CN116930378A - Detection and identification method for wild osmanthus honey - Google Patents
Detection and identification method for wild osmanthus honey Download PDFInfo
- Publication number
- CN116930378A CN116930378A CN202310925295.2A CN202310925295A CN116930378A CN 116930378 A CN116930378 A CN 116930378A CN 202310925295 A CN202310925295 A CN 202310925295A CN 116930378 A CN116930378 A CN 116930378A
- Authority
- CN
- China
- Prior art keywords
- honey
- mobile phase
- wild
- liquid chromatography
- increased
- 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.)
- Granted
Links
- 235000012907 honey Nutrition 0.000 title claims abstract description 158
- 235000019082 Osmanthus Nutrition 0.000 title claims abstract description 64
- 241000333181 Osmanthus Species 0.000 title claims abstract description 64
- 238000000034 method Methods 0.000 title claims abstract description 62
- 238000001514 detection method Methods 0.000 title claims abstract description 15
- 150000001875 compounds Chemical class 0.000 claims abstract description 64
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 41
- 238000001228 spectrum Methods 0.000 claims abstract description 18
- 244000242564 Osmanthus fragrans Species 0.000 claims abstract description 15
- 235000019083 Osmanthus fragrans Nutrition 0.000 claims abstract description 15
- 238000004445 quantitative analysis Methods 0.000 claims abstract description 9
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims abstract description 8
- 239000004480 active ingredient Substances 0.000 claims abstract description 5
- 239000012071 phase Substances 0.000 claims description 75
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 60
- JLIDBLDQVAYHNE-YKALOCIXSA-N (+)-Abscisic acid Chemical compound OC(=O)/C=C(/C)\C=C\[C@@]1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-YKALOCIXSA-N 0.000 claims description 38
- 239000000243 solution Substances 0.000 claims description 31
- 244000052158 Eurya japonica Species 0.000 claims description 27
- 235000018822 Eurya japonica Nutrition 0.000 claims description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 25
- 239000000126 substance Substances 0.000 claims description 20
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 18
- 230000014759 maintenance of location Effects 0.000 claims description 16
- 239000003480 eluent Substances 0.000 claims description 15
- 239000002253 acid Substances 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 12
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 9
- 239000008367 deionised water Substances 0.000 claims description 9
- 229910021641 deionized water Inorganic materials 0.000 claims description 9
- 235000019253 formic acid Nutrition 0.000 claims description 9
- WLYGSPLCNKYESI-RSUQVHIMSA-N Carthamin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1[C@@]1(O)C(O)=C(C(=O)\C=C\C=2C=CC(O)=CC=2)C(=O)C(\C=C\2C([C@](O)([C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)C(O)=C(C(=O)\C=C\C=3C=CC(O)=CC=3)C/2=O)=O)=C1O WLYGSPLCNKYESI-RSUQVHIMSA-N 0.000 claims description 7
- DYQVDISPPLTLLR-HJQYTNQXSA-N Carthamin Natural products CC[C@H]1O[C@H]([C@H](O)[C@@H](O)[C@@H]1O)[C@]2(O)C(=C(C=C/3C(=O)C(=C(O)[C@](O)([C@@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@H]4O)C3=O)C(=O)C=Cc5ccc(O)cc5)C(=O)C(=C2O)C(=O)C=Cc6ccc(O)cc6)O DYQVDISPPLTLLR-HJQYTNQXSA-N 0.000 claims description 7
- FCRACOPGPMPSHN-UHFFFAOYSA-N desoxyabscisic acid Natural products OC(=O)C=C(C)C=CC1C(C)=CC(=O)CC1(C)C FCRACOPGPMPSHN-UHFFFAOYSA-N 0.000 claims description 7
- 239000000523 sample Substances 0.000 claims description 7
- 238000011068 loading method Methods 0.000 claims description 6
- 239000012488 sample solution Substances 0.000 claims description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 6
- 239000012498 ultrapure water Substances 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 5
- ZCHPKWUIAASXPV-UHFFFAOYSA-N acetic acid;methanol Chemical group OC.CC(O)=O ZCHPKWUIAASXPV-UHFFFAOYSA-N 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 230000003213 activating effect Effects 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- UFLHIIWVXFIJGU-ARJAWSKDSA-N (Z)-hex-3-en-1-ol Chemical compound CC\C=C/CCO UFLHIIWVXFIJGU-ARJAWSKDSA-N 0.000 claims 1
- 241000196324 Embryophyta Species 0.000 abstract description 9
- 238000011161 development Methods 0.000 abstract description 4
- 238000004451 qualitative analysis Methods 0.000 abstract description 3
- 238000013441 quality evaluation Methods 0.000 abstract description 3
- 150000002500 ions Chemical class 0.000 description 25
- 238000010521 absorption reaction Methods 0.000 description 15
- 238000004458 analytical method Methods 0.000 description 12
- 238000001819 mass spectrum Methods 0.000 description 12
- 230000008569 process Effects 0.000 description 9
- 238000004811 liquid chromatography Methods 0.000 description 8
- 244000020518 Carthamus tinctorius Species 0.000 description 6
- 235000003255 Carthamus tinctorius Nutrition 0.000 description 6
- 239000011734 sodium Substances 0.000 description 6
- 238000004949 mass spectrometry Methods 0.000 description 4
- 240000008620 Fagopyrum esculentum Species 0.000 description 3
- 235000009419 Fagopyrum esculentum Nutrition 0.000 description 3
- 240000005323 Hoya carnosa Species 0.000 description 3
- 235000010654 Melissa officinalis Nutrition 0.000 description 3
- 235000017784 Mespilus germanica Nutrition 0.000 description 3
- 244000182216 Mimusops elengi Species 0.000 description 3
- 235000000560 Mimusops elengi Nutrition 0.000 description 3
- 240000003152 Rhus chinensis Species 0.000 description 3
- 235000014220 Rhus chinensis Nutrition 0.000 description 3
- 235000007837 Vangueria infausta Nutrition 0.000 description 3
- 238000013467 fragmentation Methods 0.000 description 3
- 238000006062 fragmentation reaction Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 210000000051 wattle Anatomy 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000207925 Leonurus Species 0.000 description 2
- 241000219071 Malvaceae Species 0.000 description 2
