CN106501393B - A method of detection hickory chick Nucleosides - Google Patents
A method of detection hickory chick Nucleosides Download PDFInfo
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- CN106501393B CN106501393B CN201610886867.0A CN201610886867A CN106501393B CN 106501393 B CN106501393 B CN 106501393B CN 201610886867 A CN201610886867 A CN 201610886867A CN 106501393 B CN106501393 B CN 106501393B
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- hickory chick
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- adenosine
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- 241000723418 Carya Species 0.000 title claims abstract description 74
- 239000002777 nucleoside Substances 0.000 title claims abstract description 70
- 125000003835 nucleoside group Chemical group 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 63
- 238000001514 detection method Methods 0.000 title claims abstract description 34
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims abstract description 94
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 claims abstract description 78
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 claims abstract description 76
- 239000000243 solution Substances 0.000 claims abstract description 59
- 238000010828 elution Methods 0.000 claims abstract description 50
- 239000002126 C01EB10 - Adenosine Substances 0.000 claims abstract description 47
- 229960005305 adenosine Drugs 0.000 claims abstract description 47
- 239000013558 reference substance Substances 0.000 claims abstract description 44
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 41
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 claims abstract description 39
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 claims abstract description 39
- 229940045145 uridine Drugs 0.000 claims abstract description 39
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 claims abstract description 38
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 claims abstract description 38
- 229940029575 guanosine Drugs 0.000 claims abstract description 38
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 34
- 239000012085 test solution Substances 0.000 claims abstract description 33
- 238000002137 ultrasound extraction Methods 0.000 claims abstract description 26
- 239000000843 powder Substances 0.000 claims abstract description 16
- 239000006228 supernatant Substances 0.000 claims abstract description 16
- 238000002360 preparation method Methods 0.000 claims abstract description 14
- 239000000523 sample Substances 0.000 claims description 62
- 238000012360 testing method Methods 0.000 claims description 35
- 239000012071 phase Substances 0.000 claims description 27
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- 239000012488 sample solution Substances 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 239000012452 mother liquor Substances 0.000 claims description 3
- 239000007791 liquid phase Substances 0.000 claims description 2
- 238000001228 spectrum Methods 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 abstract description 8
- 239000003814 drug Substances 0.000 abstract description 5
- 235000001674 Agaricus brunnescens Nutrition 0.000 abstract description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 42
- 239000007788 liquid Substances 0.000 description 35
- 238000000605 extraction Methods 0.000 description 23
- 239000002904 solvent Substances 0.000 description 17
- 239000006286 aqueous extract Substances 0.000 description 13
- 239000000284 extract Substances 0.000 description 13
- 229910021642 ultra pure water Inorganic materials 0.000 description 13
- 239000012498 ultrapure water Substances 0.000 description 13
- 239000012467 final product Substances 0.000 description 11
- 239000000401 methanolic extract Substances 0.000 description 10
- 229930182470 glycoside Natural products 0.000 description 8
- 150000002338 glycosides Chemical class 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 238000011084 recovery Methods 0.000 description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 241001248610 Ophiocordyceps sinensis Species 0.000 description 4
- 239000012490 blank solution Substances 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 239000000470 constituent Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000002538 fungal effect Effects 0.000 description 4
- 239000004615 ingredient Substances 0.000 description 4
- 150000003833 nucleoside derivatives Chemical class 0.000 description 4
- 241000233866 Fungi Species 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 210000002700 urine Anatomy 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 241000190633 Cordyceps Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 229930010555 Inosine Natural products 0.000 description 2
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 2
- 235000002779 Morchella esculenta Nutrition 0.000 description 2
- 240000002769 Morchella esculenta Species 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- ABLZXFCXXLZCGV-UHFFFAOYSA-N Phosphorous acid Chemical compound OP(O)=O ABLZXFCXXLZCGV-UHFFFAOYSA-N 0.000 description 2
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 229960003786 inosine Drugs 0.000 description 2
- -1 matter Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003809 water extraction Methods 0.000 description 2
- XWLVCCHVHXBFBC-SXBJJNBASA-N 2-amino-9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-3h-purin-6-one Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O.C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O XWLVCCHVHXBFBC-SXBJJNBASA-N 0.000 description 1
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 description 1
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 description 1
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241001558929 Sclerotium <basidiomycota> Species 0.000 description 1
- LNQVTSROQXJCDD-UHFFFAOYSA-N adenosine monophosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)C(OP(O)(O)=O)C1O LNQVTSROQXJCDD-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000008648 bailing capsule Substances 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 238000002045 capillary electrochromatography Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- SEGLCEQVOFDUPX-UHFFFAOYSA-N di-(2-ethylhexyl)phosphoric acid Chemical compound CCCCC(CC)COP(O)(=O)OCC(CC)CCCC SEGLCEQVOFDUPX-UHFFFAOYSA-N 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
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- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
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- 235000019634 flavors Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
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- 238000012417 linear regression Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000003808 methanol extraction Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- 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)
- Saccharide Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to medicine edible mushroom analysis detection fields, more particularly to a kind of method for detecting guanosine in hickory chick, 3 kinds of uridine, adenosine gradient elutions simultaneously using HPLC, described method includes following steps: hickory chick powder, add water termostat ultrasonic extraction, it shakes up, it takes supernatant to cross water system filter to filter, up to test solution to be determined after stewing process；The preparation of gradient elution reference substance solution；HPLC detects the gradient elution in hickory chick.Method stability, repeatability and the precision of detection hickory chick Nucleosides disclosed by the invention are all very well, result is reliable, method is convenient, can control for the quality of hickory chick and provide scientific basis.
