CN116953142A - Liquid chromatography-mass spectrometry combined detection method for anthocyanin in tea - Google Patents
Liquid chromatography-mass spectrometry combined detection method for anthocyanin in tea Download PDFInfo
- Publication number
- CN116953142A CN116953142A CN202310909051.5A CN202310909051A CN116953142A CN 116953142 A CN116953142 A CN 116953142A CN 202310909051 A CN202310909051 A CN 202310909051A CN 116953142 A CN116953142 A CN 116953142A
- Authority
- CN
- China
- Prior art keywords
- anthocyanin
- sample
- liquid chromatography
- mass spectrometry
- tea
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229930002877 anthocyanin Natural products 0.000 title claims abstract description 111
- 235000010208 anthocyanin Nutrition 0.000 title claims abstract description 111
- 239000004410 anthocyanin Substances 0.000 title claims abstract description 111
- 150000004636 anthocyanins Chemical class 0.000 title claims abstract description 111
- 238000001514 detection method Methods 0.000 title claims abstract description 30
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 title claims abstract description 20
- 241001122767 Theaceae Species 0.000 title claims 15
- 238000000034 method Methods 0.000 claims abstract description 63
- 239000000523 sample Substances 0.000 claims abstract description 46
- 239000000126 substance Substances 0.000 claims abstract description 40
- 239000000243 solution Substances 0.000 claims abstract description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 34
- KZMACGJDUUWFCH-UHFFFAOYSA-O malvidin Chemical compound COC1=C(O)C(OC)=CC(C=2C(=CC=3C(O)=CC(O)=CC=3[O+]=2)O)=C1 KZMACGJDUUWFCH-UHFFFAOYSA-O 0.000 claims abstract description 30
- 239000012224 working solution Substances 0.000 claims abstract description 26
- VEVZSMAEJFVWIL-UHFFFAOYSA-O cyanidin cation Chemical compound [O+]=1C2=CC(O)=CC(O)=C2C=C(O)C=1C1=CC=C(O)C(O)=C1 VEVZSMAEJFVWIL-UHFFFAOYSA-O 0.000 claims abstract description 24
- 239000000049 pigment Substances 0.000 claims abstract description 21
- YKRGDOXKVOZESV-WRJNSLSBSA-N Paeoniflorin Chemical compound C([C@]12[C@H]3O[C@]4(O)C[C@](O3)([C@]1(C[C@@H]42)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)C)OC(=O)C1=CC=CC=C1 YKRGDOXKVOZESV-WRJNSLSBSA-N 0.000 claims abstract description 17
- YKRGDOXKVOZESV-UHFFFAOYSA-N paeoniflorin Natural products O1C(C)(C2(CC34)OC5C(C(O)C(O)C(CO)O5)O)CC3(O)OC1C24COC(=O)C1=CC=CC=C1 YKRGDOXKVOZESV-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 16
- 239000012488 sample solution Substances 0.000 claims abstract description 16
- 235000009584 malvidin Nutrition 0.000 claims abstract description 15
- 235000007336 cyanidin Nutrition 0.000 claims abstract description 12
- 240000001549 Ipomoea eriocarpa Species 0.000 claims abstract description 11
- 235000005146 Ipomoea eriocarpa Nutrition 0.000 claims abstract description 11
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 claims abstract description 11
- HKUHOPQRJKPJCJ-UHFFFAOYSA-N pelargonidin Natural products OC1=Cc2c(O)cc(O)cc2OC1c1ccc(O)cc1 HKUHOPQRJKPJCJ-UHFFFAOYSA-N 0.000 claims abstract description 11
- 235000006251 pelargonidin Nutrition 0.000 claims abstract description 11
- GCPYCNBGGPHOBD-UHFFFAOYSA-N Delphinidin Natural products OC1=Cc2c(O)cc(O)cc2OC1=C3C=C(O)C(=O)C(=C3)O GCPYCNBGGPHOBD-UHFFFAOYSA-N 0.000 claims abstract description 9
- 235000007242 delphinidin Nutrition 0.000 claims abstract description 9
- 238000005303 weighing Methods 0.000 claims abstract description 7
- 238000007865 diluting Methods 0.000 claims abstract description 4
- 239000012528 membrane Substances 0.000 claims abstract description 4
- 239000006228 supernatant Substances 0.000 claims abstract description 4
- JKHRCGUTYDNCLE-UHFFFAOYSA-O delphinidin Chemical compound [O+]=1C2=CC(O)=CC(O)=C2C=C(O)C=1C1=CC(O)=C(O)C(O)=C1 JKHRCGUTYDNCLE-UHFFFAOYSA-O 0.000 claims abstract 2
- XVFMGWDSJLBXDZ-UHFFFAOYSA-O pelargonidin Chemical compound C1=CC(O)=CC=C1C(C(=C1)O)=[O+]C2=C1C(O)=CC(O)=C2 XVFMGWDSJLBXDZ-UHFFFAOYSA-O 0.000 claims abstract 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 49
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 46
- 150000002500 ions Chemical class 0.000 claims description 39
- 238000000605 extraction Methods 0.000 claims description 37
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 36
- 239000012071 phase Substances 0.000 claims description 29
- 239000007789 gas Substances 0.000 claims description 26
- 238000001035 drying Methods 0.000 claims description 17
- 238000001819 mass spectrum Methods 0.000 claims description 12
- 238000011084 recovery Methods 0.000 claims description 9
- 238000010828 elution Methods 0.000 claims description 8
- 239000007921 spray Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 229910001873 dinitrogen Inorganic materials 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 6
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims description 6
- 238000012544 monitoring process Methods 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 5
- 239000012895 dilution Substances 0.000 claims description 5
- FUKUFMFMCZIRNT-UHFFFAOYSA-N hydron;methanol;chloride Chemical compound Cl.OC FUKUFMFMCZIRNT-UHFFFAOYSA-N 0.000 claims description 5
- 238000000889 atomisation Methods 0.000 claims description 4
- 238000009210 therapy by ultrasound Methods 0.000 claims description 4
- 239000008346 aqueous phase Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000003113 dilution method Methods 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 238000004811 liquid chromatography Methods 0.000 claims description 3
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 3
- 238000000611 regression analysis Methods 0.000 claims description 3
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims description 2
- 238000004949 mass spectrometry Methods 0.000 claims description 2
- 244000269722 Thea sinensis Species 0.000 abstract description 58
- 238000004458 analytical method Methods 0.000 abstract description 5
- 239000000178 monomer Substances 0.000 abstract description 3
- 235000013616 tea Nutrition 0.000 description 37
- 230000004044 response Effects 0.000 description 28
- 230000000694 effects Effects 0.000 description 21
- 238000000926 separation method Methods 0.000 description 15