- PMKQSEYPLQIEAY-YWNRKNDBSA-N Phaseolic acid Chemical compound OC(=O)C[C@@H](C(O)=O)OC(=O)\C=C\C1=CC=C(O)C(O)=C1 PMKQSEYPLQIEAY-YWNRKNDBSA-N 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 150000001793 charged compounds Chemical class 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- RRAFCDWBNXTKKO-UHFFFAOYSA-N eugenol Chemical compound COC1=CC(CC=C)=CC=C1O RRAFCDWBNXTKKO-UHFFFAOYSA-N 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- -1 kumi A Chemical class 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- JLIDBLDQVAYHNE-WEYXYWBQSA-N 2-trans-abscisic acid Chemical compound OC(=O)\C=C(/C)\C=C\C1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-WEYXYWBQSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- NPBVQXIMTZKSBA-UHFFFAOYSA-N Chavibetol Natural products COC1=CC=C(CC=C)C=C1O NPBVQXIMTZKSBA-UHFFFAOYSA-N 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- 244000192528 Chrysanthemum parthenium Species 0.000 description 1
- 235000000604 Chrysanthemum parthenium Nutrition 0.000 description 1
- 239000005770 Eugenol Substances 0.000 description 1
- 241000610361 Eurya Species 0.000 description 1
- 206010061459 Gastrointestinal ulcer Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 235000000802 Leonurus cardiaca ssp. villosus Nutrition 0.000 description 1
- 241000307158 Penthoraceae Species 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- UVMRYBDEERADNV-UHFFFAOYSA-N Pseudoeugenol Natural products COC1=CC(C(C)=C)=CC=C1O UVMRYBDEERADNV-UHFFFAOYSA-N 0.000 description 1
- 241000793823 Tilia amurensis Species 0.000 description 1
- 238000009098 adjuvant therapy Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- WLDHEUZGFKACJH-UHFFFAOYSA-K amaranth Chemical compound [Na+].[Na+].[Na+].C12=CC=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(O)=C1N=NC1=CC=C(S([O-])(=O)=O)C2=CC=CC=C12 WLDHEUZGFKACJH-UHFFFAOYSA-K 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 206010003549 asthenia Diseases 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- PMKQSEYPLQIEAY-SNVBAGLBSA-N caffeoyl malate Natural products OC(=O)C[C@@H](OC(=O)C=Cc1ccc(O)c(O)c1)C(O)=O PMKQSEYPLQIEAY-SNVBAGLBSA-N 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- PAEVMTYBHARJPO-UHFFFAOYSA-N dec-4-en-3-one Chemical compound CCCCCC=CC(=O)CC PAEVMTYBHARJPO-UHFFFAOYSA-N 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 239000002895 emetic Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 229960002217 eugenol Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000005976 liver dysfunction Effects 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 208000008128 pulmonary tuberculosis Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 238000001196 time-of-flight mass spectrum 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/08—Preparation using an enricher
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/065—Preparation using different phases to separate parts of sample
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/08—Preparation using an enricher
- G01N2030/085—Preparation using an enricher using absorbing precolumn
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)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention provides a detection and identification method for wild osmanthus honey, which adopts a high performance liquid chromatography combined with fingerprint spectrum method and a high performance liquid chromatography quantitative analysis method to detect and identify the wild osmanthus honey. The invention adopts a solid phase extraction method to extract and separate active ingredients of the wild osmanthus honey, adopts an HPLC combined fingerprint spectrum method and an HPLC quantitative analysis method to establish a qualitative analysis and a quantitative analysis method of plant compounds in the wild osmanthus honey, and also identifies 7 representative compounds in the wild osmanthus honey, and the obtained HPLC spectrum of the plant compounds of the wild osmanthus honey is initially simulated to generate a standard fingerprint spectrum of the wild osmanthus honey with representative significance. The qualitative and quantitative analysis method is applied to the authenticity and quality evaluation of the wild osmanthus fragrans honey, and has important practical significance for protecting the legal rights and interests of honey consumers and maintaining the healthy development of the honey consumption industry.
Description
Technical Field
The invention relates to the technical field of food detection, in particular to a detection and identification method for wild osmanthus honey.
Background
Wild sweet osmanthus honey is obtained from Eurya japonica, eurya of Penthoraceae, and is mainly distributed in Hunan, guangxi, guangdong and other places. The florescence is 9-10 months, the wild sweet osmanthus is the rarest honey which is superior in quality and sweet in taste in commercial honey in China, the odor is fragrant and sweet but not greasy, the color is water white, and the crystal is fine.
The wild osmanthus flower contains free amino acids, rich proteins and has a certain metabolism enhancing effect, and the wild osmanthus flower honey is also called as honey king. Clinical application of modern medicine proves that the honey can promote digestion and absorption, promote appetite, calm and improve sleep and body resistance, and has positive effects on promoting the growth and development of infants. The honey can be used for adjuvant treatment of asthenia and recovering after illness. The old suffering from hypertension can drink 1 cup of honey water in the morning and evening every day, and the potassium in the honey has the effect of removing sodium after entering the human body, so that the electrolyte balance in blood can be maintained; the chronic hepatitis and liver dysfunction patients can improve liver function by frequently eating honey; for patients with chronic diseases such as pulmonary tuberculosis, gastrointestinal ulcer, etc., mel is a good nutritional product, and can strengthen constitution.
Therefore, the wild osmanthus honey is driven by economic benefits, has adulteration phenomenon, seriously disturbs the normal order of the honey market, and restricts the development of the characteristic economy of the wild osmanthus honey industry. Therefore, establishing a set of methods for identifying the authenticity of wild osmanthus honey is a technical problem to be solved urgently.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a detection and identification method for wild osmanthus honey.