Description
Technical field
The present invention relates to edible and medical fungi analysis detection fields, and in particular to a kind of side for detecting hickory chick Nucleosides
Method utilizes 3 kinds of guanosine, uridine and adenosine gradient elutions in HPLC detection hickory chick in particular.
Background technique
Hickory chick (Morchella esculenta) is a kind of rare edible and medicinal fungi.Traditional Chinese Medicine thinks hickory chick
It is mild-natured, it is sweet in flavor cold, it is nontoxic, have the function of to digest and help food, beneficial stomach, phlegm-eliminiating and qi-regulating, kidney tonifying, establishing-Yang, refresh the mind, has higher
Medicine edible value and wide development prospect.Modern medicine study shows albumen rich in Morchella esculenta (L.) Pers sporophore
The nutritional ingredients such as matter, nucleosides, fatty acid, carbonate compound, biotin, crude fibre.But according to investigations, living to hickory chick at present
The further investigation of property ingredient is concentrated mainly on the research to polysaccharide, and to the rarely seen report of detection of gradient elution contained in hickory chick
Road.Early period, many scholars had made intensive studies gradient elution contained in medical edible fungal, and find ucleosides at
Dividing the pharmacological actions such as has antitumor, adjusting nervous centralis, improves immunity, promotes blood circulation.As it can be seen that nucleosides in hickory chick
The detection of constituents carries out overall quality control to hickory chick and is very important.
Currently, there has been no the reports about hickory chick Nucleosides content detection.Analysis about gradient elution
Detection has more research in similar rare medicine edible mushroom, is widely used as the quality and quality evaluation index of fungi medicinal material.
If 2015 editions " Chinese Pharmacopoeia " cordyceps sinensis quality standards are using adenosine content as unique chemical Composition Control index.Also have compared with
More documents study medical edible fungal Nucleosides, but the method for detection gradient elution reported in the literature is not
The detection of nucleosides in hickory chick can be directly applied to, such as by the efficient of Bailing capsule adenosine in 2015 editions " Chinese Pharmacopoeias " and uridine
Liquid chromatogram measuring method directly applies to 3 kinds of gradient elution detections of hickory chick there are peak shape asymmetry, and baseline drift etc. is asked
Topic；" gradient elution that 4 kinds of cordyceps sinensis medicinal materials are compared in high performance liquid chromatography quantitative analysis " extracts nucleosides using 0.5% phosphoric acid solution
Constituents and uridine, inosine, guanosine and the adenosine content for determining 4 kinds of Cordyceps, but the extracting method is used to mention
The gradient elution in hickory chick is taken as a result, it has been found that nucleosides content is very low.The 10% methanol extraction side that other many documents use
Method is applied to hickory chick nucleosides and extracts the problem very low there is also content.Meanwhile inventor is in a large amount of process of experimental
It was found that there is a problem of that stability is bad, RSD cannot reach during above-mentioned analyzing detecting method carries out methodological study
To the testing requirements for being lower than 5%, it may be possible to which the method that extracts and processes of sample Nucleosides has very greatly its assay
Influence.It can mutual inversion of phases, such as " Simultaneous when ucleosides substance-measuring in early period document report Mycophyta species
determination of eleven nucleosides and related compounds in Cordyceps by
Capillary electrochromatography " speculate in article to extract gained by room temperature immersion adenosine content is lower and may be
It is converted into other compounds；" Conversion of Adenosine Study of way in cordyceps sinensis water extraction process " is as a result, it has been found that Different Extraction Method
Have on cordyceps sinensis adenosine content and significantly affects, adenylate (atriphos, Adenosine diphosphate such as under room temperature water extraction conditions
Glycosides, adenosine monophosphate) it can be converted into adenosine, adenosine can be converted into inosine.It can be seen that sample handling processes are for accurate
The content of measurement medical edible fungal medicinal material Nucleosides plays very crucial effect.
Summary of the invention
In order to detect 3 kinds of guanosine, uridine and adenosine gradient elutions simultaneously, the present invention provides a kind of HPLC detector bars
Part, measurement that can be stable simultaneously, accurate to 3 kinds of nucleoside agents in hickory chick；In addition to this, the present invention also provides extract sheep tripe
The method of nucleosides in bacterium, this is very crucial for the content of Accurate Determining medical edible fungal medicinal material Nucleosides.
The present invention is achieved through the following technical solutions:
(1) prepared by test solution: taking hickory chick powder, pure water is added, ultrasonic extraction takes supernatant to filter through water system filter
It crosses, stewing process is to get test solution to be determined；
(2) prepared by nucleosides mixed reference substance solution: precision weighs uridine, guanosine, appropriate adenosine respectively, and pure water is added and shakes
It is even, the mixed reference substance solution of uridine, guanosine and adenosine is made；
(3) HPLC is detected: by above-mentioned steps (1) and the resulting test sample of step (2) and reference substance solution, carrying out efficient liquid
The detection of phase chromatography, HPLC chromatogram condition are as follows: use ZORBAX SB-AQ150*4.6mm, 5 μm of chromatographic columns, Detection wavelength is
260nm, column temperature are kept between 24~26 DEG C, flow velocity 0.8mL/min；Using gradient elution, mobile phase A is 0.04M di(2-ethylhexyl)phosphate
Hydrogen aqueous solutions of potassium, Mobile phase B are acetonitrile；By volume percentage, the condition of gradient elution are as follows: 0~8min, mobile phase A are
100%, Mobile phase B 0%；8~45min, mobile phase A at the uniform velocity become 85% from 100%；Mobile phase B is at the uniform velocity become from 0%
15%.
In some embodiments, sample solution preparation method in the step (1) are as follows: took the hickory chick powder of No. 3 sieves
Last 0.2g takes supernatant to filter through water system filter with 10mL pure water ultrasonic extraction, and stewing process is molten to get test sample to be determined
Liquid.
In some embodiments, the ultrasonic extraction conditions in the step (1) in sample solution preparation method are as follows: 25~
35 DEG C of ultrasonic extraction 30min.
In some embodiments, the stewing process method in the step (1) in sample solution preparation method are as follows: place
16~28h under 6 DEG C of environment.
In some embodiments, in the step (2) nucleosides mixed reference substance solution the preparation method comprises the following steps: precision weighs respectively
Uridine, guanosine, adenosine into 100mL volumetric flask, are dissolved in right amount with pure water, are added water and are settled to scale, shake up to get single right
According to product solution；It is accurate respectively to draw single nucleosides reference substance mother liquor 10.00mL in 50mL volumetric flask, pure water is added and dilutes and determines
Hold to scale, shake up, the nucleosides mixing that uridine, guanosine and adenosine mass concentration are respectively 9 μ g/mL, 8 μ g/mL, 9 μ g/mL is made
Reference substance solution.
The invention has the following beneficial effects:
Contain 1. high-efficiency liquid chromatography method for detecting of the invention has detected adenosine in hickory chick, uridine and guanosine simultaneously for the first time
Amount, gained chromatogram baseline stability, peak shape is good, and separating degree is high, can control for hickory chick quality and provide scientific basis.
2. the present invention can be accurate using the pure water method that ultrasound 30min extracts nucleosides in hickory chick at 25-35 DEG C
The content of wherein adenosine, guanosine and uridine is measured, the sample recovery rate of three kinds of nucleosides is all in 98~102% ranges, and RSD value
Within 1.5%, it can be good at the real content for reflecting these three nucleosides in sample.
3. present invention discover that having when 16~28h of stewing process under 6 DEG C of environment and stablizing well after sample extraction
Property, RSD value solves the stability problem of hickory chick nucleosides detection method within 5%.
Detailed description of the invention
Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, wherein U, A, G are respectively uridine, adenosine and bird
Glycosides: Fig. 1 is the result figure of the nucleosides content of hickory chick gradient elution Different Extraction Method, wherein 1.~be 9. respectively as follows:
1. water extracts, 30 DEG C of ultrasonic extraction 30min, solid-liquid ratio 1:50；
2. 0.5%H3PO3It extracts, 30 DEG C of ultrasonic extraction 30min, solid-liquid ratio 1:50；
3. 20% methanol extracts, 30 DEG C of ultrasonic extraction 30min, solid-liquid ratio 1:50；
4. water extracts, 25 DEG C of ultrasonic extraction 25min, solid-liquid ratio 1:75；
5. 0.5%H3PO3It extracts, 35 DEG C of ultrasonic extraction 25min, solid-liquid ratio 1:75；
6. 20% methanol extracts, 35 DEG C of ultrasonic extraction 25min, solid-liquid ratio 1:75；
7. water extracts, Soakage extraction 50min, solid-liquid ratio 1:100；
8. 0.5%H3PO3It extracts, Soakage extraction 50min, solid-liquid ratio 1:100；
9. 20% methanol extracts, Soakage extraction 50min, solid-liquid ratio 1:100.
Fig. 2 is the result figure of the nucleosides content of hickory chick different solvents, wherein Extraction solventRespectively
10% methanol, 20% methanol, 40% methanol, 60% methanol, 80% methanol, 90% methanol, 100% methanol, 0.5%H3PO3,
0.05M Na2HPO4, 0.02M PBS (phosphate buffer), boiling water.
Fig. 3 is that hickory chick nucleosides Aqueous extracts stand ucleosides contained by test sample sample within 36 hours under 6 DEG C of environment
Ingredient stability variation tendency result figure.
Fig. 4 is the amplification tendency chart of hickory chick Aqueous extracts gradient elution stability change within 16~28h in Fig. 3.
Fig. 5 stands test sample sample institute within 36 hours for 20% methanol extract liquid of hickory chick nucleosides under 6 DEG C of environment
The trend result figure of stability change containing gradient elution.
Fig. 6 is that hickory chick nucleosides Aqueous extracts stand ucleosides contained by test sample sample within 36 hours under room temperature environment
Ingredient stability variation tendency result figure.
Fig. 7 is HPLC chromatogram (the respectively nucleosides mixing from the bottom up of mixture of nucleosides reference substance and test solution
Reference substance solution, two parts of parallel sample solution).
Fig. 8 is hickory chick sample system compatibility test chromatogram.
Fig. 9 is that hickory chick sample specificity tests chromatogram.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The present invention is stated, rather than is limited the scope of the invention in any way.
The optimization of 1 hickory chick gradient elution extracting method of embodiment and offer solvent
1.1 instruments and reagent:
Instrument: high performance liquid chromatograph model Agilent 1260, electronic balance (a ten thousandth and ten a ten thousandths)
Model Mettler Toledo is cleaned by ultrasonic instrument model KQ-500DB, ultrapure water instrument model MILLI-Q.
Reagent: potassium dihydrogen phosphate is analysis rank, is purchased from Chengdu section dragon；Acetonitrile is HPLC rank, is purchased from Spectrum；Urine
Glycosides, guanosine and adenosine (containing grade is surveyed) are purchased from Nat'l Pharmaceutical & Biological Products Control Institute.
Hickory chick cultivates the cultivation base that dried frozen aquatic products derive from newborn source Nanling Hao Shanhao water Food Co., Ltd.
1.2 analysis methods:
Find Different Extraction Method and different solvents to the content of hickory chick gradient elution during the experiment
All have an impact, i.e. hickory chick gradient elution has stability.Therefore, Different Extraction Method and difference have been investigated respectively
Influence of the Extraction solvent to hickory chick gradient elution content, the method for extracting hickory chick gradient elution the following are 9 kinds:
1. hickory chick the powder 0.2g, accurately weighed in 50mL triangular flask, addition ultrapure water 10mL of No. 3 sieves were taken,
Ultrasonic extraction 30min, shakes up at 30 DEG C, takes supernatant after 0.45 μm of water system filter filters, after stewing process 16h to obtain the final product.
2. taking the hickory chick powder 0.2g of No. 3 sieves, accurately weighed in 50mL triangular flask, 0.5% phosphoric acid of addition is extracted
Liquid 10mL shakes up in 30 DEG C of ultrasonic extraction 30min, takes supernatant after 0.45 μm of water system filter filters, after stewing process 16h
To obtain the final product.
3. taking the hickory chick powder 0.2g of No. 3 sieves, accurately weighed in 50mL triangular flask, 20% methanol extract liquid of addition
10mL shakes up in 30 DEG C of ultrasonic extraction 30min, takes supernatant after 0.45 μm of water system filter filters, after stewing process 16h i.e.
?.
4. hickory chick the powder 0.2g, accurately weighed in 50mL triangular flask, addition ultrapure water 15mL of No. 3 sieves were taken,
25 DEG C of ultrasonic extraction 25min, shake up, and take supernatant after 0.45 μm of water system filter filters, after stewing process 16h to obtain the final product.
5. taking the hickory chick powder 0.2g of No. 3 sieves, accurately weighed in 50mL triangular flask, 0.5% phosphoric acid of addition is extracted
Liquid 15mL shakes up in 35 DEG C of ultrasonic extraction 25min, takes supernatant after 0.45 μm of water system filter filters, after stewing process 16h
To obtain the final product.
6. taking the hickory chick powder 0.2g of No. 3 sieves, accurately weighed in 50mL triangular flask, 20% methanol extract liquid of addition
15mL shakes up in 35 DEG C of ultrasonic extraction 25min, takes supernatant after 0.45 μm of water system filter filters, after stewing process 16h i.e.
?.
7. taking the hickory chick powder 0.2g of No. 3 sieves, accurately weighed in 50mL triangular flask, addition ultrapure water 20mL soaks
Stain extracts 50min, shakes up, and takes supernatant after 0.45 μm of water system filter filters, after stewing process 16h to obtain the final product.
8. the hickory chick powder 0.2g of No. 3 sieves was taken, it is accurately weighed in 50mL triangular flask, add 0.5%H3PO3Extracting solution
20mL, Soakage extraction 50min, shakes up, and takes supernatant after 0.45 μm of water system filter filters, after stewing process 16h to obtain the final product.
9. taking the hickory chick powder 0.2g of No. 3 sieves, accurately weighed in 50mL triangular flask, 20% methanol extract liquid of addition
20mL, Soakage extraction 50min, shakes up, and takes supernatant after 0.45 μm of water system filter filters, after stewing process 16h to obtain the final product.
In addition, finding Extraction solvent not when different methods of extraction influences hickory chick gradient elution content
With to nucleosides content and stability also have an impact, therefore be solid-liquid ratio 1:50 in extraction conditions, the case where ultrasonic extraction 30min
Lower to investigate influence of the different solvents to gradient elution content, according to extracting method, 1. to prepare test sample molten for the method
Liquid, wherein Extraction solvent is respectively 1. 10% methanol, 2. 20% methanol, 3. 40% methanol, 4. 60% methanol, 5. 80% methanol,
6. 90% methanol, 7. 100% methanol, 8. 0.5%H3PO3, 9. 0.05M Na2HPO4, 10. 0.02M PBS,Boiling water.
The test solution that HPLC detection: drawing above-mentioned Different Extraction Method and different solvents obtain, is injected into height
It is detected in effect liquid phase chromatogram instrument, records the chromatogram within 40min；Wherein, HPLC chromatogram condition are as follows: use Agilent
ZORBAX SB-AQ150*4.6mm, 5 μm of chromatographic columns, Detection wavelength 260nm, column temperature are kept between 24~26 DEG C, and flow velocity is
0.8mL/min, sample volume 10 μ L, 6 DEG C of sample injection disc temperature；Using gradient elution, mobile phase A is that 0.04M potassium dihydrogen phosphate is water-soluble
Liquid, Mobile phase B are acetonitrile；By volume percentage, the gradient wash condition are as follows: 0~8min, mobile phase A 100%, stream
Dynamic phase B is 0%；8~45min, mobile phase A at the uniform velocity become 85% from 100%；Mobile phase B at the uniform velocity becomes 15% from 0%.
HPLC testing result: HPLC testing conditions are optimized, Different Extraction Method and different solvents have been obtained
Hickory chick gradient elution HPLC chromatogram, and by calculate obtain it is each under the conditions of corresponding to test solution sample
The content of gradient elution.Using gradient elution content as ordinate, extracting method is abscissa, obtain it is as shown in Figure 1 not
With nucleoside agent content results figure corresponding to extracting method, wherein abscissa indicates 9 kinds of different extracting methods in Fig. 1, according to
It is secondary correspond to it is as described in the examples 1.~9. plant method.Equally, using gradient elution content as ordinate, Extraction solvent is cross
Coordinate obtains nucleoside agent content results figure corresponding to different solvents as shown in Figure 2, and wherein abscissa indicates 11 kinds
Different Extraction solvents is corresponding in turn to what embodiment was previously mentionedKind solvent.As it can be seen that by comparing different solvents,
Extraction time and solid-liquid ratio obtain optimal extracting mode to the extraction effect of hickory chick nucleosides are as follows: using pure water as solvent, material
Liquor ratio is set as 1:50, ultrasonic extraction 30min.
The discussion of 2 hickory chick gradient elution extracting solution stability change trend of embodiment
During carrying out methodology validation, discovery carries out after the test solution of acquisition is directly placed different time
HPLC detects its precision, as a result, it has been found that partial data is unqualified, i.e., extracting solution stability is bad (less than 4h), therefore is mentioned
The investigation of liquid stability is taken, and has investigated water respectively and has proposed variation tendency with two methods of the extracting solution stability of 20% alcohol extracting.
Sample 1. and is 3. extracted according in 1 hickory chick gradient elution extracting method of embodiment, is investigated respectively in 6 DEG C of rings
36 hours are placed under border at interval of the variation of gradient elution content contained by 1 hour test sample sample, meanwhile, compare sheep
Tripe sclerotium methods of glycosides Aqueous extracts place 36 hours at interval of contained by 1 hour corresponding test sample sample under room temperature environment
The variation of gradient elution content.
HPLC detection is carried out respectively to above-mentioned test sample sample according to chromatographic test strip part described in embodiment 1.
HPLC testing result: hickory chick nucleosides Aqueous extracts and alcohol extract are obtained by the chromatographic test strip part of embodiment 1
The HPLC chromatogram and Aqueous extracts within 36 hours at interval of 1 hour test solution sample are stood under 6 DEG C of environment
The HPLC chromatogram within 36 hours at interval of 1 hour test solution sample is stood under room temperature environment.When placing
Between be abscissa, the peak area of gradient elution is ordinate, obtains Aqueous extracts, 20% methanol extract liquid mentions at 6 DEG C with water
Liquid places contained gradient elution stability change tendency chart within 36h under room temperature environment, as shown in Figure 3, Figure 5 and Figure 6.Figure
3 place 36 hours at interval of test solution sample corresponding to 1 hour for hickory chick nucleosides Aqueous extracts under 6 DEG C of environment
In contained gradient elution peak area value, Fig. 4 be hickory chick nucleosides Aqueous extracts in 16~28h gradient elution peak area become
The enlarged drawing of change, as seen from the figure under 6 DEG C of environment place 16h when start occur balance starting point, behind in 12h, can reach compared with
Good stability.Fig. 5 is that 20% methanol extract liquid of hickory chick nucleosides places 36 hours at interval of 1 hour under 6 DEG C of environment
The peak area value of contained gradient elution in corresponding test solution sample, 20% methanol extract liquid is small 36 as seen from the figure
When interior gradient elution peak area in lasting variation, ie in solution stability is poor.Fig. 6 is hickory chick nucleosides Aqueous extracts in room temperature
36 hours are placed under environment at interval of the peak area of contained gradient elution in test solution sample corresponding to 1 hour
Value, it will be appreciated from fig. 6 that the peak area of its gradient elution is also ceaselessly changing when Aqueous extracts are placed under room temperature environment, it is steady
It is qualitative that testing requirements are not achieved.
Therefore, it is obtained by this part Experiment, hickory chick nucleosides Aqueous extracts occur steady when placing 16h under 6 DEG C of environment
Starting point, and the gradient elution content within 12h behind contained by it be it is stable, be suitble to nucleosides in Accurate Determining hickory chick
Constituents simultaneously carry out methodological study to it.
The method that embodiment 3HPLC detects hickory chick Nucleosides is established
The hickory chick sample extraction best approach is determined according to Examples 1 and 2 are as follows: using pure water as solvent, solid-liquid ratio setting
For 1:50, ultrasonic extraction 30min, and the foundation of 16~28h gradient elution stability is placed under 6 DEG C of environment according to Aqueous extracts
The HPLC detection method of its hickory chick sample.
Test solution preparation: the hickory chick powder 0.2g of No. 3 sieves, accurately weighed in 50mL triangular flask, addition were taken
Ultrapure water 10mL, ultrasonic extraction 30min, shakes up at 30 DEG C, takes supernatant after 0.45 μm of water system filter filters, 6 DEG C of environment
Lower standing 16h is to get test solution to be measured.
The preparation of nucleosides mixed reference substance solution: precision weighs uridine respectively, guanosine, adenosine in right amount in 100mL volumetric flask,
It is dissolved with ultrapure water, add water and is settled to scale, shaken up to get single reference substance solution；It is accurate respectively to draw single nucleosides pair
According to product mother liquor 10.00mL in 50mL volumetric flask, be added ultrapure water dilute and be settled to scale, shake up, be made uridine, guanosine and
Adenosine mass concentration is respectively 9 μ g/mL, 8 μ g/mL, 9 μ g/mL nucleosides mixed reference substance solutions.
HPLC detection: drawing test sample and reference substance solution obtained by above-mentioned steps, be injected into high performance liquid chromatograph into
Row detection, records the chromatogram within 40min；Wherein, HPLC chromatogram condition are as follows: use Agilent ZORBAX SB-AQ150*
4.6mm, 5 μm of chromatographic columns, Detection wavelength 260nm, column temperature are kept between 24~26 DEG C, flow velocity 0.8mL/min, sample volume 10
μ L, 6 DEG C of sample injection disc temperature；Using gradient elution, mobile phase A is 0.04M potassium dihydrogen phosphate aqueous solution, and Mobile phase B is acetonitrile；It presses
Volume percentage, the gradient wash condition are as follows: 0~8min, mobile phase A 100%, Mobile phase B 0%；8~45min,
Mobile phase A at the uniform velocity becomes 85% from 100%；Mobile phase B at the uniform velocity becomes 15% from 0%.
HPLC testing result: it is illustrated in figure 7 hickory chick nucleosides mixed reference substance solution test solution parallel with two parts
The HPLC chromatogram of (sample solution -1 and sample solution -2).As seen from the figure, the chromatographic test strip part color obtained after optimization
Spectrogram separating degree and peak type are all preferable, and detect containing uridine (Uridine), guanosine (Guanosine) and adenosine
(Adenosine) 3 kinds of gradient elutions.
The methodological study of embodiment 4HPLC detection hickory chick Nucleosides
The method that following aspect is carried out to the method for the HPLC detection hickory chick Nucleosides established in embodiment 3
It learns and investigates.
4.1 system suitabilities are investigated:
The preparation of single reference substance solution:
Adenosine aligns solution: adenosine reference substance about 5mg is weighed in 100mL volumetric flask, it is molten with blank solution (ultrapure water)
It solves and is settled to scale, shake up to obtain the final product.
Guanosine aligns solution: guanosine reference substance about 5mg is weighed in 100mL volumetric flask, it is molten with blank solution (ultrapure water)
It solves and is settled to scale, shake up to obtain the final product.
Uridine aligns solution: uridine reference substance about 5mg is weighed in 100mL volumetric flask, it is molten with blank solution (ultrapure water)
It solves and is settled to scale, shake up to obtain the final product.
Test sample and nucleosides mixed reference substance solution are prepared according to 3 the method for embodiment.Take blank molten after system balancing
2 needle of liquid sample introduction, 6 needle of reference substance solution sample introduction record chromatogram.Calculate uridine, guanosine, adenosine and respective phase in reference substance solution
The separating degree at adjacent peak, symmetrical factor calculate the RSD value of uridine, guanosine, 6 each peak area of needle of adenosine single sample continuous sample introduction.Such as
It is the HPLC chromatogram of 6 sample introductions of nucleosides mixed reference substance solution shown in Fig. 8.By comparing standards of pharmacopoeia, this experiment is found
Chromatographic peak separating degree is good, and the separating degree of each main peak and respective adjacent peak is less than 1.5 in reference substance solution, and symmetrical factor is 1.30
Between~1.39, the peak area RSD value of 6 needle of single adenosine, guanosine and uridine reference substance solution continuous sample introduction is no more than 5.0%.
It can thus be appreciated that the system suitability of this experiment detects qualification.
4.2 specificities are investigated
Test solution, nucleosides mixed reference substance solution and urine are prepared according to embodiment 3 and 4.1 the method for embodiment
Glycosides, guanosine, adenosine align solution, and are detected according to HPLC chromatogram condition described in embodiment 3, record chromatogram.Meanwhile
It records uridine contraposition solution, guanosine contraposition solution, adenosine and aligns solution in the retention time of respective chromatographic peak；Nucleosides mixing control
The retention time of each main peak in product solution and test solution.
As shown in figure 9, being followed successively by blank solution, test sample sample solution, nucleosides mixed reference substance solution, urine from the bottom up
Glycosides aligns the HPLC chromatogram of solution, guanosine contraposition solution and adenosine contraposition solution.Wherein, uridine, guanosine and adenosine three are right
The retention time of position solution is respectively as follows: 8.013min, 17.147min, 30.393min.Uridine, guanosine and gland in test solution
The retention time of three main peaks of glycosides is respectively as follows: 8.033min, 17.127min, 30.380min.Uridine in nucleosides reference substance solution,
The retention time of three main peaks of guanosine and adenosine is respectively as follows: 8.040min, 17.087min, 30.347min.As it can be seen that 3 kinds of nucleosides
Absolute difference of the retention time of constituents in test solution and nucleosides mixed reference substance solution is less than 0.2min, that is, tests
Specificity testing result be qualified.
4.3 precision are investigated
The method for preparing test solution and nucleosides mixed reference substance solution according to embodiment 3.6 parts are weighed in parallel for examination
6 parts of test solutions are made in product.Each 1 needle of sample introduction of test sample liquid is taken respectively, according to HPLC chromatogram analysis item described in embodiment 3
Part is detected, and chromatogram is recorded.With before and after test sample nearby 2 needle reference substance solutions calculate uridine in 6 parts of test samples, guanosine,
The content of adenosine, and record the uridine in test sample liquid, guanosine, the peak area of adenosine, content and the RSD value for calculating content.
As a result calculate uridine mean percent content be 0.158%, RSD 2.8%；Guanosine mean percent content is 0.104%, RSD
It is 3.3%；Adenosine mean percent content is 0.130%, RSD 4.3%.
4.4 study on the stability
It is prepared according to 3 the method for embodiment and stands 16h after test solution under 6 DEG C of environment, and by test solution
It is placed in 6 DEG C of sample injection discs, in the case where not shutting down, respectively at 0,2,4,6,8,10,12h (when can be changed according to practical operation
Between) sampling, one needle of sample introduction, records chromatogram respectively.Uridine in each time point test solution, guanosine, adenosine are recorded simultaneously
The ratio of peak area and each peak area peak area corresponding with 0h.Calculate uridine peak area RSD be 0.77%, guanosine peak face
Product RSD is 1.07%, and adenosine peak area RSD is 4.08%, it is seen that sample is stable placing in the 12h after 16h.
4.5 standard curves, quantitative line and detection line are investigated
Test sample and nucleosides mixed reference substance solution are prepared according to method described in embodiment 3.Precision draws the core prepared
Glycosides mixed reference substance solution 1mL dilutes in 100mL volumetric flask, with ultrapure water and is settled to scale, shakes up up to 100 times of dilution
Mixed reference substance solution.Nucleosides mixed reference substance solution after diluting 100 times is diluted to a certain concentration with ultrapure water step by step,
The dilution (parallel 3 parts), each one needle of sample introduction are taken, sample volume is 10 μ L, is carried out according to HPLC chromatogram condition described in embodiment 3
Analysis records chromatogram.Respective concentration is determined as minimum quantitative limit when with signal-to-noise ratio (S/N) being about 10, and signal-to-noise ratio (S/N) is about
Respective concentration is determined as lowest detection line when 3.
Nucleosides mixed reference substance solution is taken, setting sample volume is respectively 5 μ L, 7.5 μ L, 10 μ L, 15 μ L, each 2 needle of sample introduction
(minimum concentration point by quantitative limit test in terms of last 2 needle), carry out analysis inspection according to HPLC chromatogram condition described in embodiment 3
It surveys.Calculate uridine, guanosine, adenosine peak area average value, with uridine, guanosine, adenosine peak area average value to its concentration into
Row binary linear regression, with peak area (Y) for ordinate, solution concentration (X) is that abscissa carries out recurrence calculating, obtains the side of recurrence
Journey, related coefficient and linear relationship (table 1).
Regression equation, related coefficient, the range of linearity, quantitative limit and the detection limit of 13 kinds of hickory chick gradient elutions of table
4.6 sample recovery rates are investigated
Test solution II preparation: weighing test sample powder about 0.1g, accurately weighed in 50mL triangular flask, and precision is added
Nucleosides mixed reference substance solution 5mL under embodiment 3, adds ultrapure water 5mL, and ultrasonic extraction 30min shakes up, takes supernatant
Liquid stands 16h to obtain the final product under 6 DEG C of environment after 0.45 μm of water system filter membrane filters, parallel 6 parts of preparation.
Example 32 parts of lower test solutions, each one needle of sample introduction；Then each test solution II, every part of sample introduction one are taken
Needle is analyzed according to HPLC chromatogram condition described in embodiment 3, records chromatogram.Calculate the peak face of uridine, guanosine, adenosine
Product, it is molten with each test sample of 2 needle reference substance solutions calculating nearby before and after 3 lower test solutions of embodiment and test solution II
Uridine, guanosine, adenosine actual measured value, theoretical value, the RSD value of the single rate of recovery and the rate of recovery in liquid II, as shown in table 2.
The sample-adding recovery test result of 23 kinds of hickory chick gradient elutions of table
By table 3 it is found that uridine, three kinds of gradient elutions of guanosine and adenosine average recovery rate range be 99.91%~
100.0%, rate of recovery RSD range is 0.96%~1.1%, i.e., the sample-adding recycling detection of three kinds nucleoside agents meets pharmacopeia and wants
It asks.
Claims (4)
1. a kind of method for detecting hickory chick Nucleosides, characterized by the following steps:
(1) prepared by test solution: hickory chick powder is taken, pure water is added, ultrasonic extraction takes supernatant to filter through water system filter,
Stewing process is to get test solution to be determined；Stewing process method in the step (1) in sample solution preparation method
Are as follows: 16~28h of stewing process under 6 DEG C of environment；
(2) prepared by nucleosides mixed reference substance solution: precision weighs uridine, guanosine, appropriate adenosine respectively, and pure water is added and shakes up, makes
At the mixed reference substance solution of uridine, guanosine and adenosine；
(3) HPLC is detected: by above-mentioned steps (1) and the resulting test sample of step (2) and reference substance solution, carrying out high-efficient liquid phase color
Spectrum detection, HPLC chromatogram condition are as follows: use ZORBAX SB-AQ150*4.6mm, 5 μm of chromatographic columns, Detection wavelength 260nm, column
24~26 DEG C of temperature, flow velocity 0.8mL/min；Using gradient elution, mobile phase A is 0.04M potassium dihydrogen phosphate aqueous solution, mobile phase
B is acetonitrile；By volume percentage, the condition of gradient elution are as follows: 0~8min, mobile phase A 100%, Mobile phase B are
0%；8~45min, mobile phase A at the uniform velocity become 85% from 100%；Mobile phase B at the uniform velocity becomes 15% from 0%.
2. detection method according to claim 1, which is characterized in that sample solution preparation method in the step (1)
Are as follows: took the hickory chick powder 0.2g of No. 3 sieves to take supernatant to filter through water system filter, at standing with 10mL pure water ultrasonic extraction
Reason is to get test solution to be determined.
3. detection method according to claim 1 or 2, which is characterized in that test solution preparation side in the step (1)
Ultrasonic extraction conditions in method are as follows: 25-35 DEG C of ultrasonic extraction 30min.
4. detection method according to claim 1, which is characterized in that nucleosides mixed reference substance solution in the step (2)
The preparation method comprises the following steps: precision weighs uridine, guanosine, adenosine in right amount into 100mL volumetric flask respectively, dissolved with pure water, adds water and fixed
Hold to scale, shakes up to get single reference substance solution；It is accurate respectively to draw single nucleosides reference substance mother liquor 10.00mL in 50mL
In volumetric flask, pure water is added and dilutes and be settled to scale, shakes up, it is respectively 9 μ g/ that uridine, guanosine and adenosine mass concentration, which is made,
The nucleosides mixed reference substance solution of mL, 8 μ g/mL, 9 μ g/mL.
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