- 239000000463 material Substances 0.000 description 14
- 239000002904 solvent Substances 0.000 description 12
- 238000005457 optimization Methods 0.000 description 11
- 235000009569 green tea Nutrition 0.000 description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 235000006468 Thea sinensis Nutrition 0.000 description 9
- 235000020279 black tea Nutrition 0.000 description 9
- YPVZJXMTXCOTJN-UHFFFAOYSA-N pelargonidin chloride Chemical compound [Cl-].C1=CC(O)=CC=C1C(C(=C1)O)=[O+]C2=C1C(O)=CC(O)=C2 YPVZJXMTXCOTJN-UHFFFAOYSA-N 0.000 description 9
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 8
- FFNDMZIBVDSQFI-UHFFFAOYSA-N delphinidin chloride Chemical compound [Cl-].[O+]=1C2=CC(O)=CC(O)=C2C=C(O)C=1C1=CC(O)=C(O)C(O)=C1 FFNDMZIBVDSQFI-UHFFFAOYSA-N 0.000 description 7
- 238000010586 diagram Methods 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 239000012086 standard solution Substances 0.000 description 6
- 239000011550 stock solution Substances 0.000 description 6
- 241000202296 Delphinium Species 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 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 4
- 235000019253 formic acid Nutrition 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 240000000982 Malva neglecta Species 0.000 description 3
- 235000000060 Malva neglecta Nutrition 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 238000013112 stability test Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 235000015459 Lycium barbarum Nutrition 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 229930014669 anthocyanidin Natural products 0.000 description 2
- 235000008758 anthocyanidins Nutrition 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- NWKFECICNXDNOQ-UHFFFAOYSA-N flavylium Chemical compound C1=CC=CC=C1C1=CC=C(C=CC=C2)C2=[O+]1 NWKFECICNXDNOQ-UHFFFAOYSA-N 0.000 description 2
- 238000010829 isocratic elution Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 235000009024 Ceanothus sanguineus Nutrition 0.000 description 1
- 244000182067 Fraxinus ornus Species 0.000 description 1
- 235000002917 Fraxinus ornus Nutrition 0.000 description 1
- JQQBXPCJFAKSPG-SVYIMCMUSA-N Geraniin Chemical compound OC1=C(O)C(O)=CC(C(=O)O[C@H]2[C@@H]3OC(=O)C=4C=C(O)C(O)=C5O[C@@]6(O)C(=O)C=C([C@@H](C5=4)C6(O)O)C(=O)O[C@H]4[C@@H]3OC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC[C@H]4O2)=C1 JQQBXPCJFAKSPG-SVYIMCMUSA-N 0.000 description 1
- 229920000061 Geraniin Polymers 0.000 description 1
- 244000017020 Ipomoea batatas Species 0.000 description 1
- 235000002678 Ipomoea batatas Nutrition 0.000 description 1
- 240000003553 Leptospermum scoparium Species 0.000 description 1
- 244000241838 Lycium barbarum Species 0.000 description 1
- 235000013939 Malva Nutrition 0.000 description 1
- 240000000851 Vaccinium corymbosum Species 0.000 description 1
- 235000003095 Vaccinium corymbosum Nutrition 0.000 description 1
- 235000017537 Vaccinium myrtillus Nutrition 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- 235000021014 blueberries Nutrition 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 150000001793 charged compounds Chemical class 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000013100 final test Methods 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- -1 flavonoid compounds Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- GJMUCSXZXBCQRZ-UHFFFAOYSA-N geraniin Natural products Oc1cc(cc(O)c1O)C(=O)OC2OC3COC(=O)c4cc(O)c(O)c(O)c4c5cc(C(=O)C67OC3C(O6)C2OC(=O)c8cc(O)c(O)c9OC%10(O)C(C(=CC(=O)C%10(O)O)C7=O)c89)c(O)c(O)c5O GJMUCSXZXBCQRZ-UHFFFAOYSA-N 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 239000013026 undiluted sample Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- 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
-
- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention provides a liquid chromatography-mass spectrometry combined detection method for anthocyanin in tea, and belongs to the technical field of quality detection. The method comprises the following steps: preparing a system standard working solution: weighing 6 anthocyanin standard substances, dissolving, and gradually diluting to obtain a series of standard working solutions; preparing a sample solution: taking a tea sample to be tested, extracting and centrifuging, passing the supernatant through a water phase filter membrane, and dividing the filtered extracting solution into two groups, wherein one group is diluted, the other group is undiluted, and the two groups of extracting solutions are sample solutions to be tested; HPLC-MS/MS detection: respectively injecting the series of standard working solutions and the sample solution to be detected into a liquid chromatography-mass spectrometer under the same condition for analysis, so as to determine the content of each anthocyanin in the sample; the 6 anthocyanin are as follows: pelargonidin, cyanidin, paeoniflorin, delphinidin, morning glory pigment, and malvidin. The method can accurately detect the content of 6 common anthocyanin monomers in food.
Description
Technical Field
The invention belongs to the technical field of quality detection, and particularly relates to a liquid chromatography-mass spectrometry combined detection method for anthocyanin in tea.
Background
Anthocyanin is a common natural plant water-soluble pigment, belongs to flavonoid compounds, and is red or purple in an acidic environment and blue in an alkaline environment, so that plants such as fruits, vegetables and flowers can be provided with colorful colors, and the color depth is proportional to the content of anthocyanin. As a natural edible pigment, the anthocyanin has rich content, no toxicity, safety and high nutritive value and pharmacological action, and can be used for preventing and treating partial diseases. Since tea leaf anthocyanin has a bitter taste, and tea prepared from purple bud leaves rich in anthocyanin has poor quality, the previous researches have been mostly biased to reduce anthocyanin accumulation. However, along with the change of market demands, the tea with high anthocyanin content is valued by virtue of the unique quality characteristics and character characteristics, and the development of tea products rich in anthocyanin becomes a current hot spot.