In order to achieve the object, the technical scheme of the invention is as follows:
in a first aspect of the invention, a method for detecting and identifying wild osmanthus honey is provided, which adopts a high performance liquid chromatography combined with fingerprint spectrum method and a high performance liquid chromatography quantitative analysis method to detect and identify the wild osmanthus honey,
wherein, the high performance liquid chromatography combined with fingerprint spectrum indicates that the wild osmanthus fragrans honey contains Eurya japonica alcohol A, which has a structure shown in formula (1):
further, the retention time of the Eurya japonica alcohol A is 42.60min;
further, in the high performance liquid chromatography, the mobile phase A is an aqueous solution of acetic acid with the concentration of 0.18-0.22 v/v%, and the mobile phase B is a methanol solution of acetic acid with the concentration of 0.18-0.22 v/v%;
further, in the high performance liquid chromatography, the gradient elution procedure is: 0-11 min, the mobile phase B is increased from 9v/v% to 14v/v%; 11-14 min, the mobile phase B is increased from 14v/v% to 15v/v%; the mobile phase B is kept at 15v/v% for 14-17 min; 17-24 min, the mobile phase B is increased from 15v/v% to 16v/v%; the mobile phase B rises from 16v/v% to 17v/v% after 24-28 min; 28-30 min, the mobile phase B rises from 17v/v% to 22v/v%; 30-41 min, and the mobile phase B is kept at 22v/v%; 41-46 min, the mobile phase B is increased from 22v/v% to 23v/v%; 46-55 min, the mobile phase B is increased from 23v/v% to 25v/v%; 55-60 min, the mobile phase B is increased from 25v/v% to 28v/v%; 60-70 min, the mobile phase B is increased from 28v/v% to 30v/v%; 70-80 min, and the mobile phase B is increased from 30v/v% to 40v/v%; 80-90 min, the mobile phase B is increased from 40v/v% to 45v/v%; 90-100 min, the mobile phase B is increased from 45v/v% to 52v/v%; 100-110 min, the mobile phase B is increased from 52v/v% to 57v/v%; 110-120 min, the mobile phase B is increased from 57v/v% to 65v/v%; 120-130 min, the mobile phase B rises from 65v/v% to 70v/v%; 130-135 min, the mobile phase B is increased from 70v/v% to 80v/v%; mobile phase a changes with the volume change of mobile phase B, and the sum of the volumes of the mobile phase a and the mobile phase B is 100%;
further, the high performance liquid chromatography further includes the following chromatographic conditions:
the chromatographic column is Phenomenex Gemini C 18 The chromatographic column has the flow rate of 0.6-0.8 mL/min, the sample injection amount of 20 mu L, the column temperature of 34-36 ℃ and the detection wavelength of 270nm; further preferably, the flow rate is 0.7mL/min and the column temperature is 35 ℃;
further preferably, in the high performance liquid chromatography, the mobile phase A is an aqueous acetic acid solution with a concentration of 0.2v/v%, and the mobile phase B is a methanol acetic acid solution with a concentration of 0.2 v/v%;
further, the high performance liquid chromatography further includes a pretreatment step:
s1: mixing honey to be detected with a proper amount of water, fully dissolving the honey to obtain aqueous honey solution, regulating the pH value of the aqueous honey solution to be 6.5-7, centrifuging the aqueous honey solution at a rotating speed of 8800-9200rpm for 18-22 min, and taking supernatant for later use;
s2: extracting the supernatant obtained in S1 by using a Strata-X-A solid phase extraction column, activating the solid phase extraction column by using methanol, balancing the extraction column by using ultrapure water, loading the supernatant, eluting by using deionized water, eluting by using a methanol solution containing 9-11 v/v% formic acid, extracting active ingredients in the supernatant to obtain an eluent, drying the obtained eluent, re-dissolving by using a proper amount of methanol, and filtering to obtain the product, wherein the dosage of the methanol, the ultrapure water, the deionized water and the methanol solution containing 9-11 v/v% formic acid is 2-4 times of the column volume;
further, in S1, the pH value is regulated to 6.8, the centrifugal rotating speed is 9000rpm, and the centrifugal time is 20min;
further, in S2, the pore diameter of the filtering membrane is 0.22 μm;
further, in S2, the eluent contains 10v/v% formic acid in methanol;
in S2, the dosage of the methanol, the ultrapure water, the deionized water and the methanol solution containing 9-11 v/v percent of formic acid is 3 times of the column volume;
furthermore, the high performance liquid chromatography combined with the fingerprint spectrum indicates that the wild osmanthus fragrans honey also contains kumi-seng A, 6R, 9S-toyol, carthamin acid, trans-abscisic acid, winter honey and cis, trans-abscisic acid;
further, retention time of the kumi element A, 6R, 9S-toxenol, carthamin acid, trans-abscisic acid, winter honey element and cis, trans-abscisic acid is 31.41min,44.61min, 50.47min, 65.37min, 67.79min and 75.45min respectively;
further, the quantitative analysis method of high performance liquid chromatography comprises the following steps:
(1) Chromatographic conditions are the same as the high performance liquid chromatography combined fingerprint method;
(2) Preparing wild sweet osmanthus honey standard substance solution: weighing a proper amount of kumi element A, 6R, 9S-toyophyllol, eurya japonica thunb alcohol A, winter honey element and abscisic acid to prepare standard substance solutions, wherein the concentrations of the standard substance solutions are respectively 0.4-0.6 mg/ml, 0.7-0.8 mg/ml, 0.5-0.7 mg/ml, 0.5-0.6 mg/ml and 0.4-0.5 mg/ml, drawing a wild osmanthus fragrans honey standard curve as shown in table 1, and correcting according to the loading quantity x and the peak area y and the gradient sampling quantity of 5-10 uL by adopting a 5-point method; preferably, the concentrations of the standard solutions of the kumi element A, the 6R, the 9S-toyol, the Eurya japonica thunb alcohol A, the winter honey element and the abscisic acid are respectively 0.5mg/ml,0.72mg/ml,0.6mg/ml,0.56mg/ml and 0.48mg/ml;
(3) Detecting a sample: injecting the sample solution according to the method in the step (1), and injecting the sample solution according to the corresponding t R Obtaining chromatographic peak area and substituting the chromatographic peak area into a corresponding standard curve, and obtaining the content of the compound in the honey, wherein the content of Eurya japonica linn A is not less than 0.40mg/kg, the content of winter honey is not less than 2.50mg/kg, and the content of 6R, 9S-toyophyllol is not less than 12.30mg/kg.
TABLE 1 information about standards
The second aspect of the invention provides application of the Eurya japonica alcohol A in identifying or detecting wild osmanthus fragrans honey.
Advantageous effects
1. The invention adopts a solid phase extraction method to extract and separate the active ingredients of the wild osmanthus honey, and adopts an HPLC combined fingerprint spectrum method and an HPLC quantitative analysis method to establish a qualitative analysis and a quantitative analysis method of the plant compounds in the wild osmanthus honey.
2. The analysis method identifies 7 compounds which are representative in the wild osmanthus honey, namely, the kumi element A, the Eurya japonica alcohol A, the 6R, the 9S-toyophyllol, the carthamin acid, the trans-abscisic acid, the winter honey element and the cis, trans-abscisic acid respectively, and the obtained HPLC (high performance liquid chromatography) spectrum of the wild osmanthus honey plant compound is obtained. The spectrum is imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the wild osmanthus honey standard fingerprint with representative significance is generated through preliminary simulation.
By comparing with HPLC patterns of four large amounts of single nectar such as locust honey, basswood honey, wattle honey and rape honey and analyzing and comparing plant compounds such as collected other masses of honey such as safflower honey, chinese gall honey, buckwheat honey, leonurus honey, medlar honey and the like, compounds such as kumi A, euryan alcohol A, 6R, 9S-emetic alcohol, safflower vigor acid, trans-abscisic acid, winter honey and cis, trans-abscisic acid are not detected in other single nectar except wild osmanthus honey. The seven components are proved to be used as characteristic plant compound markers of the wild osmanthus honey, and are applied to the authenticity and quality evaluation of the wild osmanthus honey, thereby having important practical significance for protecting the legal rights and interests of honey consumers and maintaining the healthy development of the honey consumption industry.
Drawings
FIG. 1 is a first-order mass spectrum of Eurya japonica alcohol A in positive ion mode
FIG. 2 is a secondary mass spectrum of Eurya japonica alcohol A in ion mode
FIG. 3 shows the mass fragmentation pathway of Eurya japonica alcohol A.
Fig. 4 is a chromatogram of the wild osmanthus honey composition of example 1.
Fig. 5 is an HPLC overlay of the sweet osmanthus raw honey of ten different producing areas in example 2. In the figure, R represents a standard fingerprint generated based on the wild osmanthus raw material honey of ten different producing areas, and S1-S10 represent the wild osmanthus raw material honey of ten different producing areas.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1: method for separating, analyzing and measuring content of wild osmanthus honey components and establishing fingerprint
1) The analysis method of the wild osmanthus honey comprises the following steps:
s1, preparing wild osmanthus fragrans honey solution: accurately weighing 20.0g of wild osmanthus honey in a beaker, adding 80mL of deionized water, stirring with a glass rod until the honey is fully dissolved, adding 5v/v% ammonia water solution to adjust the pH value to about 6.8, centrifuging at 9000rpm for 20min, and taking supernatant for later use;
s2, extracting and enriching wild osmanthus honey plant compounds: extracting the supernatant obtained in the step S1 by using a Strata-X-A solid phase extraction column, treating the Strata-X-A solid phase extraction column by using 3mL of methanol as an activating agent and 3mL of deionized water as a balancing agent, sampling the supernatant, eluting with 3mL of deionized water, eluting with 3mL of methanol solution containing 10v/v% formic acid, extracting active ingredients in the supernatant to obtain an eluent, drying the obtained eluent, re-dissolving with 2.0mL of methanol, and filtering with a 0.22 mu m filter membrane to obtain an extract for later use;
s3, separating and detecting wild osmanthus honey plant compounds: and (3) separating and detecting the extracting solution obtained in the step S2 by using high performance liquid chromatography, wherein the detection conditions comprise:
chromatographic column: phenomenex Gemini C 18 A chromatographic column;
mobile phase: the mobile phase A is acetic acid aqueous solution with the concentration of 0.2v/v%, and the mobile phase B is acetic acid methanol solution with the concentration of 0.2 v/v%;
the gradient elution procedure was: 0-11 min, the mobile phase B is increased from 9v/v% to 14v/v%; 11-14 min, the mobile phase B is increased from 14v/v% to 15v/v%; the mobile phase B is kept at 15v/v% for 14-17 min; 17-24 min, the mobile phase B is increased from 15v/v% to 16v/v%; the mobile phase B rises from 16v/v% to 17v/v% after 24-28 min; 28-30 min, the mobile phase B rises from 17v/v% to 22v/v%; 30-41 min, and the mobile phase B is kept at 22v/v%; 41-46 min, the mobile phase B is increased from 22v/v% to 23v/v%; 46-55 min, the mobile phase B is increased from 23v/v% to 25v/v%; 55-60 min, the mobile phase B is increased from 25v/v% to 28v/v%; 60-70 min, the mobile phase B is increased from 28v/v% to 30v/v%; 70-80 min, and the mobile phase B is increased from 30v/v% to 40v/v%; 80-90 min, the mobile phase B is increased from 40v/v% to 45v/v%; 90-100 min, the mobile phase B is increased from 45v/v% to 52v/v%; 100-110 min, the mobile phase B is increased from 52v/v% to 57v/v%; 110-120 min, the mobile phase B is increased from 57v/v% to 65v/v%; 120-130 min, the mobile phase B rises from 65v/v% to 70v/v%; 130-135 min, the mobile phase B is increased from 70v/v% to 80v/v%; mobile phase a changes with the volume change of mobile phase B, and the sum of the volumes of the mobile phase a and the mobile phase B is 100%;
flow rate: the flow rate is 0.7mL/min, the sample injection amount is 20 mu L, the column temperature is 35 ℃, and the detection wavelength is 270nm;
2) The content determination method comprises the following steps:
(1) Chromatographic conditions are the same as the high performance liquid chromatography combined fingerprint method;
(2) Preparing wild sweet osmanthus honey standard substance solution: weighing appropriate amount of kumi-seng A, 6R, 9S-toyophyllol, eurya japonica thunb alcohol A, winter honey element and abscisic acid to prepare standard substance solutions with the concentration of 0.5mg/ml,0.72mg/ml,0.6mg/ml,0.56mg/ml and 0.48mg/ml respectively; drawing a standard curve of wild osmanthus honey as shown in table 1, and correcting according to the sample loading amount x and the peak area y and the gradient sample loading amount of 5-10 uL by adopting a 5-point method;
TABLE 1 information about standards
(3) Detecting a sample: injecting the sample solution according to the method in the step (1), and injecting the sample solution according to the corresponding t R Obtaining chromatographic peak area and substituting the chromatographic peak area into a corresponding standard curve, and obtaining the content of the compound in the honey, wherein the content of Eurya japonica linn A is not less than 0.40mg/kg, the content of winter honey is not less than 2.50mg/kg, and the content of 6R, 9S-toyophyllol is not less than 12.30mg/kg;
3) The structure identification method comprises the following steps: the method for detecting the tandem diode array detector (HPLC-PDA) by the liquid chromatography and detecting the tandem quadrupole/time-of-flight mass spectrum (HPLC-QTOF) by the liquid chromatography is specifically adopted to carry out category identification on the plant compounds obtained by effective separation and enrichment.
The specific identification method is described in detail as follows:
1. carrying out structural identification on plant compound components in the wild osmanthus honey detected under the liquid chromatography conditions:
liquid phase mass spectrometry conditions: the ion source is ESI source (electrospray), the ion source spray voltage is 4kV, the outlet voltage is 130V, the heating temperature is 350 ℃, and nitrogen (N) 2 ) The flow rate is 11L/min, the collision gas is helium, the flow rate of the atomization gas is 80kPa, the pressure of the atomizer is 40psi, and the mass scanning range m/z=100-900 Da.
2. The structure identification analysis process and the result of the plant compound in the wild osmanthus honey:
(1) The structural analysis process of the compound in the eluent with retention time of 31.41min is as follows, the specific primary mass spectrum is shown in figure 1, and the secondary mass spectrum is shown in figure 2:
the compound has a molecular weight of 241.1444M/z [ M+H ] in positive ion mode]+and 263.1266M/z [ M+Na]Molecular ion peak of + 239.0567M/z [ M-H ] in negative ion mode]Molecular ion peak, judging the molecular weight of the compound to be 240 according to the first-order mass spectrum information, wherein the compound has maximum ultraviolet absorption at 232nm wavelengthA peak. Combining the secondary mass spectrum result and the maximum ultraviolet absorption wavelength of the compound to determine the molecular formula as C 13 H 20 O 4 The structure of the compound is shown as a formula II:
the chemical name of the compound is 3,4-dihydroxy-2,6,10,10-tetramethyl-1-oxapipro- [4.5] dec-6-en-8-one, and the compound is named as amaranth A.
(2) The structure analysis process of the compound in the eluent with the retention time of 42.60min is as follows:
the compound has m/z 225.1362[ M+H ] in positive ion mode]Ion peak of +m/z 207.1248[ M-H ] 2 O]The molecular weight of this compound was thus determined to be 224. The compound has a maximum ultraviolet absorption peak at a wavelength of 286 nm. Combining mass spectrum data, the retention time of Eurya japonica alcohol standard substance spectrum and the maximum ultraviolet absorption wavelength, and simultaneously verifying according to Eurya japonica alcohol standard substance liquid chromatography to finally determine the compound Eurya japonica alcohol.
The secondary mass spectrometry analysis process is as follows, the mass spectrometry fragmentation path of the compound is shown in figure 3, and the mass spectrometry fragmentation path of the Eurya japonica alcohol A is identified as shown in the formula (I):
the chemical name is (E) -4- (2, 3-dihydroxybutyl) -3, 5-trimethyl-cyclohex-2-en-1-one, which is named Eurya japonica alcohol A in the present invention.
(3) The structure analysis process of the compound in the eluent with the retention time of 44.61min is as follows: the compound had an ion peak of m/z 225.1485[ M+H ] + and an ion peak of m/z471.2707[2M+Na ] + in the positive ion mode, and thus it was found that the molecular weight of the compound was 224, and the compound had a maximum ultraviolet absorption peak at a wavelength of 268 nm. And combining mass spectrum data, consulting related documents and determining that the compound is 6R, 9S-toyol finally.
(4) The structure analysis process of the compound in the eluent with the retention time of 50.47min is as follows:
the compound has m/z 303.2367[ M+Na ] in positive ion state in positive ion mode] + Has an ion peak of 279.0977[ M-H ] in the negative ion state] - Ion peak sum 559.1507[2M-H ]] - The molecular weight of the compound can be judged to be 280. The maximum wavelength of ultraviolet absorption is 264, and the compound of the carthamic acid is finally determined by combining mass spectrum data, the retention time of carthamic acid standard substance spectrum and the maximum wavelength of ultraviolet absorption and simultaneously according to liquid chromatography verification of the carthamic acid standard substance.
(5) The structure analysis process of the compound in the eluent with the retention time of 65.37min is as follows:
the compound has m/z 265.1392[ M+H ] in positive ion mode] + Has an ion peak of 263.1054[ M-H ] in the negative ion state] - And therefore the molecular weight of the compound can be judged to be 263. The maximum wavelength of ultraviolet absorption of the compound is 263, and the compound has a maximum ultraviolet absorption peak at 263nm wavelength. And combining mass spectrum data, retention time of a trans-abscisic acid standard substance spectrum and maximum ultraviolet absorption wavelength, and simultaneously verifying according to a liquid chromatography of the trans-abscisic acid standard substance, and finally determining that the compound is the trans-abscisic acid.
(6) The structure analysis process of the compound in the eluent with the retention time of 67.79min is as follows:
the compound has m/z 241.1376[ M+H ] in positive ion mode] + Ion peaks of (2 M+H) m/z481.2754[2M+H ]] + Ion peaks of (2); 239.1035[ M-H ] in the negative ion state] - And therefore the molecular weight of the compound can be judged to be 240. The maximum wavelength of ultraviolet absorption of the compound is 242, and the compound is finally determined to be winter honey element by combining mass spectrum data, retention time of winter honey element standard substance spectrum and maximum ultraviolet absorption wavelength and simultaneously according to liquid chromatography verification of winter honey element standard substance.
(7) The structure analysis process of the compound in the eluent with the retention time of 75.45min is as follows:
the compound has m/z 287.1203[ M+Na ] in positive ion mode]Ion peak of +263.1023 [ M-H ] in negative ion state] - And therefore the molecular weight of the compound can be judged to be 263. The compound has a maximum ultraviolet absorption peak at 263nm wavelength. And combining mass spectrum data, retention time of a standard substance spectrum of cis-trans abscisic acid and maximum ultraviolet absorption wavelength, and verifying according to liquid chromatography of the standard substance of cis-trans abscisic acid to finally determine that the compound is cis-trans abscisic acid.
Example 2
The high performance liquid chromatography of example 1 was used to detect the wild osmanthus honey in ten fields, and the detected components of the wild osmanthus honey were quantitatively analyzed by an external standard method. The pretreatment and detection methods of the samples were the same as in example 1.
Specific honey source information is as follows table 1:
table 1 wild sweet osmanthus raw honey
The obtained chromatographic superposition patterns of the wild osmanthus honey of ten different producing areas are shown in figure 5.
Ten honey samples, wherein the average content of the kumi-sodium A is 2.46+/-0.91 mg/kg; the average content of Eurya japonica alcohol A is 1.11+/-0.47/kg; the average content of the toyol (6R, 9S) is 34.96 +/-20.21 mg/kg; the average content of the carthamin acid is 0.41+/-0.19 mg/kg; the average content of trans-abscisic acid is 2.74+/-1.14 mg/kg; the average content of winter honey element is 5.76+/-2.13 mg/kg; the average content of cis, trans-abscisic acid is 6.85+/-2.46 mg/kg. The detailed results are shown in Table 2.
Table 2 results of measuring the content of Honey compounds of wild osmanthus flower (mg/kg)
Example 3
In this example, the high performance liquid chromatography of example 1 was used to analyze the components of locust honey, tilia amurensis honey, wattle honey, rape honey, safflower honey, chinese gall honey, buckwheat honey, leonurus honey and medlar honey (see table 3 for specific raw material honey source information), and the above honey was compared with the HPLC profile analysis of wild osmanthus honey of example 1. The results show that the compounds of kumi A, eurya japonica alcohol, 6R, 9S-toyol, carthamic acid, trans-abscisic acid, winter honey and cis, trans-abscisic acid are not detected in other single nectar except wild osmanthus honey. The specific detection results are shown in Table 4.
TABLE 3 other raw Honey Source information Table
TABLE 4 information about whether other honey contains five marker compounds
Example 4
In this example, the high performance liquid chromatography of example 1 was used to detect and analyze eight commercial (different brands of goods from different sales platforms) wild osmanthus honey cases using a high performance liquid chromatograph. The quality of the wild osmanthus honey of five brands is better through HPLC chromatogram comparison analysis, the compounds of Eurya japonica alcohol A, 6R, 9S-toyophyllol and winter honey are detected, the contents are respectively in the ranges recorded in Table 2, the specific average contents are respectively 3.22mg/kg, 42.06mg/kg and 4.97mg/kg of the other 3 brands of wild osmanthus honey, the three compounds are not detected at the same time, and the product is judged to be not the wild osmanthus honey.
In summary, through the comparison of the high performance liquid chromatography of the wild osmanthus honey and the rape honey, the locust honey, the wattle honey and the basswood honey, and the analysis and comparison of the plant compound components of other collected public honey such as the motherwort honey, the Chinese gall honey, the safflower honey, the buckwheat honey, the medlar honey and the like, the compounds of the kumi-seng A, the eugenol, the 6R, 9S-toyol, the safflower soy acid, the trans-abscisic acid, the winter honey, the cis-abscisic acid are not detected in other single nectar except the wild osmanthus honey. Therefore, the kumi element A, the Eurya japonica alcohol, the 6R, 9S-toxenol, the safflower phaseolic acid, the trans, trans-abscisic acid, the winter honey element and the cis, trans-abscisic acid can be used as specific markers of the wild osmanthus honey component compounds and can be applied to the construction of a wild osmanthus honey authenticity and quality evaluation system.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. A detection and identification method for wild osmanthus honey is characterized in that the method adopts a high performance liquid chromatography combined with fingerprint spectrum method and a high performance liquid chromatography quantitative analysis method to detect and identify the wild osmanthus honey,
wherein, the high performance liquid chromatography combined with fingerprint spectrum indicates that the wild osmanthus fragrans honey contains Eurya japonica alcohol A, which has a structure shown in formula (1):
further, the retention time of Eurya japonica alcohol A is 42.60min.
2. The method for detecting and identifying wild osmanthus honey according to claim 1, wherein in the high performance liquid chromatography, a mobile phase A is an acetic acid aqueous solution with the concentration of 0.18-0.22 v/v%, and a mobile phase B is an acetic acid methanol solution with the concentration of 0.18-0.22 v/v%; preferably, in the high performance liquid chromatography, the mobile phase A is an aqueous acetic acid solution with a concentration of 0.2v/v%, and the mobile phase B is a methanol acetic acid solution with a concentration of 0.2 v/v%.
3. The method for detecting and identifying wild osmanthus honey according to claim 1, wherein in the high performance liquid chromatography, the gradient elution procedure is as follows: 0-11 min, the mobile phase B is increased from 9v/v% to 14v/v%; 11-14 min, the mobile phase B is increased from 14v/v% to 15v/v%; the mobile phase B is kept at 15v/v% for 14-17 min; 17-24 min, the mobile phase B is increased from 15v/v% to 16v/v%; the mobile phase B rises from 16v/v% to 17v/v% after 24-28 min; 28-30 min, the mobile phase B rises from 17v/v% to 22v/v%; 30-41 min, and the mobile phase B is kept at 22v/v%; 41-46 min, the mobile phase B is increased from 22v/v% to 23v/v%; 46-55 min, the mobile phase B is increased from 23v/v% to 25v/v%; 55-60 min, the mobile phase B is increased from 25v/v% to 28v/v%; 60-70 min, the mobile phase B is increased from 28v/v% to 30v/v%; 70-80 min, and the mobile phase B is increased from 30v/v% to 40v/v%; 80-90 min, the mobile phase B is increased from 40v/v% to 45v/v%; 90-100 min, the mobile phase B is increased from 45v/v% to 52v/v%; 100-110 min, the mobile phase B is increased from 52v/v% to 57v/v%; 110-120 min, the mobile phase B is increased from 57v/v% to 65v/v%; 120-130 min, the mobile phase B rises from 65v/v% to 70v/v%; 130-135 min, the mobile phase B is increased from 70v/v% to 80v/v%; mobile phase a varied with the volume of mobile phase B, and the sum of the volumes was 100%.
4. The method for detecting and identifying wild osmanthus honey according to claim 1, wherein the high performance liquid chromatography further comprises the following chromatographic conditions:
the chromatographic column is Phenomenex Gemini C 18 The chromatographic column has the flow rate of 0.6-0.8 mL/min, the sample injection amount of 20 mu L, the column temperature of 34-36 ℃ and the detection wavelength of 270nm;further preferably, the flow rate is 0.7mL/min and the column temperature is 35 ℃.
5. The method for detecting and identifying wild osmanthus honey according to claim 1, wherein the high performance liquid chromatography further comprises the following pretreatment steps:
s1: mixing honey to be detected with a proper amount of water, fully dissolving the honey to obtain aqueous honey solution, regulating the pH value of the aqueous honey solution to be 6.5-7, centrifuging the aqueous honey solution at a rotating speed of 8800-9200rpm for 18-22 min, and taking supernatant for later use; preferably, the pH value is regulated to 6.8, the centrifugal rotating speed is 9000rpm, and the centrifugal time is 20min;
s2: extracting the supernatant obtained in S1 by using a Strata-X-A solid phase extraction column, activating the solid phase extraction column by using methanol, balancing the extraction column by using ultrapure water, loading the supernatant, eluting by using deionized water, eluting by using a methanol solution containing 9-11 v/v% formic acid, extracting active ingredients in the supernatant to obtain an eluent, drying the obtained eluent, re-dissolving by using a proper amount of methanol, and filtering to obtain the product, wherein the dosage of the methanol, the ultrapure water, the deionized water and the methanol solution containing 9-11 v/v% formic acid is 2-4 times of the column volume;
further, in S2, the pore diameter of the filtering membrane is 0.22 μm;
further, in S2, the eluent contains 10v/v% formic acid in methanol;
in S2, the amount of methanol, ultrapure water, deionized water and a methanol solution containing 9-11 v/v% formic acid is 3 times the column volume.
6. The method for detecting and identifying wild osmanthus honey according to any one of claims 1 to 5, wherein the high performance liquid chromatography combined with fingerprint spectrum indicates that the wild osmanthus honey further comprises kumi-seng A, 6R, 9S-toyol, carthamin acid, trans-abscisic acid, winter honey and cis, trans-abscisic acid.
7. The method for detecting and identifying wild osmanthus honey according to claim 6, wherein retention time of kumi-seng A, 6R, 9S-toxenol, carthamin acid, trans-abscisic acid, winter honey element and cis, trans-abscisic acid is 31.41min,44.61min, 50.47min, 65.37min, 67.79min and 75.45min respectively.
8. The method for detecting and identifying wild osmanthus honey according to claim 1, wherein the method for quantitatively analyzing the wild osmanthus honey by high performance liquid chromatography comprises the following steps:
(1) Chromatographic conditions: combining with the high performance liquid chromatography and fingerprint method;
(2) Preparing wild sweet osmanthus honey standard substance solution: weighing a proper amount of kumi element A, 6R, 9S-toyophyllol, eurya japonica thunb alcohol A, winter honey element and abscisic acid, preparing into standard substance solutions, wherein the concentrations of the standard substance solutions are respectively 0.4-0.6 mg/ml, 0.7-0.8 mg/ml, 0.5-0.7 mg/ml, 0.5-0.6 mg/ml and 0.4-0.5 mg/ml, drawing a wild osmanthus fragrans honey standard curve as shown in table 1, and correcting according to the loading quantity x and the peak area y and the gradient sampling quantity of 5-10 uL by adopting a 5-point method;
TABLE 1 information about standards
(3) Content determination method: injecting the sample solution according to the method in the step (1), and injecting the sample solution according to the corresponding t R Obtaining chromatographic peak area and substituting the chromatographic peak area into a corresponding standard curve, and obtaining the content of the compound in the honey, wherein the content of Eurya japonica linn A is not less than 0.40mg/kg, the content of winter honey is not less than 2.50mg/kg, and the content of 6R, 9S-toyophyllol is not less than 12.30mg/kg.
9. The application of Eurya japonica alcohol A in the identification or detection of wild osmanthus honey is characterized in that the detection and identification method of the wild osmanthus honey is adopted.
10. The use according to claim 9, wherein the detection and identification method also simultaneously identifies kumi-manitol a, 6r,9 s-vomit leaf alcohol, carthamin acid, trans-abscisic acid, winter honey and cis, trans-abscisic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310925295.2A CN116930378B (en) | 2023-07-25 | 2023-07-25 | Detection and identification method for wild osmanthus honey |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310925295.2A CN116930378B (en) | 2023-07-25 | 2023-07-25 | Detection and identification method for wild osmanthus honey |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116930378A true CN116930378A (en) | 2023-10-24 |
| CN116930378B CN116930378B (en) | 2024-07-16 |
Family
ID=88376838
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310925295.2A Active CN116930378B (en) | 2023-07-25 | 2023-07-25 | Detection and identification method for wild osmanthus honey |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116930378B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109212086A (en) * | 2018-10-22 | 2019-01-15 | 中国农业科学院蜜蜂研究所 | A method of identifying acacia honey authenticity |
| CN110702832A (en) * | 2019-12-13 | 2020-01-17 | 中国农业科学院蜜蜂研究所 | Application of high-content 4-hydroxyquinoline as characteristic marker of jujube honey |
| AU2019232766A1 (en) * | 2018-09-14 | 2020-04-02 | Comvita Limited | Honey analysis |
| CN112162054A (en) * | 2020-08-31 | 2021-01-01 | 中国农业科学院蜜蜂研究所 | Authenticity evaluation method of sophora moorcroftianain honey |
| CN113109466A (en) * | 2021-03-27 | 2021-07-13 | 广东互信生物科技有限公司 | Fingerprint spectrum detection method for wasp venom of black peltate wasp |
-
2023
- 2023-07-25 CN CN202310925295.2A patent/CN116930378B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2019232766A1 (en) * | 2018-09-14 | 2020-04-02 | Comvita Limited | Honey analysis |
| CN109212086A (en) * | 2018-10-22 | 2019-01-15 | 中国农业科学院蜜蜂研究所 | A method of identifying acacia honey authenticity |
| CN110702832A (en) * | 2019-12-13 | 2020-01-17 | 中国农业科学院蜜蜂研究所 | Application of high-content 4-hydroxyquinoline as characteristic marker of jujube honey |
| CN112162054A (en) * | 2020-08-31 | 2021-01-01 | 中国农业科学院蜜蜂研究所 | Authenticity evaluation method of sophora moorcroftianain honey |
| CN113109466A (en) * | 2021-03-27 | 2021-07-13 | 广东互信生物科技有限公司 | Fingerprint spectrum detection method for wasp venom of black peltate wasp |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116930378B (en) | 2024-07-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Maragou et al. | Determination of bisphenol A in milk by solid phase extraction and liquid chromatography–mass spectrometry | |
| CN102818865A (en) | Method for Analyzing Oligomeric Proanthocyanidin (OPC) | |
| WO2009100229A1 (en) | Methods for analysis of vitamins | |
| CN111398499B (en) | Application of 3-amino-2-naphthoic acid in identifying apis cerana honey and apis mellifera honey | |
| KR102202225B1 (en) | Biomarker for the Discriminating Geographical Origins of Sesame and Method for Discriminating Geographical Origin Using the Same | |
| CN116908352B (en) | Detection and identification method for loquat honey | |
| CN114181176B (en) | Shishan kunmiki A and preparation method thereof, and identification method of bauhinia championii honey | |
| CN116930378B (en) | Detection and identification method for wild osmanthus honey | |
| CN113820420A (en) | Method for measuring chemical components in intelligence-developing wine by using UPLC-Q-TOF-MS | |
| Du et al. | Determination of 43 pesticide residues in intact grape berries (Vitis vinifera L.) by using an ultrasound-assisted acetonitrile extraction method followed by LC–MS/MS | |
| CN111337605B (en) | Method for evaluating authenticity of lotus bee pollen | |
| Li et al. | Simultaneous Determination of Fifteen Polyphenols in Fruit Juice Using Ultrahigh‐Performance Liquid Chromatography‐Tandem Mass Spectrometry Combining Dispersive Liquid‐Liquid Microextraction | |
| CN106918655A (en) | A kind of polygala UPLC assay methods | |
| Perestrelo et al. | Volatile flavour constituent patterns of terras madeirenses red wines extracted by dynamic headspace solid‐phase microextraction | |
| Yao et al. | HILIC‐UPLC‐MS/MS combined with hierarchical clustering analysis to rapidly analyze and evaluate nucleobases and nucleosides in Ginkgo biloba leaves | |
| Leloux et al. | Doping analysis of beta-blocking drugs using high-performance liquid chromatography | |
| CN115611840B (en) | Cold winter honey element and preparation method and application thereof | |
| CN111398497B (en) | Application of kynurenine in identifying Chinese bee honey and apis mellifera honey | |
| CN114315806A (en) | Preparation method of ester catechin-theanine adduct | |
| CN114487197A (en) | Method for detecting content of peanut lycopene in food and application thereof | |
| Tsai et al. | Disposition of asarone after intravenous administration to rabbits assessed using HPLC | |
| CN110780005A (en) | Analysis method of Cribolol raw material and synthetic intermediate thereof | |
| CN113848261B (en) | Identification method of fungi based on lipidomic | |
| CN114384183B (en) | Method for detecting trigonelline and application of trigonelline in honey identification | |
| CN117263790A (en) | Longan essence, preparation method thereof and identification method of longan honey |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |