 280.05892, 265.03713 and 236.04102 which are respectively [ M + H-H ]2O]+，[M+H-H2O-CO2]+，[M+H-H2O-CO2-CH2O]+、[M+H-H2O-CO2-CH2O-CO]+So as to generate; according to molecular weight, ionFragment information and compound database, the fact that the compound is aristolochic acid I is inferred, and other aristolochic alkaloid compounds are inferred by the method.
Among them, 18 aconitine alkaloids were identified by co-separation, taking compound 11 as an example (FIG. 2), and the primary mass spectrum M/z of compound 11 is 646.3199 [ M + H ] in positive ion mode]+The fragment ions M/z observed in the secondary mass spectrogram are 614.29416, 586.29921, 570.30447, 554.27359, 522.28237 and 368.18411 which are respectively [ M + H-CH [)3OH]+、[M+H-CH3COOH]+、[M+H-CH3COOH-H2O]+、［M+H-CH3COOH-CH3OH]+、[M+H-CH3COOH-3CH3OH-C6H5COOH]The generated compound is inferred to be aconitine according to the molecular weight, the ion fragment information and the compound database, and the rest aconitine alkaloid compounds are inferred according to the method.
Among them, 9 pyrrolizidine alkaloids were identified by co-separation, and compound 26 (FIG. 3) was used as an example, and M/z thereof was 366.19088 [ M + H ] in positive ion mode]+The fragment ions M/z can be seen from the secondary mass spectrum to be 348.18072, 168.10164, 150.09096 and 122.05917, and the corresponding ions are [ M + H-H ] respectively2O]+、[M+H-C10H14O4(necic acid)]+、[M+H-C10H14O4(necic acid)-H2O]+、[M+H-C10H14O4(necic acid)-H2O-CO]+According to the molecular weight, the ion fragment information and the compound database, the compound is deduced to be crimson-gehridine, and the rest pyrrolizidine alkaloid compounds are deduced according to the method.
Among them, the co-separation identified 14 other types of alkaloids, exemplified by compound 46 (FIG. 4), whose M/z was 868.59372 [ M + H ] in positive ion mode]+The fragment ions are 850.49265, 722.44697, 704.43586 and 558.37915, and the corresponding ions are [ M + H-H ] respectively2O]+、[M+H-Rha]+、[M+H-Rha-H2O]+、[M+H-Rha-Glu]+The compound is presumed to be solanine according to the molecular weight, the ion fragment information and the compound database, and the rest types of alkaloid compounds are deduced according to the method.
TABLE 2 alkaloid isolation and identification from 43 Chinese herbs
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (9)
1. A method for rapidly screening toxic alkaloids in Chinese herbal medicines based on UPLC-QTOF-MS technology combined with UNIFI is characterized by comprising the following steps:
step a), crushing and grinding dry Chinese herbal medicines into powder, and sieving the powder; mixing the Chinese herbal medicine powder with a solvent, performing ultrasonic extraction at room temperature, centrifuging, filtering supernate with an organic filter membrane to obtain a sample, and storing the sample in a sample bottle for sample injection;
and b) separating and detecting the sample prepared in the step a) by using an ultra-high performance liquid chromatography tandem mass spectrometer to obtain an alkaloid component substance peak, and qualitatively analyzing the substance peak by using UNIFI software.
2. The method as claimed in claim 1, wherein in step a), the solvent is methanol, and the ratio of the herbal powder to the solvent is 1:100 m/v.
3. The method according to claim 1, wherein in step a), the ultrasonic extraction time is 30min, the ultrasonic power is 200W, and the ultrasonic frequency is 40 kHz.
4. The method according to claim 1, wherein in step a), the centrifugation conditions are 10000 rpm and 10 min.
5. The method according to claim 1, characterized in that in step a), the supernatant is filtered through a 0.45 μm organic filter membrane and a 0.22 μm organic filter membrane in this order.
6. The method according to claim 1, wherein in step b), the chromatographic separation conditions are: the chromatographic column is ACQUITY UPLC BEH C18The specification of the chromatographic column is 2.1 mm multiplied by 100 mm and 1.7 mu m; the sample size is 1 mu L, the flow rate is 0.2 mL/min, and the column temperature is 40 ℃; the mobile phase of the elution system is as follows: mobile phase a was water containing 10 mM ammonium formate and mobile phase B was methanol; the procedure for gradient elution was: 0-1 min, 95% A; 1-3 min, 95% A → 70% A; 3-12 min, 75% A → 55% A; 12-20 min, 55% A → 1% A; 20-25min, 1% A; 25-25.1min, 1% A-95% A; 25.1-30min, 95% A.
7. The method according to claim 1, wherein in step b), the mass spectrometric detection conditions are: electrospray ion source, MSE positive ion scan mode; the nitrogen is cone hole back-blowing gas, desolventizing gas and atomizing gas, and the argon is collision gas; the capillary tube voltage is 2.00 kV, the cone hole voltage is 40V, the ion source temperature is 150 ℃, the desolvation gas flow is 600L/h, the desolvation gas temperature is 350 ℃, the cone hole gas flow is 50L/h, the low-energy collision voltage is 6V, the high-energy collision voltage is 30-50V, the mass scanning range is 50-1200 Da, the scanning time is 0.250 s, and the scanning time interval is 0.250 s.
8. The method according to claim 1, wherein in step b), the model number of the UNIFI software is Waters UNIFI 1.8.
9. Use of the method of claim 1 for the differential testing of alkaloid components in a sample of Chinese medicinal herbs.
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| CN112444592A (en) * | 2020-11-11 | 2021-03-05 | 谱尼测试集团北京检验认证科学研究院有限公司 | Method for rapidly screening active ingredients in liquorice by UPLC-Q-TOF-MS |
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