With the recent progress of scientific research, various methods for quantitative analysis of anthocyanin have been reported, among which the most widely used methods include spectrophotometry, HPLC method and HPLC-MS/MS method. The spectrophotometry can be used for detecting the total amount of the anthocyanin, the HPLC method is suitable for analyzing different anthocyanin in a sample, and the HPLC-MS/MS can not only quantitatively detect the anthocyanin, but also identify the type of the anthocyanin. However, many studies on anthocyanin content detection have focused on plants with a high anthocyanin content, such as blueberry, purple sweet potato and black matrimony vine, which are well known. Anthocyanin is also an important substance in tea, and 80% of tea contains anthocyanin, especially purple bud leaves which are studied more in recent years, and the anthocyanin content of the purple bud leaves accounts for 0.5% -1.0% of dry substance. Therefore, the HPLC-MS/MS method is also of great significance in detecting the anthocyanin content in the tea.
Disclosure of Invention
In view of the above, the invention provides a liquid chromatography-mass spectrometry combined detection method for anthocyanin in tea, which can accurately detect the content of 6 anthocyanin monomers including pelargonidin, cyanidin, paeoniflorin, delphinidin, morning glory pigment and malvidin, and has the characteristics of simplicity, rapidness and accuracy in operation.
The technical scheme of the invention is as follows:
the invention provides a liquid chromatography-mass spectrometry combined detection method for anthocyanin in tea, which comprises the following steps:
s1, preparing a system standard working solution: weighing 6 anthocyanin standard substances, dissolving, and gradually diluting to obtain a series of standard working solutions;
s2, preparing a sample solution: taking a tea sample to be tested, extracting and centrifuging, passing the supernatant through a water phase filter membrane, and dividing the filtered extracting solution into two groups, wherein one group is diluted, the other group is undiluted, and the two groups of extracting solutions are sample solutions to be tested;
s3, HPLC-MS/MS detection: sequentially injecting the series of standard working solutions in the step S1 into a liquid chromatography-mass spectrometry instrument, carrying out regression analysis on the corresponding concentrations of the anthocyanin by using the multi-reaction monitoring mode ion peak areas of the anthocyanin to obtain a standard working curve, and under the same condition, injecting the sample solution to be detected in the step S2 into the liquid chromatography-mass spectrometry instrument to obtain the multi-reaction monitoring mode ion peak areas of each anthocyanin, and substituting the multi-reaction monitoring mode ion peak areas into the standard working curve to obtain the content of 6 anthocyanin in the sample;
the 6 anthocyanin are as follows: pelargonidin, cyanidin, paeoniflorin, delphinidin, morning glory pigment, and malvidin.
The diluted sample solution to be tested is used for measuring the content of 3 anthocyanin types of delphinidin, cyanidin and pelargonidin, and the undiluted sample solution to be tested is used for measuring the content of 3 anthocyanin types of paeoniflorin, morning glory pigment and malvidin.
In one embodiment, in step S1, the dissolution process uses 10% methanol hydrochloride; in step S1 and step S2, methanol is used for the dilution process.
Anthocyanin is easily dissolved in methanol, ethanol, acetone, water or their mixed solvent, and in order to prevent degradation of non-acylated anthocyanin during extraction, hydrochloric acid or formic acid with a certain concentration is usually added into the extraction solvent.
Preparing a series of standard solutions, preferably weighing 6 anthocyanin standard substances, dissolving the standard substances with 10% hydrochloric acid methanol solution to prepare 6 anthocyanin single standard substance stock solutions, then sucking the 6 anthocyanin single standard substance stock solutions to prepare a mixed standard solution, and then gradually diluting with methanol to obtain a series of standard working solutions.
In one embodiment, in step S1, the concentration of 6 anthocyanins in the series of standard working fluids is 25 μg/L, 50 μg/L, 100 μg/L, 250 μg/L, 500 μg/L, 1000 μg/L, 2500 μg/L.
1. In a specific embodiment, in step S2, the specific steps of tea sample extraction are as follows: adding acidified ethanol solution into tea leaf sample, performing ultrasonic treatment for 50min, and performing water bath at 100deg.C for 75min; the volume ratio of ethanol, hydrochloric acid and water in the acidified ethanol solution is 2:1:1; in step S2, the model of the aqueous phase filter is 0.22 μm.
In the specific steps of tea extraction, adding an acidified ethanol solution, carrying out ultrasonic treatment and carrying out water bath, and uniformly mixing; preferably vortex mixing.
In one embodiment, the feed ratio of the tea leaf sample to the acidified ethanol solution is 1:100 (g: mL).
In a specific embodiment, in step S2, the centrifugation is at 7500r/min for 3min.
In one embodiment, in step S2, the dilution factor of the extract is 100 times.
The dilution factor of the extract can be properly adjusted according to the anthocyanin content in the sample.
In a specific embodiment, in step S3, the chromatographic conditions are:
the chromatographic column model is as follows: agilent Poroshell 120SB-C18,2.1 mm. Times.100 mm,2.7 μm;
chromatographic column temperature: 40 ℃;
flow rate: 0.35mL/min;
sample injection amount: 2. Mu.L;
mobile phase: mobile phase A is 1% formic acid water solution, mobile phase B is methanol;
mobile phase gradient elution procedure table:
in a specific embodiment, in step S3, the conditions of the mass spectrum are:
ion source: electrospray ion source (ESI);
scanning mode: a positive ion scanning mode;
the detection mode is as follows: multiple reaction monitoring mode (MRM);
drying gas: nitrogen gas;
atomizing gas: nitrogen gas;
atomization gas pressure: 50psi;
ion spray voltage: 2000V;
drying gas temperature: 350 ℃;
drying gas flow rate: 11L/min;
the mass spectrum acquisition parameters are shown in the table:
in a specific embodiment, the average standard recovery rate of the method is in the range of 91.95% -106.99%, RSD is less than 10%, the detection limit is 0.02-17 mg/kg, and the quantitative limit is 0.07-55 mg/kg.
The detection limit in the present invention means the lowest concentration at which the method can detect the substance. The term "limit" as used herein means the minimum concentration at which the method can accurately determine the substance.
Compared with the prior art, the invention has the following technical effects:
1. compared with the existing high performance liquid chromatography which can only carry out qualitative and quantitative detection through retention time, the method can simultaneously and more accurately detect the content of 6 anthocyanin monomers of pelargonidin, cyanidin, paeoniflorin, delphinidin and morning glory pigment in a sample to be detected by adopting a liquid chromatography mass spectrometry method.
2. The method has good linear relation in the concentration range of 25-2500 mug/L, the average standard adding recovery rate range of 91.95-106.99%, RSD of less than 10%, detection limit of 0.02-17 mg/kg, quantitative limit of 0.07-55 mg/kg, and stability and precision all meet the requirements of related standards.
3. The method is simple to operate, good in sensitivity and high in accuracy, can be suitable for related analysis and detection work, can provide reference for the establishment of a standard method for detecting anthocyanin content in tea at home and abroad, and can also provide a certain technical support for purple bud tea tree breeding work.
Drawings
FIG. 1 shows the effect of the ratio of the sample to be tested to the acidified ethanol solution of the extraction solvent on the response value;
FIG. 2 is a graph showing the effect of extraction solvent species on response values during extraction of a sample to be tested;
FIG. 3 is a graph showing the effect of hydrochloric acid concentration in an extraction solvent acidified ethanol solution of a sample to be tested on the response value;
FIG. 4 is a graph showing the effect of ultrasonic time on response values during extraction of a sample to be tested;
FIG. 5 is a graph showing the effect of water bath time on response values during extraction of a sample to be tested;
FIG. 6 is a TIC diagram of standard solutions of different types of chromatographic columns for separating anthocyanin substances;
FIG. 7 is a TIC diagram showing separation of anthocyanin-based materials in different mobile phase systems;
FIG. 8 is a TIC diagram showing separation of anthocyanin standard solutions at different column temperatures;
FIG. 9 TIC graphs of different elution procedures.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Universal materials and methods
(1) Apparatus and device
(2) Materials and reagents
6 anthocyanin standard: pelargonidin, cyanidin, paeoniflorin, delphinidin, morning glory pigment, and malvidin (purity not less than 98%) were purchased from Chengdodernikoku biotechnology Co.
Reagent: chromatographic pure formic acid, high grade pure hydrochloric acid, high grade pure absolute ethanol, high grade pure ammonium formate, all purchased from Colon Chemicals Co., ltd
Sample: tea samples used in the experiments are purchased from tea enterprises in Sichuan province and foreign province.
The experimental water is super-stored water.
(3) Preparation of standard working solution
Accurately weighing 10.00mg of each of the standard substances of pelargonidin, cyanidin, paeoniflorin, delphinidin, morning glory pigment and malvidin, respectively dissolving with 10% methanol hydrochloride, fixing volume to 10mL, mixing to obtain different single standard substance stock solutions with concentration of 1000mg/L, and storing at-20deg.C in dark for use with effective period of 1 month.
100 mu L of each of the 6 anthocyanin single standard substance stock solutions are accurately absorbed respectively to prepare 6 anthocyanin single standard substance stock solutions with the concentration of 10mg/L, and then the mixed standard solutions are gradually diluted by methanol to prepare 6 anthocyanin single standard substance stock solutions with the concentration of 25 mu g/L, 50 mu g/L, 100 mu g/L, 250 mu g/L, 500 mu g/L, 1000 mu g/L and 2500 mu g/L.
(4) Sample pretreatment
Weighing 0.1000g of tea powder, putting the tea powder into a 15mL centrifuge tube, adding 10mL of acidified ethanol solution (ethanol: hydrochloric acid: water=2:1:1), carrying out vortex mixing uniformly, carrying out ultrasonic treatment for 50min, taking out, carrying out vortex mixing uniformly, carrying out water bath at 100 ℃ for 75min, taking out, carrying out vortex mixing uniformly, carrying out centrifugation at 7500r/min for 3min, pouring the supernatant into a 10mL volumetric flask, carrying out constant volume to 10mL, passing through a 0.22 mu m water phase filter membrane, dividing the filtered extract into two groups, wherein one group is diluted by methanol by 100 times, the other group is not diluted, and the two groups of extracts are samples to be tested.
Because the method has lower detection limit, when three anthocyanin substances (delphinidin, cyanidin and pelargonidin) with higher content in tea are detected, the sample needs to be diluted 100 times, and the other three anthocyanin substances do not need to be diluted. The dilution factor of the extract can be properly adjusted according to the anthocyanin content in the sample.
(5) Instrument method
The liquid chromatography conditions were:
the chromatographic column model is as follows: agilent Poroshell 120SB-C18,2.1 mm. Times.100 mm,2.7 μm;
chromatographic column temperature: 40 ℃;
flow rate: 0.35mL/min;
sample injection amount: 2. Mu.L;
mobile phase: mobile phase A is 1% formic acid water solution, mobile phase B is methanol;
the mobile phase gradient elution procedure is shown in table 1:
TABLE 1 gradient elution procedure for mobile phases
| Time (min) | 1% formic acid water (%) | Methanol (%) |
| 0 | 70 | 30 |
| 5 | 70 | 30 |
| 10 | 65 | 35 |
| 20 | 60 | 40 |
| 20.1 | 5 | 95 |
| 25 | 5 | 95 |
| 25.1 | 70 | 30 |
| 33 | 70 | 30 |
The mass spectrum conditions are as follows:
ion source: electrospray ion source (ESI);
scanning mode: a positive ion scanning mode;
the detection mode is as follows: multiple reaction monitoring mode (MRM);
drying gas: nitrogen gas;
atomizing gas: nitrogen gas;
atomization gas pressure: 50psi;
ion spray voltage: 2000V;
drying gas temperature: 350 ℃;
drying gas flow rate: 11L/min;
the mass spectrum acquisition parameters are shown in table 2.
