CN115184442B - Method for determining anthraquinone components by combining salting-out auxiliary liquid-liquid extraction and capillary electrophoresis on-line enrichment method - Google Patents
Method for determining anthraquinone components by combining salting-out auxiliary liquid-liquid extraction and capillary electrophoresis on-line enrichment method Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 57
- 238000005251 capillar electrophoresis Methods 0.000 title claims abstract description 41
- 238000005185 salting out Methods 0.000 title claims abstract description 34
- PYKYMHQGRFAEBM-UHFFFAOYSA-N anthraquinone Natural products CCC(=O)c1c(O)c2C(=O)C3C(C=CC=C3O)C(=O)c2cc1CC(=O)OC PYKYMHQGRFAEBM-UHFFFAOYSA-N 0.000 title claims abstract description 24
- 238000000638 solvent extraction Methods 0.000 title claims abstract description 18
- 238000000622 liquid--liquid extraction Methods 0.000 title claims abstract description 17
- 150000004056 anthraquinones Chemical class 0.000 title claims abstract description 13
- LQGUBLBATBMXHT-UHFFFAOYSA-N chrysophanol Chemical compound C1=CC=C2C(=O)C3=CC(C)=CC(O)=C3C(=O)C2=C1O LQGUBLBATBMXHT-UHFFFAOYSA-N 0.000 claims abstract description 46
- 238000004458 analytical method Methods 0.000 claims abstract description 23
- NZPQWZZXRKZCDU-UHFFFAOYSA-N chrysophanol Natural products Cc1cc(O)c2C(=O)c3c(O)cccc3Oc2c1 NZPQWZZXRKZCDU-UHFFFAOYSA-N 0.000 claims abstract description 23
- VWDXGKUTGQJJHJ-UHFFFAOYSA-N Catenarin Natural products C1=C(O)C=C2C(=O)C3=C(O)C(C)=CC(O)=C3C(=O)C2=C1O VWDXGKUTGQJJHJ-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000010282 Emodin Substances 0.000 claims abstract description 22
- RBLJKYCRSCQLRP-UHFFFAOYSA-N Emodin-dianthron Natural products O=C1C2=CC(C)=CC(O)=C2C(=O)C2=C1CC(=O)C=C2O RBLJKYCRSCQLRP-UHFFFAOYSA-N 0.000 claims abstract description 22
- YOOXNSPYGCZLAX-UHFFFAOYSA-N Helminthosporin Natural products C1=CC(O)=C2C(=O)C3=CC(C)=CC(O)=C3C(=O)C2=C1O YOOXNSPYGCZLAX-UHFFFAOYSA-N 0.000 claims abstract description 22
- NTGIIKCGBNGQAR-UHFFFAOYSA-N Rheoemodin Natural products C1=C(O)C=C2C(=O)C3=CC(O)=CC(O)=C3C(=O)C2=C1O NTGIIKCGBNGQAR-UHFFFAOYSA-N 0.000 claims abstract description 22
- RHMXXJGYXNZAPX-UHFFFAOYSA-N emodin Chemical compound C1=C(O)C=C2C(=O)C3=CC(C)=CC(O)=C3C(=O)C2=C1O RHMXXJGYXNZAPX-UHFFFAOYSA-N 0.000 claims abstract description 22
- VASFLQKDXBAWEL-UHFFFAOYSA-N emodin Natural products OC1=C(OC2=C(C=CC(=C2C1=O)O)O)C1=CC=C(C=C1)O VASFLQKDXBAWEL-UHFFFAOYSA-N 0.000 claims abstract description 22
- PKUBGLYEOAJPEG-UHFFFAOYSA-N physcion Natural products C1=C(C)C=C2C(=O)C3=CC(C)=CC(O)=C3C(=O)C2=C1O PKUBGLYEOAJPEG-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000003960 organic solvent Substances 0.000 claims abstract description 16
- 238000000926 separation method Methods 0.000 claims abstract description 15
- 238000001514 detection method Methods 0.000 claims abstract description 13
- -1 anthraquinone compounds Chemical class 0.000 claims abstract description 12
- 239000000523 sample Substances 0.000 claims description 62
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 45
- 238000002347 injection Methods 0.000 claims description 22
- 239000007924 injection Substances 0.000 claims description 22
- 239000012488 sample solution Substances 0.000 claims description 21
- ODLHGICHYURWBS-LKONHMLTSA-N trappsol cyclo Chemical compound CC(O)COC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)COCC(O)C)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1COCC(C)O ODLHGICHYURWBS-LKONHMLTSA-N 0.000 claims description 20
- 239000007853 buffer solution Substances 0.000 claims description 19
- BFBPISPWJZMWJN-UHFFFAOYSA-N methyl 2-[(7-hydroxy-3,7-dimethyloctylidene)amino]benzoate Chemical compound COC(=O)C1=CC=CC=C1N=CCC(C)CCCC(C)(C)O BFBPISPWJZMWJN-UHFFFAOYSA-N 0.000 claims description 17
- 229910021538 borax Inorganic materials 0.000 claims description 16
- 239000003795 chemical substances by application Substances 0.000 claims description 16
- 239000011159 matrix material Substances 0.000 claims description 16
- 239000004328 sodium tetraborate Substances 0.000 claims description 16
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 claims description 14
- 241001122767 Theaceae Species 0.000 claims description 13
- CFLNHFUPWNRWJA-UHFFFAOYSA-N Obtusin Chemical compound O=C1C2=CC(C)=C(O)C(OC)=C2C(=O)C2=C1C=C(OC)C(OC)=C2O CFLNHFUPWNRWJA-UHFFFAOYSA-N 0.000 claims description 12
- OBBJQZSMXOJMCN-UHFFFAOYSA-N Obtusin Natural products COc1cc2C=CC(=O)Oc2c3OCC(Oc13)C(=C)C OBBJQZSMXOJMCN-UHFFFAOYSA-N 0.000 claims description 12
- 235000002639 sodium chloride Nutrition 0.000 claims description 12
- 239000012086 standard solution Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 11
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 11
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 11
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 10
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 10
- 238000001962 electrophoresis Methods 0.000 claims description 10
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 8
- 210000000582 semen Anatomy 0.000 claims description 8
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 5
- 239000012044 organic layer Substances 0.000 claims description 5
- 239000001103 potassium chloride Substances 0.000 claims description 5
- 235000011164 potassium chloride Nutrition 0.000 claims description 5
- 210000002700 urine Anatomy 0.000 claims description 5
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 4
- 235000011181 potassium carbonates Nutrition 0.000 claims description 4
- 239000001632 sodium acetate Substances 0.000 claims description 4
- 235000017281 sodium acetate Nutrition 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 3
- 238000010276 construction Methods 0.000 claims description 3
- 238000005259 measurement Methods 0.000 claims description 3
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 3
- 235000017550 sodium carbonate Nutrition 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 238000002203 pretreatment Methods 0.000 claims description 2
- 238000011002 quantification Methods 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims 1
- 244000037364 Cinnamomum aromaticum Species 0.000 abstract description 9
- 235000014489 Cinnamomum aromaticum Nutrition 0.000 abstract description 9
- 238000005516 engineering process Methods 0.000 abstract description 8
- 239000012472 biological sample Substances 0.000 abstract description 6
- 238000004094 preconcentration Methods 0.000 abstract description 5
- 238000005515 capillary zone electrophoresis Methods 0.000 abstract description 3
- 244000269722 Thea sinensis Species 0.000 abstract 1
- QUQPHWDTPGMPEX-UTWYECKDSA-N aurantiamarin Natural products COc1ccc(cc1O)[C@H]1CC(=O)c2c(O)cc(O[C@@H]3O[C@H](CO[C@@H]4O[C@@H](C)[C@H](O)[C@@H](O)[C@H]4O)[C@@H](O)[C@H](O)[C@H]3O)cc2O1 QUQPHWDTPGMPEX-UTWYECKDSA-N 0.000 abstract 1
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- 150000001875 compounds Chemical class 0.000 description 4
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- 239000012266 salt solution Substances 0.000 description 4
- 238000010408 sweeping Methods 0.000 description 4
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- 238000002474 experimental method Methods 0.000 description 3
- 230000005012 migration Effects 0.000 description 3
- 238000013508 migration Methods 0.000 description 3
- DFPMSGMNTNDNHN-ZPHOTFPESA-N naringin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](OC=2C=C3O[C@@H](CC(=O)C3=C(O)C=2)C=2C=CC(O)=CC=2)O[C@H](CO)[C@@H](O)[C@@H]1O DFPMSGMNTNDNHN-ZPHOTFPESA-N 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 229920000858 Cyclodextrin Polymers 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- RWZYAGGXGHYGMB-UHFFFAOYSA-N anthranilic acid Chemical compound NC1=CC=CC=C1C(O)=O RWZYAGGXGHYGMB-UHFFFAOYSA-N 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
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- 239000002773 nucleotide Substances 0.000 description 2
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- 239000002516 radical scavenger Substances 0.000 description 2
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 2
- 241000894007 species Species 0.000 description 2
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- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 description 1
- 229930182837 (R)-adrenaline Natural products 0.000 description 1
- RRLUFPHCTSFKNR-DUXPYHPUSA-N (e)-3-(3,4-dichlorophenyl)prop-2-enoic acid Chemical compound OC(=O)\C=C\C1=CC=C(Cl)C(Cl)=C1 RRLUFPHCTSFKNR-DUXPYHPUSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 229940126678 chinese medicines Drugs 0.000 description 1
- 238000005557 chiral recognition Methods 0.000 description 1
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- 239000012895 dilution Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 229960005139 epinephrine Drugs 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000005350 fused silica glass Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000008240 homogeneous mixture Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
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- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4055—Concentrating samples by solubility techniques
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4055—Concentrating samples by solubility techniques
- G01N2001/4061—Solvent extraction
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for measuring anthraquinone components by combining salting-out auxiliary liquid-liquid extraction and capillary electrophoresis on-line enrichment, which combines salting-out auxiliary liquid-liquid extraction off-line preconcentration technology with dynamic pH connection-sweeping-LVSS capillary on-line technology for the first time under a capillary zone electrophoresis mode and is successfully applied to separation and detection of anthraquinone compounds (chrysophanol, emodin and aurantiamarin) in cassia seed tea and biological sample matrixes; the method has the advantages of simple operation, good reproducibility, short analysis time, high separation efficiency and small organic solvent consumption, meets the requirements of green chemistry better, and simultaneously can enrich target analytes up to 468 times.
Description
Technical Field
The invention relates to a method for measuring anthraquinone components in cassia seed tea or biological samples by using a capillary electrophoresis method based on a pre-concentration strategy combining salting-out auxiliary liquid-liquid extraction and an online enrichment method.
Background
The cassia seed tea is a tea prepared from stir-fried cassia seeds, and is used as a healthy drink, and has the effects of protecting liver, protecting eyes, losing weight and the like. This makes it popular in china, korea and japan. A plurality of researches show that the biological active ingredients of the semen cassiae mainly comprise anthraquinone compounds (AQs) such as chrysophanol, emodin, aurantium obtusin and the like, and the semen cassiae tea has the effects of reducing blood fat, protecting nerves, protecting liver, resisting bacteria, resisting mutation and the like, so the semen cassiae tea is widely applied to the health industry. In order to ensure safe use thereof, researchers have increased attention to quality evaluation of cassia tea. Among many quantitative analysis methods of AQs (TLC, HPLC, CE, etc.), HPLC is still the most commonly used method. However, in HPLC, the solvent consumption is large and the resource waste is serious due to the long retention time of some AQs. The Capillary Electrophoresis (CE) has the advantages of high resolution, short analysis time, low solvent consumption and the like, can make up the defects of the high performance liquid chromatography, and has been used for AQs quantitative analysis. However, in the conventional literature, there is a problem that the sensitivity is low when AQs in plants or Chinese medicines is detected by capillary electrophoresis. This is due to the small sample volume of capillary electrophoresis itself and the short optical path of the mounted ultraviolet detector. Thus, pre-concentration of analytes to increase sensitivity of CE using an on-line enrichment strategy is of interest to researchers. Many on-line enrichment techniques, such as field amplified sample stacking (FASI), electro-pressurizing (EKS), micelle-to-solvent stacking (MSS), cation selective exhaustive injection (CESI), anion selective exhaustive injection (AESI) have been proposed for sensitive determination of trace analytes. However, most of them, such as FASI, EKS, CESI and AESI, rely on electric injection (EKI), and are more suitable for enrichment of charged analytes. In contrast, weakly ionized analytes (e.g., AQs) are difficult to effectively increase sensitivity by these methods when analyzed by capillary electrophoresis.
The methods reported so far for enriching weakly ionized substances in CE analysis mainly include electroosmotic flow pump bulk stacking (LVSEP) and dynamic pH ligation (DYNAMIC PH junction, dypH). Literature reports LVSEP that when trans-3, 4-dichloro-cinnamic acid, 2-aminobenzoic acid, tryptophan, etc. are analyzed, separation can be performed within 8min and the Sensitivity Enhancing Factor (SEF) reaches 170 times. However, in this enrichment method, in order to obtain a stable enrichment effect, the capillary column must be modified by a dynamic coating method or the like. DypH is another enrichment method for analysis of weakly acidic substances, which has been reported in the literature to be applied to the enrichment of epinephrine and weakly acidic compounds, but the improvement in sensitivity is not significant.
In recent years, the combination of various enrichment methods, such as two-step enrichment (FASI-sweeping) and three-step enrichment (FASI-sweeping-MSS), plays a crucial role in increasing the sensitivity of CE. Rageh a new three-step enrichment method is developed, dypH, large-volume sample superposition (LVSS) and sweep (sweeping) are combined, and the nucleotide is quantitatively analyzed by an ultraviolet detector, so that the effect equivalent to the sensitivity of MS detection is achieved, namely LOD is as low as 10ng/mL. This demonstrates the great potential this strategy has in enriching zwitterionic compounds. Meanwhile, a new idea is provided for on-line enrichment of weak acid and weak alkaline substances in CE. In this method, the complexing of borax with the ortho-dihydroxyl groups is used to sweep large volumes of nucleotides into a relatively narrow band of borax. Therefore, the method is suitable for compounds containing an ortho-dihydroxyl structure, and has a limited application range. The method developed by the invention is used for enriching emodin, chrysophanol and aurantium obtusin, and the three compounds have no structure of ortho-dihydroxyl, so that other pseudo stationary phases such as surfactant, cyclodextrin and the like are required to be added into a background buffer solution. Cyclodextrin has been successfully used in chromatographic and electrophoretic methods because of its chiral recognition. The application range of the analyte can be greatly expanded (namely, compared with the literature, the application range of the invention is greatly increased by not only being limited to the compound with the specific ortho-dihydroxyl) by innovatively taking the hydroxypropyl-beta-cyclodextrin as the pseudo stationary phase scavenger.
The presence of the trace AQs in a complex matrix (e.g., cassia tea or biological sample) is a significant challenge for its effective analysis. Thus, in order to exclude interference of other substances with the analyte, the sample pretreatment technique plays an important role in purifying the sample and enriching the target analyte. Salting-out assisted liquid-liquid extraction (SALLE) is one of the homogeneous liquid-liquid extractions, i.e. the use of salt in a homogeneous mixture to induce phase separation, followed by extraction of the target analyte into the organic layer. In literature reports, it has been implemented in conjunction with capillary on-line enrichment techniques and is widely used for pretreatment of trace analytes in various sample matrices (e.g., water, food, biological samples, etc.). And SALLE, when combined with capillary single step enrichment such as field amplified stacking (FASS)/LVSS, the sensitivity is only improved by 61-265 times. Thus, combining the proposed on-line three-step enrichment with SALLE may further increase the sensitivity of the analysis of the liquid sample.
Disclosure of Invention
The invention aims to solve the problems of long pretreatment time and low sensitivity when the anthraquinone components in a complex matrix are analyzed by utilizing a capillary electrophoresis method, and a simple and sensitive method for measuring anthraquinone compounds (chrysophanol, emodin and aurantium obtusin) in cassia seed tea or biological samples by utilizing a preconcentration strategy combining salting-out auxiliary liquid-liquid extraction based on hydroxypropyl-beta-cyclodextrin and on-line dynamic pH connection-sweeping-LVSS is established for the first time.
The technical scheme of the invention is as follows:
a method for determining anthraquinone components by combining salting-out auxiliary liquid-liquid extraction and capillary electrophoresis on-line enrichment, which comprises the following steps:
(1) Salting-out auxiliary liquid-liquid extraction for extracting anthraquinone compounds in sample to be detected
Adding an organic solvent and a salting-out agent into a sample to be tested, swirling the mixture to form two phases, separating an organic layer, volatilizing the organic solvent, and re-dissolving the organic solvent with a sample matrix to obtain a sample solution;
The sample to be tested is for example: semen Cassiae tea or standard urine sample;
the organic solvent is acetonitrile;
the salting-out agent is an aqueous solution of sodium carbonate, potassium carbonate, ammonium sulfate, magnesium sulfate, sodium acetate, sodium chloride or potassium chloride, particularly preferably a 2mol/L aqueous solution of ammonium sulfate;
the volume ratio of the salting-out agent to the organic solvent is 2:1-1:5, and particularly preferably 1:1;
The swirling time is 20 to 40s, particularly preferably 30s;
(2) Three-step enrichment method-capillary electrophoresis for detecting anthraquinone compounds in sample
Pretreating a capillary tube, and analyzing and detecting the sample solution obtained in the step (1) based on a three-step enrichment method-capillary electrophoresis to obtain a capillary electrophoresis spectrogram of the sample solution;
The electrophoresis analysis conditions were: 15-30 mmol/L sodium tetraborate is used as background buffer solution, and 15-50 mmol/L hydroxypropyl-beta-cyclodextrin and 10-20% methanol (pH is adjusted to 9-9.5) are added into the background buffer solution; 12mmol/L disodium hydrogen phosphate buffer (pH=4.1) was used as sample matrix; injecting a sample solution at the pressure of 50mbar, wherein the sample injection time of the sample solution is 100-400 s, the separation voltage is applied to the sample solution at +20kV after the sample injection is finished, the temperature is 25 ℃, and the detection wavelength is 250nm;
Preferred electrophoresis analysis conditions are: taking 25mmol/L sodium tetraborate as a background buffer, and adding 35mmol/L hydroxypropyl-beta-cyclodextrin and 15% methanol (adjusting pH to 9.3) in volume fraction to the background buffer; 12mmol/L disodium hydrogen phosphate buffer (pH=4.1) was used as sample matrix; injecting the sample solution at 50mbar pressure for 300s, wherein the separation voltage is +20kV, the temperature is 25 ℃, and the detection wavelength is 250nm;
The pretreatment method of the capillary tube comprises the following steps: the new capillary column is washed for 10 to 20 minutes respectively with 1mol/L NaOH, 0.1mol/L NaOH and pure water in sequence before being used for activation; before daily sample injection, the capillary column is sequentially washed for 5-10 min by 0.1mol/L NaOH, pure water and background buffer solution respectively; between every two sample analysis operations, the capillary column is sequentially washed for 3-5 min by 0.1mol/L NaOH, pure water and background buffer solution respectively so as to keep the reproducibility of analysis, and a new background buffer solution is timely replaced after every two operations;
(3) Quantification of anthraquinone compounds in a sample
Preparing a mixed standard solution of chrysophanol, emodin and aurantium with the concentration range of 100-3000 ng/mL, detecting under the electrophoresis analysis condition of the step (2), and carrying out parallel measurement for three times to obtain a capillary electrophoresis spectrogram of the mixed standard solution, and respectively drawing standard curves of chrysophanol, emodin and aurantium by taking the peak area of each standard in the spectrogram as an ordinate and the concentration of the standard in the mixed standard solution as an abscissa to complete the construction of the standard curves;
Substituting peak areas of chrysophanol, emodin and aurantium obtusin in a capillary electrophoresis spectrogram of the sample solution obtained in the step (2) into a standard curve, and further calculating to obtain the content of chrysophanol, emodin and aurantium obtusin in the sample.
In the method, SALLE technology and dynamic pH connection-sweeping-LVSS technology are combined, firstly, in the SALLE method, a salting-out agent breaks the combination of target analytes and water, so that the analytes are rapidly distributed into an organic phase, and finally, an organic layer containing the target analytes is obtained.
The mechanism of the dynamic pH connection-sweeping-LVSS technology in the method of the invention is as follows: the capillary is first filled with a high conductivity background buffer followed by pressure injection of a large section of sample solution. After injection of the sample solution, a positive voltage is applied across the CE column, at which stage the hydroxide ions in the background buffer move towards the positive electrode, and the pH in front of the sample solution suddenly increases, causing the analyte to ionize and move towards the positive electrode. The migration rate of the analyte is suddenly slowed down and enriched at the boundary when an acidic sample matrix is encountered. Meanwhile, the hydroxypropyl-beta-cyclodextrin in the background buffer solution moves towards the negative electrode to sweep the sample area under the influence of electroosmotic flow, so that the analyte is further enriched. The enriched sample zone migrates towards the detector under the action of electroosmotic flow, encounters a background buffer solution of high conductivity, suddenly slows down the migration velocity, and is enriched again. Finally, the separation is completed in CZE mode.
Compared with the prior art, the invention has the following advantages:
1. The invention combines the salting-out auxiliary liquid-liquid extraction off-line preconcentration technology with the dynamic pH connection-sweeping-LVSS capillary on-line technology for the first time under the capillary zone electrophoresis mode, and is successfully applied to separation and detection of anthraquinone compounds (chrysophanol, emodin and aurantium obtusin) in cassia seed tea and biological sample matrixes.
2. The method has the advantages of simple operation, good reproducibility, short analysis time, high separation efficiency and small organic solvent consumption, meets the requirements of green chemistry better, and simultaneously can enrich target analytes up to 468 times.
Drawings
FIG. 1 shows structural formulas of chrysophanol, emodin and aurantiin.
FIG. 2 shows the effect of salting-out agent type on SALLE extraction effect.
FIG. 3 is a graph showing the effect of salting-out agent concentration on SALLE.
FIG. 4 shows the effect of salting-out agent concentration on SALLE extraction.
FIG. 5 is a graph showing the effect of salting-out agent volume on SALLE extraction.
FIG. 6 is a graph showing the effect of vortex time on SALLE extraction.
FIG. 7 is a graph showing the effect of sodium tetraborate concentration in background buffer on the accumulation effect of the enrichment method.
FIG. 8 is a graph showing the effect of concentration of hydroxypropyl-beta-cyclodextrin in background buffer on the stacking effect of the enrichment method.
FIG. 9 is a graph showing the effect of methanol content in the background buffer on the stacking effect of the enrichment method.
FIG. 10 is a graph showing the effect of pH of the sample matrix on the stacking effect of the enrichment method.
FIG. 11 is a graph showing the effect of sample injection time on the accumulation effect of the enrichment method.
FIG. 12 shows the analysis of actual samples, (a) tea of Cassia Torae semen, (b) labeled urine sample.
Detailed Description
The present invention is further described below by way of specific examples, but the scope of the present invention is not limited thereto.
In the following examples of the present invention,
Sodium tetraborate, sodium hydroxide, sodium dihydrogen phosphate were purchased from national pharmaceutical chemicals (Shanghai) limited. Ammonium sulfate, sodium acetate, potassium carbonate are supplied by Chengkolong chemical Co. Magnesium sulfate, potassium chloride, sodium sulfate were purchased from Tianjin Yongda chemical company, inc. Hydroxypropyl-beta-cyclodextrin (HP-beta-CD) was produced by Shandong Zibo thousand Hui Biotechnology Co. Purified water was obtained from ha group limited of child, hangzhou, china. HPLC grade methanol and acetonitrile are supplied by Tedia company (Fairfield, US). The standard products of chrysophanol, emodin and aurantium obtusin (purity more than 99%) were purchased from Chengdu Mansite company of China. Semen Cassiae sample was purchased from Hangzhou drug store. Urine samples from healthy rats (university of Zhejiang industries) were stored in a-4 ℃ freezer.
The preparation method of the reference substance solution comprises the following specific steps: respectively precisely weighing appropriate amount of emodin, chrysophanol and aurantiin, adding methanol to dilute into reference solution containing 1mg of emodin, 1mg of emodin and 1mg of aurantiin per 1mL, and shaking.
The apparatus employed in the embodiments of the present invention is equipped with an ultraviolet detector for an Agilent capillary electrophoresis apparatus (AGILENT CE 7100,Agilent Technologies,Waldbronn,Germany). Electrophoresis experiments were performed using fused silica capillaries with an inner diameter of 50 μm provided by Yongnian Rayleigh Feng Sepu equipment Co. The total length and effective length of the capillary were 50cm and 42cm, respectively. Detection wavelength: 250nm.
Example 1
Mixing 0.5mL of semen cassiae tea or a standard urine sample with 0.5mL of acetonitrile, adding 0.5mL of ammonium sulfate solution (2 mol/L) to vortex for 30s, taking an upper organic layer, volatilizing acetonitrile, and redissolving with 1mL of sample matrix to obtain a sample solution.
Pretreatment of the capillary: the new capillary column is washed by 1mol/L NaOH for 20min, 0.1mol/L NaOH for 10min and pure water for 10min in turn before being used for activation; before daily sample injection, washing with 0.1mol/L NaOH, pure water and background buffer solution for 10min respectively; between every two sample analysis runs, 0.1mol/L NaOH, pure water and background buffer solution are sequentially used for flushing for 3min so as to keep the reproducibility of analysis, and a new background buffer solution needs to be replaced in time after every two runs.
The electrophoresis analysis conditions were: 25mmol/L sodium tetraborate as background buffer (pH adjusted to 9.3 using 1mol/L NaOH solution); the sample matrix was 12mmol/L disodium hydrogen phosphate. Injecting a sample solution for 300s at the pressure of 50mbar, and applying a separation voltage of +25kV after sample injection is finished; detecting the temperature: 25 ℃; detection wavelength: 250nm.
Condition optimization of example 2 SALLE
(1) Optimization of the species of salting-out agent
A suitable SALLE condition not only requires that as much analyte as possible be transferred from the aqueous phase to the organic phase, but also that the degree of mixing of the organic phase with the aqueous phase be as low as possible. Therefore, extraction efficiency (E) and phase ratio (r) are introduced as indexes, and the optimal conditions of SALLE are studied. E and r represent the ratio of the mass of the analyte in the organic phase to the initial total mass of the analyte and the ratio of the organic phase to the aqueous phase, respectively.
In order to optimize the salt type, the present invention examined the usual salting-out agents (sodium carbonate, potassium carbonate, ammonium sulfate, magnesium sulfate, sodium acetate, sodium chloride and potassium chloride). Experimental salt solutions presented different r and E in SALLE, these salts can be easily classified into three classes (r >1, r=1, r <1, respectively) according to the r value. It is not difficult to find that sulfate extraction efficiency is far better than chloride and other salts, probably because the ability of anions to precipitate hydrophilic species is reduced in the order SO4 2->CH3COO->Cl->NO3 ->Br->I->CNS-. The optimal salt type and salt concentration were then selected by comparing the effect of three different concentrations of salt (1.0, 1.5, 2.0, 2.5, 3.0 mol/L) on r and E. Notably, low concentrations of KCl (1.0, 1.5 mol/L) do not allow separation of the two phases. The effect of magnesium sulfate and ammonium sulfate on E is similar, but ammonium sulfate exhibits a larger r-value, which is detrimental to concentrating the sample solution. Therefore, a 2mol/L ammonium sulfate solution is preferable as the final salting-out solution.
(2) Optimization of salting-out agent volume
The volume of the organic solvent was fixed at 0.5mL, and the ratio of salt solution to organic solvent was optimized at 2:1, 1:1, 1:2, 1:3, 1:4, and 1:5. Wherein, when the ratio of the salt solution to the organic solvent is 2:1, the two-phase separation cannot be achieved. When the ratio is 1:1, the extraction efficiency is optimal. This is due to insufficient salt-induced separation when the salt volume is low or the organic phase volume is high. Therefore, a ratio of the salt solution (2 mol/L ammonium sulfate solution) to the organic solution of 1:1 is preferred.
(3) Optimization of vortex time
In SALLE, the vortexes result in rapid and efficient transfer of analytes due to the large surface contact between the aqueous and organic phases. Thus, the extraction efficiency was tested over a vortex time of 0 to 100 s. As the vortex time increases from 0s to 30s, the peak area of all three analytes increases. This is probably due to the very fast initial mass transfer, and then the equilibrium state is reached around 30s. After 30s, longer vortex times resulted in reduced analyte extraction, probably due to higher miscibility of the two phases at longer contact times. Preferably the vortex time is 30s.
Example 3 optimization of enrichment Process
(1) Concentration of sodium tetraborate in background buffer
Background buffer is one of the most important parameters in capillary electrophoresis that affect target analyte sensitivity and separation efficiency. Since the analyte is acidic, the boric acid system was chosen as the background buffer system for subsequent experiments. The concentration of buffer salt directly affects the conductivity of BGS, and thus the enrichment effect. The concentration of sodium tetraborate in BGS is optimized (15-30 mmol/L). As the concentration of sodium tetraborate increases, the peak area of the target analyte increases. And when the concentration of sodium tetraborate reaches 30mmol/L, the resolution is significantly reduced. This is probably due to the joule heating effect caused by the higher conductivity of sodium tetraborate at higher concentrations and large operating currents. The concentration of sodium tetraborate is preferably 25mmol/L in view of resolution and peak shape.
(2) Concentration of HP-beta-CD and methanol content in background buffer
The role of HP-beta-CD addition to BGS is mainly to form a pseudo stationary phase in the column. When a voltage is applied to the capillary column, the HP-beta-CD sweeps across the sample region, enriching the sample zone. Thus, the concentration of HP-beta-CD in BGS directly affects sweeping performance. In the present invention, several concentrations of HP-beta-CD (15-50 mmol/L) were evaluated. As the HP-beta-CD concentration increases, the peak area gradually increases and gradually decreases beyond 35 mmol/L. This may be due to the fact that when the HP-beta-CD concentration is too high, the affinity between the analyte and HP-beta-CD is too strong to release the analyte. Therefore, preferably 35mmol/L HP-beta-CD is added as a scavenger to the BGS.
Methanol is typically added to BGS to increase the resolution of the analyte. Therefore, the invention examines the influence of methanol content (0%, 5%, 10%, 15%, 20%) (mL: mL) in BGS on separation effect. When the methanol content is increased from 0% to 15%, the resolution is gradually increased, and when the methanol content exceeds 15%, the resolution is gradually decreased. The migration time increases with increasing methanol content. Therefore, 15% methanol is preferable as the addition amount of methanol in BGS.
(3) Sample matrix pH
In DypH, the pH of the BGS and sample matrices need to be different. Therefore, it is necessary to optimize the pH of the sample matrix. When the pH is too low (pH < 3.7), the enrichment of chrysophanol is poor, probably due to the fact that when the pH of the sample matrix is below 3.7, the final capillary column conditions are not suitable for enrichment of analytes such as chrysophanol, resulting in a reduced peak area. However, when the pH is higher (pH > 5.7), the resolution drops significantly and the enrichment is poor. In order to ensure a stable enrichment effect, it is finally preferred to carry out subsequent experiments on a sample matrix with a pH of 4.1.
(4) Sample injection time
Increasing the injection time can significantly increase the sensitivity of capillary electrophoresis. The influence of the sample injection time on the sensitivity was examined with sample injection times of 100s, 150s, 200s, 250s, 300s and 400 s. It is not difficult to find that as the injection time increases, the peak area increases significantly and the resolution decreases. In order to ensure good resolution and peak shape, a 300s sample injection time is preferred.
Example 4 comparison of three-step enrichment with one-step and two-step enrichment
The sensitivity of DypH-sweeping-LVSS was compared with those of DypH and DypH-LVSS. In DypH, enrichment was not ideal due to the limitation of the injection time, and peak broadening of chrysophanol was severe. Peak shape disruption was more severe, although the fold enrichment of DypH-LVSS was increased compared to DypH. In DypH-sweeping-LVSS, the analyte was concentrated in a narrower band, indicating that binding sweeping can improve peak shape to a large extent.
Example 5 comparison of the three-step enrichment method with other reported methods
DypH-sweeping-LVSS-CE-UV was compared with the methods in the literature reported (CE-DAD; NACE-DAD; CE-AD; CE-UV). Compared with other methods, the DypH-sweeping-LVSS-CE-UV strategy provided by the invention can obviously improve the sensitivity of capillary electrophoresis analysis AQs.
Table 1 comparison of three-step enrichment with other reported enrichment methods
Example 6 establishment of a Standard Curve, linear Range of method, detection Limit, reproducibility and enrichment factors
And respectively taking a proper amount of 1mg/mL standard solution, accurately preparing a mixed standard solution of chrysophanol, emodin and aurantium obtusin with the concentration range of 100-3000ng/mL, carrying out electrophoresis analysis based on a three-step enrichment method under the optimized detection condition, carrying out parallel measurement for three times to obtain a capillary electrophoresis spectrogram of the mixed standard solution, and respectively drawing standard curves of chrysophanol, emodin and aurantium obtusin by taking the peak area of each standard in the obtained spectrogram as an ordinate and the concentration of the standard in the mixed standard solution as an abscissa to complete the construction of the standard curves. The results are shown in Table 1, where all three anthraquinone analytes exhibit good linearity and a correlation coefficient (R 2) between 0.993 and 0.998. The 1 mug/mL mixed standard solution is continuously injected for 6 times in one day to evaluate the daily precision, 3 times in 3 days to evaluate the daily precision, and finally, the Relative Standard Deviation (RSD) of the area of the obtained peak is calculated to be lower than 4.80 percent, which indicates that the reproducibility of the method is better.
Table 2 Linear Range, detection Limit, reproducibility and enrichment factors of the method
Enrichment factor= (peak area of the sample to be measured/peak area of the sample to be measured in conventional manner) x dilution factor.
The conventional sample injection conditions are as follows: the background buffer contained 25mmol/L sodium tetraborate, 35mmol/L HP-beta-CD and 15% methanol (pH adjusted using NaOH solution=9.3), and 100. Mu.g/mL of a mixed standard solution of chrysophanol, emodin and aurantium was injected at 50mbar for 3s. The optimized on-line enrichment strategy is calculated to ensure that the enrichment times of chrysophanol, emodin and aurantium obtusin can reach up to 468 times, thereby effectively improving the detection sensitivity of capillary electrophoresis to anthraquinone compounds in biological matrixes.
Claims (6)
1. A method for determining anthraquinone components by combining salting-out auxiliary liquid-liquid extraction and capillary electrophoresis on-line enrichment method is characterized by comprising the following steps:
(1) Salting-out auxiliary liquid-liquid extraction for extracting anthraquinone compounds in sample to be detected
Adding an organic solvent and a salting-out agent into a sample to be tested, swirling the mixture to form two phases, separating an organic layer, volatilizing the organic solvent, and re-dissolving the organic solvent with a sample matrix to obtain a sample solution;
the organic solvent is acetonitrile;
The salting-out agent is aqueous solution of sodium carbonate, potassium carbonate, ammonium sulfate, magnesium sulfate, sodium acetate, sodium chloride or potassium chloride;
The volume ratio of the salting-out agent to the organic solvent is 2:1-1:5;
the vortex time is 20-40 s;
(2) Three-step enrichment method-capillary electrophoresis for detecting anthraquinone compounds in sample
Pretreating a capillary tube, and analyzing and detecting the sample solution obtained in the step (1) based on a three-step enrichment method-capillary electrophoresis to obtain a capillary electrophoresis spectrogram of the sample solution;
the pretreatment method of the capillary tube comprises the following steps: the new capillary column is washed for 10 to 20 minutes respectively with 1mol/L NaOH, 0.1mol/L NaOH and pure water in turn before being used for activation; before daily sample injection, the capillary column is sequentially washed for 5-10 min by 0.1mol/L NaOH, pure water and background buffer solution respectively; between every two sample analysis operations, the capillary column is sequentially washed for 3-5 min by 0.1mol/L NaOH, pure water and background buffer solution respectively so as to keep the reproducibility of analysis, and a new background buffer solution is timely replaced after every two operations;
The electrophoresis analysis conditions were: 15-30 mmol/L sodium tetraborate is used as a background buffer solution, 15-50 mmol/L hydroxypropyl-beta-cyclodextrin and 10-20% methanol in volume fraction are added into the background buffer solution, and the pH is regulated to 9-9.5; 12 mmol/L disodium hydrogen phosphate buffer was used as sample matrix, ph=4.1; 50 Injecting a sample solution at the mbar pressure, wherein the sample injection time of the sample solution is 100-400 s, and after the sample injection is finished, applying a separation voltage of +20 kV, wherein the temperature is 25 ℃, and the detection wavelength is 250 nm;
(3) Quantification of anthraquinone compounds in a sample
Preparing a mixed standard solution with the concentration range of 100-3000 ng/mL of chrysophanol, emodin and aurantium, detecting under the electrophoresis analysis condition of the step (2), and carrying out parallel measurement for three times to obtain a capillary electrophoresis spectrogram of the mixed standard solution, and respectively drawing standard curves of the chrysophanol, the emodin and the aurantium by taking the peak area of each standard in the spectrogram as an ordinate and the concentration of the standard in the mixed standard solution as an abscissa to complete the construction of the standard curves;
Substituting peak areas of chrysophanol, emodin and aurantium obtusin in a capillary electrophoresis spectrogram of the sample solution obtained in the step (2) into a standard curve, and further calculating to obtain the content of chrysophanol, emodin and aurantium obtusin in the sample.
2. The method for determining anthraquinone components by combining salting-out auxiliary liquid-liquid extraction and capillary electrophoresis on-line enrichment method as claimed in claim 1, wherein in the step (1), the sample to be determined is: semen Cassiae tea or standard urine sample.
3. The method for determining anthraquinone components by combining salting-out auxiliary liquid-liquid extraction with capillary electrophoresis on-line enrichment method as claimed in claim 1, wherein in the step (1), the salting-out agent is 2 mol/L ammonium sulfate aqueous solution.
4. The method for determining anthraquinone components by combining salting-out auxiliary liquid-liquid extraction and capillary electrophoresis on-line enrichment method according to claim 1, wherein in the step (1), the volume ratio of the salting-out agent to the organic solvent is 1:1.
5. The method for determining anthraquinone components by combining salting-out auxiliary liquid-liquid extraction with capillary electrophoresis on-line enrichment method as claimed in claim 1, wherein in the step (1), the time of vortex is 30 s.
6. The method for determining anthraquinone components by combining salting-out auxiliary liquid-liquid extraction with capillary electrophoresis on-line enrichment method as claimed in claim 1, wherein in the step (2), the electrophoresis analysis conditions are as follows: taking 25 mmol/L sodium tetraborate as a background buffer solution, adding 35 mmol/L hydroxypropyl-beta-cyclodextrin and 15% methanol in volume fraction into the background buffer solution, and adjusting the pH to 9.3; 12 mmol/L disodium hydrogen phosphate buffer was used as sample matrix, ph=4.1; 50 The sample solution was injected at a pressure of mbar for 300s with a separation voltage of +20 kV and a temperature of 25℃and a detection wavelength of 250 nm.
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