Table 2 mass spectrum acquisition parameters
And (3) measuring:
the prepared serial mixed standard working solution is injected into HPLC-MS/MS, regression analysis is carried out on the peak areas of MRM ions of 6 anthocyanin and the corresponding concentrations, and a standard working curve is obtained, and the standard working curve is shown in the following table 3. Under the same conditions, injecting the sample solution to be measured into HPLC-MS/MS to obtain the MRM ion peak area of each anthocyanin, and substituting the MRM ion peak area into a standard working curve to obtain the content of each anthocyanin in the sample to be measured.
Table 3 6 linear equation, correlation coefficient, linear range, dilution factor for anthocyanidins
Example 1 optimization of sample pretreatment conditions
1. Optimization of feed-liquid ratio
To examine the effect of feed-to-liquid ratio on extraction rate, tea leaves were extracted at ratios (g: mL) of 1:25, 1:50, 1:75, 1:100, 1:125, respectively, of tea leaves to acidified ethanol solution (ethanol: hydrochloric acid: water=2:1:1), each group was assayed 3 times in parallel. The response values (peak areas) of anthocyanin in the liquid to be tested under the condition of each liquid-to-liquid ratio are shown in figure 1. As shown in fig. 1, with increasing of the extraction solution ratio, the extraction efficiency is gradually increased, the extraction rate is highest under the condition of a feed-liquid ratio of 1:100, and the extraction efficiency is not obviously improved again by increasing the solution ratio, so the feed-liquid ratio is selected to be 1:100.
2. Optimization of extraction solvent species
To examine the effect of the extraction solvent type on the extraction yield, tea leaves were extracted with acidified ethanol solution (ethanol: hydrochloric acid: water=2:1:1), acidified methanol solution (methanol: hydrochloric acid: water=2:1:1), acidified acetonitrile solution (acetonitrile: hydrochloric acid: water=2:1:1), and acidified aqueous solution (hydrochloric acid: water=1:3) as extraction solvents, respectively, and each group was assayed 3 times in parallel. The response values (peak areas) of anthocyanin in the liquid to be tested under each extraction solvent are shown in figure 2. As can be seen from fig. 2, the extraction efficiency is highest when the acidified ethanol solution (ethanol: hydrochloric acid: water=2:1:1) is used as the extraction solvent, and thus the extraction solvent is selected as the acidified ethanol solution (ethanol: hydrochloric acid: water=2:1:1).
3. Optimization of hydrochloric acid concentration
To examine the effect of the hydrochloric acid concentration in the extraction solvent on the extraction yield, tea leaves were extracted with aqueous ethanol solutions (ethanol: water=2:1) having hydrochloric acid concentrations of 0.5%, 1.0%, 2.0%, 5.0%, 8.0%, 10.0%, 20.0%, 25.0%, 30.0% as the extraction solvents, respectively, and each group was measured 3 times in parallel. The response values (peak areas) of anthocyanin in the test solution under each hydrochloric acid concentration are shown in figure 3. As can be seen from fig. 3, the extraction efficiency was improved to a different extent with an increase in the hydrochloric acid concentration, and the extraction efficiency was high for each sample at the hydrochloric acid concentrations of 25% and 30%. Considering that the too high acid concentration is harmful to the mass spectrometer, the hydrochloric acid concentration is not increased any more. Among the 6 test substances, the content of paeoniflorin and malvidin is relatively low, and the hydrochloric acid concentration with high extraction rate of paeoniflorin and malvidin should be preferentially considered, and when the hydrochloric acid concentration is 25%, the extraction rate of paeoniflorin and malvidin is relatively high, so that the hydrochloric acid concentration is 25%.
4. Optimization of ultrasound time
To examine the effect of the ultrasonic time on the extraction rate, the tea leaves were extracted at ultrasonic times of 0, 10, 20, 30, 40, 50, 60min, respectively, and each group was measured 3 times in parallel. The response value (peak area) of anthocyanin in the liquid to be tested under each ultrasonic time condition is shown in fig. 4. As can be seen from fig. 4, the extraction efficiency is highest when the ultrasonic time is 50min, and thus the ultrasonic time is selected to be 50min.
5. Optimization of the Water bath time
To examine the effect of the water bath time on the extraction rate, the tea leaves were extracted at water bath times of 30, 45, 60, 75, 90min, respectively, and each group was measured 3 times in parallel. The response values (peak areas) of anthocyanin in the liquid to be tested under the water bath time conditions are shown in figure 5. As can be seen from FIG. 5, the extraction efficiency of each analyte was high at the water bath times of 75 and 90 minutes. Among the 6 samples, the content of paeoniflorin and malvidin is relatively low, the water bath time with high extraction rate of paeoniflorin and malvidin should be considered preferentially, and the extraction rate of paeoniflorin and malvidin is relatively high when the water bath time is 75min. The water bath time was therefore chosen to be 75min.
Example 2 optimization of liquid chromatography conditions
1. Selection of chromatographic columns
Because the anthocyanin is stable under the condition that the pH is less than 3, a low-pH-resistant chromatographic column is selected for ensuring the stability of the column effect of the chromatographic column, and the separation effect of 2 types of specification chromatographic columns on anthocyanin substances is totally examined aiming at the structural characteristics of the existing chromatographic column and anthocyanin substances, wherein the types and the specifications of the chromatographic columns are shown in table 4. The total ion flow diagram (TIC diagram) of the standard solution of the anthocyanin class substances separated by the different types of chromatographic columns is shown in fig. 6, wherein 1: delphinium pigment; 2: cyanidin; 3: morning glory pigment; 4: pelargonidin; 5: paeoniflorin; 6: malvidin. As can be seen from fig. 6: the separation effect and peak shape of Agilent Poroshell EC-C18 type chromatographic column are obviously better than those of XSELECTM HSS T3 type chromatographic column, and Agilent Poroshell EC-C18 (2.1 mm multiplied by 100mm,2.7 μm) type chromatographic column is selected as the detection chromatographic column by the method after comprehensive consideration.
TABLE 4 model, specification and TIC diagram of chromatography column for separating anthocyanin substances
2. Optimization of mobile phase species
After the chromatographic column was selected, the effect of 8 different mobile phase systems on anthocyanin class response and separation was examined with mixed standard working solutions as shown in table 5 and fig. 7. As can be seen from FIG. 7, the anthocyanin substances have early peak time and poor separation condition under the acetonitrile system; the higher the acidity of the aqueous phase is, the better the peak separation condition and peak shape are, and the highest concentration of the formic acid water is set to be 1% in consideration of the acid resistance of the chromatographic column; after salt is added into the water phase, the difference of peak appearance, peak shape and the like is small compared with that of the water phase without salt, so that 1% of formic acid water is directly selected as the water phase. In general, the separation condition, peak shape and response of each object to be detected under the 1% formic acid water-methanol system are better than those of other mobile phase systems, so that the 1% formic acid water-methanol system is selected as the mobile phase type of the method.
Table 5 8 different mobile phase types
Note that: the ion response is low in the positive ion mode when the used instrument collects the positive and negative ion modes at the same time, so that the positive and negative ion mode scanning is respectively carried out on 8 mobile phase systems.
3. Optimization of chromatographic column temperature
The influence of the chromatographic column temperature on the separation degree and the response of the to-be-detected object is different, and the influence of the chromatographic column on the separation and the response of anthocyanin substances at the temperature of 35 ℃, 40 ℃, 45 ℃ and 50 ℃ is examined respectively, as shown in fig. 8, wherein 1: delphinium pigment; 2: cyanidin; 3: morning glory pigment; 4: pelargonidin; 5: paeoniflorin; 6: malvidin; A. b, C, D the column temperature was 35 ℃, 40 ℃, 45 ℃ and 50 ℃, respectively. As can be seen from fig. 8, as the temperature of the chromatographic column increases, the peak time of the analyte becomes faster, but the separation between the analytes is not affected; the effect of the chromatographic column on the response of the test substance is not particularly great. In order to improve analysis efficiency as much as possible, reduce column pressure of the chromatographic column, prolong service life of the chromatographic column, and comprehensively consider that the temperature of the chromatographic column is 40 ℃.
4. Optimization of mobile phase gradients
To ensure good separation and response of the anthocyanidins, different initial mobile phase ratios were examined, as shown in table 6, the effect on anthocyanin retention time, separation effect and response was shown, and the results are shown in fig. 9. As can be seen from table 6 and fig. 9, under the condition of low-proportion organic isocratic elution, the peak time of the object to be detected is late and the peak width is wider; under the condition of high-proportion organic isocratic elution, the object to be detected has fast peak and poor peak separation condition; the gradient elution is carried out, and the peak time and the separation condition of each object to be detected can be effectively changed by changing the initial proportion of the organic phase and the increasing rate of the organic phase; number 4 was chosen as the mobile phase gradient elution procedure after comprehensive consideration.
TABLE 6 gradient elution procedure
Example 3 optimization of Mass Spectrometry conditions
Preparing a single standard working solution of 6 anthocyanin types by using a 10% hydrochloric acid methanol solution, wherein the concentration is 500 mug/L, and performing primary mass spectrum scanning on the working solution in ESI positive ion and negative ion modes respectively to obtain parent ions of an object to be detected. The anthocyanin substance is found to respond well in the positive ion mode. And then optimizing to obtain the optimal fragmentation voltage of the primary mass spectrum of each anthocyanin substance, performing secondary mass spectrum analysis on the molecular ion peak of each anthocyanin substance to obtain fragment ion information, and finally optimizing to obtain the optimal collision voltage of the secondary mass spectrum of each anthocyanin substance. The MRM information of the optimized anthocyanin substances is shown in Table 2.
Meanwhile, the influence of the temperature of the drying gas, the flow rate of the drying gas, the pressure of the atomizing gas and the voltage of the ion spray on the response (peak area) of anthocyanin substances is examined in the recommended setting range of instrument parameters, and the results are shown in tables 7-10. It can be seen from the table that under all experimental conditions, the optimal response conditions of the drying gas temperature, the atomizing gas pressure and the ion spray voltage of all the objects to be tested are respectively concentrated at 350 ℃, 50psi and 2000V, and only the optimal response conditions of all the objects of the drying gas flow rate parameters are uniformly distributed in two conditions of 10L/min and 11L/min, and the ion source parameters with higher response values of the delphinium pigment and the mallow pigment are preferentially selected because the response values of the delphinium pigment and the mallow pigment are relatively lower. After comprehensively comparing the response values of the parameters, the final mass spectrum ion source condition is determined as the drying gas temperature of 350 ℃, the drying gas flow rate of 11L/min, the atomization air pressure of 50psi and the ion spray voltage of 2000V.
TABLE 7 influence of different drying gas temperatures on anthocyanin response values
TABLE 8 influence of different drying gas flow rates on anthocyanin response values
TABLE 9 influence of different atomizing gas pressures on anthocyanin class substance response values
TABLE 10 influence of different ion spray voltages on anthocyanin response values
Example 4 evaluation of Performance by HPLC-MS/MS detection method
1. Linearity and sensitivity of the method
The standard working curve obtained in the general material and method shows that each target has good linear relationship within the range of 25-2500 mug/L, and the correlation coefficient R is more than 0.99. In order to examine the sensitivity of the method, the concentration with the signal to noise ratio S/N more than or equal to 3 is taken as the detection limit concentration, and the concentration with the signal to noise ratio S/N more than or equal to 10 is taken as the quantitative limit concentration, and specific results are shown in Table 11. As can be seen from Table 11, the method for measuring anthocyanin substances has good sensitivity, and can well meet the measurement requirements of 6 anthocyanin substances in tea.
Table 11 results of linearity and sensitivity experiments of the method
2. Accuracy and precision of the method
And accurately weighing 12 parts of green tea and black tea samples, wherein 3 parts of the green tea and black tea samples are completely prepared into a liquid to be tested according to the sample pretreatment method of the general material and method, 9 parts of the mixed standard working solution of the general material and method are respectively added into the mixed standard working solution of the general material and method to ensure that the final test liquid concentration forms three different concentration levels of low concentration, medium concentration and high concentration, the standard samples are added, then the liquid to be tested is prepared according to the sample pretreatment method of the general material and method, the detection condition of the general material and method is adopted, and the standard recovery rate and the precision condition of the method are examined, and the specific results are shown in tables 12-14. The table shows that the average recovery rate of anthocyanin substances in the tea is 91.95% -106.99%, and the RSD is less than 10%, so that the requirements of the related standard on the recovery rate and precision of the method are met, and the method can accurately detect the content of anthocyanin substances in the tea.
Because the method has lower detection limit, according to the comprehensive consideration of the content of the sample and the linear range of the anthocyanin on the instrument, the sample needs to be diluted 100 times when the sample is detected for three anthocyanin substances with higher content in the tea, and meanwhile, the matrix effect can be reduced by a dilution method, and the matrix effect is indeed overcome by the marked recovery result, so that the matrix effect of the sample does not need to be specially optimized.
Table 12 green tea sample labeled recovery experimental results (n=3)
Table 13 black tea sample addition recovery experimental results (n=3)
Table 14 results of precision test of method
3. Solution stability verification
Respectively sucking 2 mu L of black tea and green tea sample solutions and corresponding low, medium and high-concentration standard adding solutions of tea, and respectively injecting the black tea and green tea sample solutions into a liquid chromatograph tandem mass spectrometer for analysis when the black tea and green tea sample solutions are placed for 0h, 2h, 4h, 8h, 12h, 16h, 20h and 24h according to the detection conditions described in the general materials and methods; when each single standard working solution is placed for 7, 15, 30, 45 and 60 days in a dark environment at the temperature of minus 20 ℃,2 mu L of mixed standard working solution (prepared by the single standard working solution) with the concentration of 1000 mu g/L is respectively sucked up according to the detection conditions of the general materials and the method, and is injected into a liquid chromatograph tandem mass spectrometer for analysis and investigation of the stability of the standard working solution, and specific results are shown in tables 15-17. The table shows that the sample solution to be measured has no obvious change within 24 hours and the single standard working solution has no obvious change within 60 days, which indicates that the solution to be measured prepared by the method has good stability within 24 hours and the single standard working solution stored in the dark environment at-20 ℃ has good stability within 60 days.
TABLE 15 results of green tea solution stability test
Table 16 results of black tea solution stability test
TABLE 17 Single Standard working solution stability test results
| Substance name | Single standard working solution RSD (%) |
| Delphinium pigment | 6.40 |
| Cyanidin | 4.07 |
| Geraniin | 6.92 |
| Morning glory pigment | 8.30 |
| Paeoniflorin | 6.15 |
| Malva pigment | 8.80 |
Example 5
In this example, the method described in "Universal materials and methods" was used to determine the content of 6 anthocyanins in five tea samples.
The 5 tea samples were: the method comprises the steps of processing Yunnan big-leaf kungfu black tea (sample 1), west lake Longjing green tea (sample 2), mengding manna green tea (sample 3), junlian black tea (sample 4) and Xufu Long Ya green tea (sample 5) according to the sample pretreatment steps of general materials and methods, and obtaining sample solutions to be detected of the five tea samples.
Under the working condition of HPLC-MS/MS described in general materials and methods, injecting sample solutions to be tested of five tea samples into the HPLC-MS/MS to obtain MRM ion peak areas of the anthocyanin, and substituting the MRM ion peak areas into a standard working curve to obtain the content of the anthocyanin in the sample. The results of the content measurement are shown in Table 18.
Table 18 test results of the sample to be tested in this example
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. The method for detecting anthocyanin in tea leaves by liquid chromatography and mass spectrometry is characterized by comprising the following steps of:
s1, preparing a system standard working solution: weighing 6 anthocyanin standard substances, dissolving, and gradually diluting to obtain a series of standard working solutions;
s2, preparing a sample solution: taking a tea sample to be tested, extracting and centrifuging, passing the supernatant through a water phase filter membrane, and dividing the filtered extracting solution into two groups, wherein one group is diluted, the other group is undiluted, and the two groups of extracting solutions are sample solutions to be tested;
s3, HPLC-MS/MS detection: sequentially injecting the series of standard working solutions in the step S1 into a liquid chromatography-mass spectrometry instrument, carrying out regression analysis on the corresponding concentrations of the anthocyanin by using the multi-reaction monitoring mode ion peak areas of the anthocyanin to obtain a standard working curve, and under the same condition, injecting the sample solution to be detected in the step S2 into the liquid chromatography-mass spectrometry instrument to obtain the multi-reaction monitoring mode ion peak areas of each anthocyanin, and substituting the multi-reaction monitoring mode ion peak areas into the standard working curve to obtain the content of 6 anthocyanin in the sample;
the 6 anthocyanin are as follows: pelargonidin, cyanidin, paeoniflorin, delphinidin, morning glory pigment, and malvidin.
2. The method for detecting anthocyanin in tea leaves by liquid chromatography-mass spectrometry according to claim 1, wherein in step S1, 10% methanol hydrochloride is used for dissolution; in step S1 and step S2, methanol is used for the dilution process.
3. The method for detecting anthocyanin in tea leaves by liquid chromatography-mass spectrometry according to claim 1, wherein in step S1, the concentration of 6 anthocyanin in the series of standard working solutions is 25 μg/L, 50 μg/L, 100 μg/L, 250 μg/L, 500 μg/L, 1000 μg/L, 2500 μg/L.
4. The method for detecting anthocyanin in tea leaves by liquid chromatography-mass spectrometry according to claim 1, wherein in step S2, the specific steps of tea leaf sample extraction are as follows: adding acidified ethanol solution into tea leaf sample, performing ultrasonic treatment for 50min, and performing water bath at 100deg.C for 75min; the volume ratio of ethanol, hydrochloric acid and water in the acidified ethanol solution is 2:1:1; in step S2, the model of the aqueous phase filter is 0.22 μm.
5. The method for liquid chromatography-mass spectrometry detection of anthocyanin in tea leaves as claimed in claim 4, wherein the feed liquid ratio of tea leaf sample to acidified ethanol solution is 1:100 (g: mL).
6. The method for detecting anthocyanin in tea leaves by liquid chromatography-mass spectrometry combined with claim 1, wherein in step S2, the centrifugation is at 7500r/min for 3min.
7. The method for detecting anthocyanin in tea leaves by liquid chromatography-mass spectrometry according to claim 1, wherein in step S2, the dilution factor of the extract is 100 times.
8. The method for detecting anthocyanin in tea leaves by liquid chromatography-mass spectrometry according to claim 1, wherein in step S3, the conditions of the chromatography are:
the chromatographic column model is as follows: agilent Poroshell 120SB-C18,2.1 mm. Times.100 mm,2.7 μm;
chromatographic column temperature: 40 ℃;
flow rate: 0.35mL/min;
sample injection amount: 2. Mu.L;
mobile phase: mobile phase A is 1% formic acid water solution, mobile phase B is methanol;
mobile phase gradient elution procedure table:
9. the method for detecting anthocyanin in tea leaves by liquid chromatography-mass spectrometry according to claim 1, wherein the conditions of the mass spectrum are as follows:
ion source: an electrospray ion source;
scanning mode: a positive ion scanning mode;
the detection mode is as follows: a multiple reaction monitoring mode;
drying gas: nitrogen gas;
atomizing gas: nitrogen gas;
atomization gas pressure: 50psi;
ion spray voltage: 2000V;
drying gas temperature: 350 ℃;
drying gas flow rate: 11L/min;
the mass spectrum acquisition parameters are shown in the table:
10. the method for detecting anthocyanin in tea leaves by liquid chromatography-mass spectrometry according to claim 1, wherein the average standard adding recovery rate of the method is 91.95% -106.99%, RSD is less than 10%, detection limit is 0.02-17 mg/kg, and quantitative limit is 0.07-55 mg/kg.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310909051.5A CN116953142A (en) | 2023-07-24 | 2023-07-24 | Liquid chromatography-mass spectrometry combined detection method for anthocyanin in tea |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310909051.5A CN116953142A (en) | 2023-07-24 | 2023-07-24 | Liquid chromatography-mass spectrometry combined detection method for anthocyanin in tea |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116953142A true CN116953142A (en) | 2023-10-27 |
Family
ID=88452394
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310909051.5A Pending CN116953142A (en) | 2023-07-24 | 2023-07-24 | Liquid chromatography-mass spectrometry combined detection method for anthocyanin in tea |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116953142A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117607066A (en) * | 2023-12-27 | 2024-02-27 | 中国热带农业科学院南亚热带作物研究所 | Method for determining anthocyanin concentration in eggplant peel based on hyperspectral imaging |
-
2023
- 2023-07-24 CN CN202310909051.5A patent/CN116953142A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117607066A (en) * | 2023-12-27 | 2024-02-27 | 中国热带农业科学院南亚热带作物研究所 | Method for determining anthocyanin concentration in eggplant peel based on hyperspectral imaging |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107543877A (en) | A kind of method that SPE liquid chromatography tandem mass spectrometry determines six 24 kinds of antibiotic of class in water body simultaneously | |
| CN108918711B (en) | Detection method of polyphenol compounds in tobacco leaves | |
| CN107505405B (en) | Method for efficiently and rapidly extracting and measuring flavonoid pigment in Chinese rose petals | |
| CN110108830B (en) | Method for simultaneously carrying out qualitative and quantitative detection on 9 anthocyanidins in lonicera edulis | |
| CN116953142A (en) | Liquid chromatography-mass spectrometry combined detection method for anthocyanin in tea | |
| CN104316638B (en) | Detect the LC-MS/MS assay method of 7 kinds of invertebrate poisons in vegetables and fruit simultaneously | |
| CN113325103A (en) | Analysis method for simultaneously measuring gelsemium, gelsemine and gelsemine in hair | |
| CN111798937B (en) | Method for establishing metabonomics database of wolfberry tissue and application thereof | |
| CN113820435B (en) | Application of stachydrine and levo-stachydrine as characteristic markers of Leucosceptrum Nostoc honey | |
| CN114002355B (en) | A solid phase extraction post that is arranged in agricultural product nine kinds of mycotoxins to enrich purification simultaneously | |
| CN115728408A (en) | Method for simultaneously determining seven artemisinin related compounds in artemisia annua | |
| Miao et al. | Comparison of second-derivative spectrophotometry and HPLC for determination of amygdalin in wild apricot kernels | |
| CN114441671A (en) | Method for determining content of platinum impurity cyclohexanediamine dihydrate in oxaliplatin by combination of HPLC-ICP-MS | |
| CN111751466A (en) | Method for simultaneously determining DON toxin and NIV toxin | |
| CN111208227A (en) | Method for simultaneously determining content of 8 carbonyl compounds in cigarette blasting beads | |
| CN111413393A (en) | Method for rapidly detecting formaldehyde content in food by high-throughput reaction paper spray mass spectrometry | |
| CN116106467B (en) | Method for identifying raw pinellia tuber in Huoxiang Zhengqi water | |
| CN112730574B (en) | Method for rapidly and high-throughput detecting aldicarb, aldicarb sulfoxide and aldicarb sulfone in environmental water body | |
| CN115015446B (en) | Method for simultaneously measuring L-theanine, caffeine, tea polyphenol and hexadecanoic acid in green tea | |
| CN110672771B (en) | Detection method of 11 pigments in reconstituted tobacco | |
| CN114636768B (en) | Detection method for thiamethoxam residues in jasmine flowers | |
| CN115236259B (en) | High performance liquid chromatography determination method for residual citric acid in Fmoc-amino acid | |
| CN116359367B (en) | Method for detecting gamma-aminobutyric acid in food by isotope internal elevation high performance liquid chromatography-tandem mass spectrometry | |
| CN114487189B (en) | Method for determining aflatoxin B1 in food based on dispersion extraction technology | |
| CN110618212B (en) | Method for simultaneously detecting residual quantity of multiple plant growth regulators in vegetables |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |