CN106053662A - Method for measuring aflatoxin B1 content in traditional Chinese medicine - Google Patents
Method for measuring aflatoxin B1 content in traditional Chinese medicine Download PDFInfo
- Publication number
- CN106053662A CN106053662A CN201610528482.7A CN201610528482A CN106053662A CN 106053662 A CN106053662 A CN 106053662A CN 201610528482 A CN201610528482 A CN 201610528482A CN 106053662 A CN106053662 A CN 106053662A
- Authority
- CN
- China
- Prior art keywords
- chinese medicine
- flavacin
- assay
- magnetic field
- fecl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for measuring aflatoxin B1 content in traditional Chinese medicine. The method includes: regulating the pH of a traditional Chinese medicine solution to 2-5, adding magnetic nano particles, evenly mixing, allowing the mixed solution to be layered under an external magnetic field, removing supernate, adding the lower-layer sediment into mixed liquor of dichloromethane and acetone with the volume ratio being 2:1, stirring for desorption, allowing for layering under the external magnetic field, collecting supernate, naturally volatilizing and drying, redissolving with absolute methanol, filtering with a 0.22-micrometer filter membrane, and using high performance liquid chromatography to measure the aflatoxin B1 of the filtrate. The method has the advantages that the magnetic solid-phase extraction technology is applied to the aflatoxin content measuring for the first time, and the method is simple to operate, low in cost, high in accuracy and the like.
Description
(1) technical field
The present invention relates to a kind of flavacin B1The method of assay, particularly to flavacin B in a kind of Chinese medicine1Contain
Amount method for measuring.
(2) background technology
Flavacin is the fungal secondary metabolite that one group of chemical constitution is similar, and its basic structure is two furan nucleus blending
Legumin, mainly includes flavacin B1(AFB1)、B2(AFB2)、G1(AFG1)、G2(AFG2), there is serious carcinogenic, teratogenesis, cause
The effects such as sudden change, endanger huge.Wherein, AFB1Toxicity is the most most commonly seen, is ground by World Health Organization's cancer in 1993
Study carefully mechanism delimiting is I class carcinogen.
Flavacin is not only widely present in the agricultural product such as Semen Maydis, wheat and barley, Oryza glutinosa and the multiple eating such as nut, Semen arachidis hypogaeae is planted
In thing, exist in the medicinal plants such as Chinese medicine.Chinese crude drug is of a great variety, and extensively, most medical materials are producing, adding in plantation area
Work, preserve, transport during, it is easy to go mouldy and polluted by flavacin.There is scholar to Chinese medicine flavacin
Finding when pollution condition is investigated, the situation that Chinese crude drug, decoction pieces and Chinese patent medicine are polluted by flavacin is the most universal.Such as detection
25 batches of different lot numbers are containing AFB in the Chinese medicine preparation such as Semen Sojae Preparatum, bent class1Content, testing result show 100% pollution AFB1, Lignum Aquilariae Resinatum
Change the AFB of stagnant ball, antitoxic bolus of honeysuckle flower and forsythia, mind-easing tonic bolus with arborvitate seed1Mass fraction is all at more than 600ng/g.
Because of its hypertoxic and strong carcinogenecity, and the weakness that its pollution prevention is studied, flavacin becomes domestic and international government
With consumer's supervision and focal point.Control is taked to arrange for agricultural product and food, China and countries in the world with regard to its pollution problem
Execute, strict limit standard is i.e. set to ensure agricultural product quality and safety.And in comparison, for the Environmental capacity in Chinese medicine very
For weakness." Chinese Pharmacopoeia " version in 2010 has been recorded in the minority Chinese crude drugs such as Semen Persicae, Bombyx Batryticatus, Semen Ziziphi Spinosae, Semen Sterculiae Lychnophorae, Pericarpium Citri Reticulatae first
Flavacin residue limits detects, it is stipulated that wherein AFB1The highest allowance must not exceed 5 μ g/kg, flavacin (B1+B2+G1+
G2) content must not exceed 10 μ g/kg.Chinese patent medicine, as the Major Clinical type of service of Chinese medicine, not yet has standard to its Aspergillus flavus
The limitation of element specifies.So imperative for the supervision of Chinese native medicine security and the control of flavacin pollution, and for
Highly sensitive flavacin assay method set up by Chinese medicine, especially Chinese patent medicine urgent demand.
Isolation technics based on magnetic material is a hot fields of recent domestic research, and this technology is divided at cell
From, transport of drug, enzyme immobilizatio, be catalyzed, adsorb-separate, the numerous areas such as material science, environment all show application before
Scape.Magnetic Nano material can extract the adsorbent of (MSPE) as Magnetic solid phases.With conventional Solid-Phase Extraction (SPE) column packing phase
Ratio, the specific surface area of nanoparticle is big, diffusion length is short, and a small amount of adsorbent and shorter equilibration time just energy only need to be used real
Existing extract and separate, therefore has higher extracting power and extraction efficiency.Through functional modification, it is right that magnetic adsorbent is expected to realize
The selective extraction of analyte.It addition, magnetic adsorbent can recycle after suitable cleaning.MSPE is only by applying one
Individual external magnetic field can realize being separated, the most simple to operate, save time quickly, without troublesome operation such as centrifugal filtrations, it is to avoid
Tradition SPE adsorbent need to fill the time-consuming problem such as post and sample loading, and does not haves SPE when processing biology, environmental sample
In run into post blocking problem.MSPE technology is applied, for trace analysis in contaminant trace species extract and separate field
The preenrichment of thing opens a new window of fan.But the magnetic nanometer particles of currently used functionalization sample pre-treatments in Chinese medicine
Applied research is less, is concentrated mainly in the enrichment to metabolite in biological sample or metal ion.Not yet there is this skill of application
The report that art flavacin in Chinese medicine measures.
To sum up, a sample pre-treatments difficult problem in the urgent need to address in analyzing for Chinese patent medicine flavacin, the present invention intends choosing
Taking and representing Chinese patent medicine SHENGMAI YIN (Radix Codonopsis side) simply is object of study, and novel functional magnetic Nano material is used for it
In the detection research of flavacin.The functional magnetic nano-particle strong by finding selectivity, the bar to Magnetic solid phases extraction
Part is optimized, and sets up the method for highly sensitive Magnetic solid phases extraction-high performance liquid chromatography-fluorescence for SHENGMAI YIN
Flavacin assay in (Radix Codonopsis side), provides technology to prop up for the control of Chinese medicine flavacin and the formulation of limit standard
Hold, provide foundation for a kind of low cost of exploitation, high selective flavacin sample pretreatment material.
(3) summary of the invention
It is an object of the present invention to provide a kind of method quick, sensitive, low cost and magnetic nanoparticle is used in Chinese medicine Huang
Aspergillin B1Assay.
The technical solution used in the present invention is:
The present invention provides flavacin B in a kind of Chinese medicine1The method of assay, described method is: by molten for Chinese medicine to be measured
Liquid regulation pH value, to 2～5 (preferably 3.5), adds magnetic nanoparticle, stirring and evenly mixing, and mixed liquor divides under additional the action of a magnetic field
Layer, removes the supernatant, and lower sediment adds dichloromethane and the acetone mixture of volume ratio 2:1, stirs desorption, in magnetic field
Effect lower leaf, collects the supernatant, redissolves with absolute methanol, cross 0.22 μm filter membrane after naturally volatilizing, and filtrate uses high-efficient liquid
Phase chromatograph carries out flavacin B1Measure；Described magnetic nanoparticle is 2-amino-5-sulfydryl-1,3,4-thiadiazoles 3-trimethyl
Silane-1-propanethiol modifies ferroferric oxide magnetic nanoparticle, i.e. AMT-TMSPT-MNPs, and being called for short MMNPs, AMT is 2-ammonia
Base-5-sulfydryl-1,3,4-thiadiazoles, TMSPT is 3-trimethyl silane-1-propanethiol, and MNPs is ferroferric oxide magnetic nano
Granule.
Further, described magnetic nanoparticle addition is calculated as 10-20g/L with Chinese medicine liquor capacity to be measured, preferably 15g/
L。
Further, described dichloromethane and acetone mixture and Chinese medicine liquor capacity to be measured are than for 0.3-1.2:1, preferably
0.9:1。
Further, described adsorption time (i.e. stirring and evenly mixing time) is 5～15min, preferably 10min.
Further, described desorption time (i.e. stirring desorption time) is 2～10min, preferably 8min.
Further, described high performance liquid chromatography testing conditions is: chromatographic column: C18Post, 4.6mm × 250mm ,≤5 μm；Flowing
Phase: acetonitrile, methanol, volumetric concentration 0.1% aqueous formic acid, volume ratio 10-25:10-25:50-80；Flow velocity: 0.8-1.2mL/
min；Column temperature: 20-30 DEG C；Fluorescence detector.Number of theoretical plate presses flavacin B1Peak calculates should be not less than 3000.
Further, described externally-applied magnetic field refers to using Magnet as externally-applied magnetic field source.
Further, described C18Carbon octadecylsilane chemically bonded silica particle diameter used by post is 5 μm or 3.5 μm.
Further, described flowing phase: acetonitrile, methanol, volumetric concentration 0.1% aqueous formic acid, volume ratio 20:20:60.
Further, described column temperature: 25 DEG C.
Further, described fluorescence detector excitation wavelength: 360nm, launches wavelength: 440nm.
Further, described magnetic nanoparticle is prepared as follows:
(1) by FeCl3·6H2O and FeCl2·4H2O mixes with deionized water, 85 DEG C of water-baths under conditions of nitrogen is protected
Heat and stir 30min, being slowly injected into the ammonia of mass concentration 25-28%, after addition ammonia terminates, continue reacting by heating
30min, centrifugal, take precipitate with deionized water and wash 2 times, then wash 1 time with 0.02mol/L sodium-chloride water solution, be deposited in 40
～be dried at 60 DEG C, obtain ferroferric oxide magnetic nanoparticle；Described FeCl3·6H2O and FeCl2·4H2O mass ratio is 2
～3:1, described deionized water volumetric usage is with FeCl3·6H2O and FeCl2·4H2O gross mass is calculated as 12～13mL/g, described
Ammonia volumetric usage is with FeCl3·6H2O and FeCl2·4H2O gross mass is calculated as 1.4～1.8mL/g；
(2) step (1) ferroferric oxide magnetic nanoparticle deionized water is configured to the storing solution of 40g/L, will storage
Standby liquid, in additional the action of a magnetic field lower leaf, removes the supernatant, and lower sediment adds mass concentration 10%3-trimethyl silane-1-
Propanethiol aqueous solution, stirs, and adds glycerol, heats and be stirred vigorously 2h under nitrogen protection in 90 DEG C of water-baths, cold
But to after room temperature, in additional the action of a magnetic field lower leaf, removing the supernatant, lower sediment is washed with deionized 3 times successively, nothing
Water methanol washs 3 times, deionized water wash 5 times, and precipitation granule is dried at 40～60 DEG C, obtains 3-trimethyl silane-1-third
Mercaptan modified by magnetic nanoparticles thing；Described 3-trimethyl silane-1-propanethiol aqueous solution and storing solution volume ratio are 4～5:1,
Described glycerol and storing solution volume ratio are 3～4:1；
(3) step (2) 3-trimethyl silane-1-propanethiol modified by magnetic nanoparticles thing deionized water is configured to
40g/L storing solution, washs 2 times with methanol, addition mass concentration 1%2-amino-5-sulfydryl-1,3,4-thiadiazoles aqueous solutions,
Ultrasonic power is ultrasonic 2h under conditions of 600W, obtains granule precipitate with deionized water and washs 3 times, then washs with absolute methanol
2 times, at 40～60 DEG C, it is dried 12h, i.e. obtains 2-amino-5-sulfydryl-1,3,4-thiadiazoles 3-trimethyl silane-1-propanethiols
Modified by magnetic nanoparticles thing, is magnetic nanoparticle (MMNPs)；Described 2-amino-5-sulfydryl-1,3,4-thiadiazoles is water-soluble
Liquid and storing solution volume ratio are 5～8:1, preferably 6:1.
Compared with prior art, the present invention has the following advantages and beneficial effect:
(1) there is for Chinese medicine the substance system of complexity, and flavacin content this feature extremely low, set up high sensitivity
Magnetic nanoparticle-stationary phases for HPLC to aflatoxin B1Carry out assay research, there is low cost, choosing
Selecting property advantages of higher.
(2) functional magnetic nano-particle is mainly concentrated use in the metabolite in biological sample and food or gold at present
Belonging in the enrichment of ion, the present invention is that Magnetic solid phases abstraction technique is used for the assay of aflatoxin first in Chinese medicine,
There is easy and simple to handle, low cost, accuracy advantages of higher.
(4) accompanying drawing explanation
Fig. 1: different factors are to flavacin B1Response rate influence curve figure, (a) sample solution pH, (b) magnetic Nano
Grain addition, (c) enrichment time, (d) effluent volume, (e) elution time.
Fig. 2: flavacin B1The liquid chromatogram of standard substance.
The liquid chromatogram of Fig. 3: embodiment 4 need testing solution.
Fig. 4: the liquid chromatogram of SHENGMAI YIN (Radix Codonopsis side) under different flow phase system.
Fig. 5: the liquid chromatogram of SHENGMAI YIN (Radix Codonopsis side) under the conditions of different column temperatures.
Fig. 6: the liquid chromatogram of different enterprises' SHENGMAI YIN (Radix Codonopsis side), A is A enterprise SHENGMAI YIN (Radix Codonopsis side), and B is B enterprise
Industry SHENGMAI YIN (Radix Codonopsis side), C is C enterprise SHENGMAI YIN (Radix Codonopsis side), and D is D enterprise SHENGMAI YIN (Radix Codonopsis side), and E is E enterprise SHENGMAI YIN
(Radix Codonopsis side).
(5) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This:
SHENGMAI YIN (Radix Codonopsis side) used by embodiment of the present invention 2-3 is purchased from Jiangxi Huiren Pharmaceutical Co., Ltd.
Externally-applied magnetic field described in the embodiment of the present invention all presses close to round-bottomed flask as additional using the magnet piece of 150*100*25mm
Magnetic Field Source.
The preparation of embodiment 1 magnetic nanoparticle
(1) FeCl of 11.68g is weighed3·6H2The FeCl of O and 4.30g2·4H2O is placed in there-necked flask, adds 200mL
Deionized water, is filled with after nitrogen and seals, 85 DEG C of heating in water bath stir 30min under conditions of nitrogen is protected, and is slowly injected into matter
The ammonia 23mL of amount concentration 25-28%, makes solution colour change.After addition ammonia terminates, continue heating, 85 DEG C of reactions
30min, centrifugal, obtained precipitate with deionized water is washed 2 times, then washs 1 time with 0.02mol/L sodium-chloride water solution,
To granule be deposited at 45 DEG C be dried 36h, i.e. obtain ferroferric oxide magnetic nanoparticle (MNPs) 1.5g, by it with 40g/
The concentration of L is stored in deionized water, makes MNPs storing solution, standby.
(2) take 20mL MNPs storing solution and pour in round-bottomed flask, make its fast hierarchical with externally-applied magnetic field, remove upper strata clear
Liquid, lower sediment is initially charged 80mL mass concentration 10%3-trimethyl silane-1-propanethiol (TMSPT) aqueous solution, stirs,
Add 60mL glycerol, seal after being filled with nitrogen, heat in 90 DEG C of water-baths under nitrogen protection and be stirred vigorously 2h, being cooled to
After room temperature, in additional the action of a magnetic field lower leaf, removing the supernatant, lower sediment is washed with deionized 3 times successively, without water beetle
Alcohol washs 3 times, deionized water wash 5 times, is deposited at 45 DEG C and is dried 12h, obtains 3-trimethyl silane-1-propanethiol magnetic and receive
Rice grain trim (TMSPT-MNPs) about 1.0g, stores it in deionized water with the concentration of 40g/L, makes TMSPT-
MNPs storing solution, standby.
(3) take above-mentioned TMSPT-MNPs storing solution 25mL absolute methanol to wash 2 times, be uniformly added into 150mL mass concentration
1%2-amino-5-sulfydryl-1, in the aqueous solution of 3,4-thiadiazoles (AMT), gained solution is transferred in beaker, in ultrasound wave merit
Rate is ultrasonic 2h under conditions of 600W, the solution obtained, and makes its fast hierarchical with externally-applied magnetic field, removes supernatant, obtains granule
Precipitate with deionized water is washed 3 times, then washs 2 times with absolute methanol, is dried 12h, i.e. obtains 2-amino-5-mercapto at 45 DEG C
Base-1,3,4-thiadiazoles 3-trimethyl silane-1-propanethiol modified by magnetic nanoparticles thing AMT-TMSPT-MNPs (MMNPs)
0.8g, for sample pre-treatments.
The optimization of embodiment 2 chromatographic condition
1, sample pre-treatments: take SHENGMAI YIN (Radix Codonopsis side) 10mL, adds MMNPs prepared by 150mg embodiment 1 method, room
Stirring 10min under temperature, separated with supernatant by MMNPs with externally-applied magnetic field, pour out supernatant, precipitation adds 9mL CH2Cl2:
Me2CO (2:1, v/v), stirs desorption 8min, in the action of a magnetic field lower leaf, collects the supernatant, with 300 μ L after naturally volatilizing
Absolute methanol redissolves, and crosses 0.22 μm filter membrane, takes filtrate, prepares sample solution.
2, flowing selects mutually
Use stationary phases for HPLC that sample solution is carried out flavacin B1Assay.
(1) instrument and reagent:
Agilent Technologies 1290infinity Ultra Performance Liquid Chromatography system (includes quaternary gradient pump
(G4204A, 1290Quat Pump), online degasser, automatic sampler (G4226A, 1290ALS), column oven (G1330B,
1290TCC), fluorescence detector (G1321B, 1260FLD), Data Processing in Chromatography Workstation；Ao Haosi AR224w (ten thousand/electronics
Balance)；100000/electronic balance (METTLER XS205)；Aflatoxin B1 reference substance is purchased from U.S. Sigma
Chemical company, through efficient Liquid Detection, purity is all more than 98%；Formic acid (chromatographically pure, TEDIA company of the U.S.), acetonitrile
(chromatographically pure, TEDIA company of the U.S.), water is pure water.
(2) selection of chromatographic condition
Chromatographic column: Agilent SB-C18Post (4.6mm × 250mm, 5 μm)；
Flowing phase: A be acetonitrile, B is mass concentration 0.1% aqueous formic acid, and C is absolute methanol, by acetonitrile-methanol-
0.1% formic acid water (20:20:60, v/v/v) carries out isocratic elution；
Flow velocity: 1.0mL/min；
Column temperature: 25 DEG C；
Detector: fluorescence detector；
Detection wavelength: excitation wavelength: 360nm, launches wavelength: 440nm；
Sample size: 20 μ L.
Flowing is respectively as follows: acetonitrile-methanol-0.1% formic acid water (10:10:80, v/v/v), acetonitrile-methanol-0.1% first mutually
Sour water (15:15:70, v/v/v), acetonitrile-methanol-0.1% formic acid water (20:20:60, v/v/v).
Result shows that (see Fig. 4) washes for flowing equality with acetonitrile-methanol-0.1% formic acid water (20:20:60, v/v/v)
De-effect is more excellent, and each chromatographic peak separating degree is good, and peak shape is sharp-pointed.
3, the impact of column temperature
With reference to the selection of flowing phase, column temperature is respectively as follows: 20 DEG C, 25 DEG C, 30 DEG C；Flowing is mutually: acetonitrile-methanol-0.1% first
Sour water (20:20:60, v/v/v).
Result shows (see Fig. 5), has investigated the separation feelings of chromatographic peak under different column temperature (20 DEG C, 25 DEG C, 30 DEG C) in test
Condition is similar to, and the separation of chromatographic peak is affected little by temperature.
The optimization of embodiment 3 magnetic nanoparticle extraction conditions
1, sample solution pH: take each 10mL of SHENGMAI YIN (Radix Codonopsis side), by its pH respectively furnishing 2.0,3.0,3.5,4.0,
5.0, in sample solution, it is uniformly added into MMNPs prepared by 100mg embodiment 1 method, stirs 10min, will with externally-applied magnetic field
MMNPs separates with supernatant, pours out the supernatant, lower sediment 9mL CH2Cl2:Me2CO (2:1, v/v), stirs desorption
10min, in the action of a magnetic field lower leaf, collects the supernatant, redissolves with 300 μ L absolute methanols after naturally volatilizing, and crosses 0.22 μm filter
Film, takes filtrate, prepare sample solution, use embodiment 2 method carry out high performance liquid chromatograph detection (column temperature: 25 DEG C, acetonitrile-
Methanol-0.1% formic acid water (20:20:60, v/v/v) is flowing phase), result shows the solution condition of (see a in Fig. 1) pH=3.5
Flavacin B1Adsorption effect more excellent.
2, magnetic nanoparticle consumption: take each 10mL of SHENGMAI YIN (Radix Codonopsis side), regulates pH=3.5, is separately added into embodiment 1
MMNPs100mg, 120mg, 150mg, 170mg, 200mg prepared by method, stirs 10min, with externally-applied magnetic field by MMNPs with upper
Clear liquid separates, and pours out the supernatant, lower sediment 9mL CH2Cl2:Me2CO (2:1, v/v), stirs desorption 10min, outside
Add the action of a magnetic field lower leaf, collect the supernatant, redissolve with 300 μ L absolute methanols after naturally volatilizing, cross 0.22 μm filter membrane, take filter
Liquid prepares sample solution, uses embodiment 2 method high performance liquid chromatograph detection (column temperature: 25 DEG C, acetonitrile-methanol-0.1% first
Sour water (20:20:60, v/v/v) for flowing phase, result show (see b in Fig. 1) when treating that test sample consumption is 10mL, amount of particles
During 150mg, can make aflatoxin B1Absorption is completely.
3, magnetic nanoparticle adsorption time: take each 10mL of SHENGMAI YIN (Radix Codonopsis side), regulates pH=3.5, respectively adds 150mg
MMNPs prepared by embodiment 1 method, stirs 5min, 8min, 10min, 12min, 15min respectively, with externally-applied magnetic field by MMNPs
Separate with supernatant, pour out the supernatant, lower sediment 9mL CH2Cl2:Me2CO (2:1, v/v), stirs desorption 10min,
In additional the action of a magnetic field lower leaf, collect the supernatant, redissolve with 300 μ L absolute methanols after naturally volatilizing, cross 0.22 μm filter membrane,
Prepare sample solution, use embodiment 2 method high performance liquid chromatograph detection (column temperature: 25 DEG C, acetonitrile-methanol-0.1% formic acid
Water (20:20:60, v/v/v) is flowing phase), result shows that (see c in Fig. 1) adsorption time is that 10min is more excellent.
4, effluent volume: take each 10mL of SHENGMAI YIN (Radix Codonopsis side), regulates pH=3.5, each addition 150mg embodiment 1 side
MMNPs prepared by method, stirs 10min, is separated with supernatant by MMNPs with externally-applied magnetic field, pours out supernatant, and precipitation is used respectively
The CH of 3mL, 6mL, 9mL, 12mL2Cl2:Me2CO (2:1, v/v), stirs desorption 10min, in additional the action of a magnetic field lower leaf,
Collect the supernatant, redissolve with 300 μ L absolute methanols after naturally volatilizing, cross 0.22 μm filter membrane, take filtrate, prepare sample solution,
Use embodiment 2 method high performance liquid chromatograph detection (column temperature: 25 DEG C, acetonitrile-methanol-0.1% formic acid water (20:20:60, v/
V/v) for flowing phase), result shows that (see d in Fig. 1) effluent volume is that 9mL is more excellent.
5, the desorption time: take each 10mL of SHENGMAI YIN (Radix Codonopsis side), pH=3.5, each addition 150mg embodiment 1 side are regulated
MMNPs prepared by method, stirs 10min, is separated with supernatant by MMNPs with externally-applied magnetic field, pours out the supernatant, and lower sediment is used
The CH of 9mL2Cl2:Me2CO (2:1, v/v), stirs desorption 2min, and 4min, 6min, 8min, 10min, at additional the action of a magnetic field
Lower leaf, collects the supernatant, redissolves with 300 μ L absolute methanols, crosses 0.22 μm filter membrane, take filtrate, prepare sample after naturally volatilizing
Product solution, employing embodiment 2 method high performance liquid chromatograph detection (column temperature: 25 DEG C, acetonitrile-methanol-0.1% formic acid water (20:
20:60, v/v/v) be flowing phase), result shows that (see e in Fig. 1) the desorption time is that 8min is more excellent.
Flavacin B in embodiment 4 Chinese medicine1Assay
(1) chromatographic condition and system suitability test
(1) instrument and reagent are with embodiment 2.
(2) selection of chromatographic condition
On the basis of instrument, flowing phase, detection wavelength, column temperature isochromatic spectrum condition are optimized investigation, it is thus achieved that
Can be suitably used for flavacin B1The chromatographic condition of assay:
Chromatographic column: Agilent SB-C18Post (4.6mm × 250mm, 5 μm)；
Flowing phase: A be acetonitrile, B is mass concentration 0.1% aqueous formic acid, and C is absolute methanol, by acetonitrile-methanol-
0.1% formic acid water (20:20:60, v/v/v) carries out isocratic elution；
Flow velocity: 1.0mL/min；
Column temperature: 25 DEG C；
Detector: fluorescence detector；
Detection wavelength: excitation wavelength: 360nm, launches wavelength: 440nm；
Sample size: 20 μ L.
(3) preparation of standard solution:
It is appropriate that precision weighs aflatoxin B1 standard substance, and being configured to concentration with methanol is 10ng/mL standard solution, efficiently
Liquid chromatogram is shown in Fig. 2.
(4) preparation of need testing solution:
Precision measures SHENGMAI YIN (Radix Codonopsis side) 10mL, is uniformly added into MMNPs prepared by 150mg embodiment 1 method, stirring
10min, separates MMNPs with supernatant with externally-applied magnetic field, pours out the supernatant, lower sediment 9mL CH2Cl2:Me2CO(2:
1, v/v) eluting, stirs desorbing 8min, in the action of a magnetic field lower leaf, collects eluent, volatilizes, and residue redissolves with 300 μ L methanol,
Cross 0.22 μm filter membrane, prepare need testing solution, enter chromatogram such as Fig. 3 of Liquid Detection.
(5) drafting of standard curve
Precision draws standard solution 1 μ L, 2 μ L, 5 μ L, 10 μ L, 20 μ L injection high performance liquid chromatographs respectively, records peak
Area.With sample size (X, ng/mL) for abscissa, peak area (Y, mAU) is vertical coordinate, draws standard curve, linearly returns
Return, obtain regression equation and be shown in Table 1.
Table 1 flavacin B1Standard curve
(6) precision test
Under above-mentioned chromatographic condition, accurate absorption standard solution is interior on the same day repeats sample introduction 6 times.Result calculates Aspergillus flavus
Element B1The RSD of peak area is 1.2%.Under above-mentioned chromatographic condition, accurate absorption standard solution, for three days on end sample introduction every day 1
Secondary.Result calculates flavacin B1The RSD of peak area is 1.2%.Result above shows that instrument precision is good.
(7) stability test
Accurate draw same need testing solution, respectively at 2,4,8,16,24h measures peak area, result is 24 little mensuration
Time interior, calculate flavacin B1Peak area RSD value is 1.1%.Show that need testing solution is good at 24h internal stability.
(8) replica test
Taking 6 parts of same SHENGMAI YIN (Radix Codonopsis side) sample, prepared by the method according to step (four) need testing solution, measurement result meter
Calculate flavacin B1The RSD of peak area is 1.2%, shows that the repeatability of sample is good.
(9) recovery test
Use sample-adding recovery experiment, take with 6 parts of a collection of SHENGMAI YIN (Radix Codonopsis side) sample, be separately added into the flavacin of equivalent
B1Standard solution, is prepared by the method for step (four) need testing solution, measures, and calculates the response rate, and average recovery is 97.67
± 2.1%, show that the method accuracy is good.
(10) sample size measures
The sample of the SHENGMAI YIN (Radix Codonopsis side) deriving from 5 producers is carried out flavacin B1Measure, the results are shown in Table 2.5
The chromatogram of producer's SHENGMAI YIN (Radix Codonopsis side) is shown in Fig. 6.
Different enterprise SHENGMAI YIN (Radix Codonopsis side) the flavacin B of table 21Assay sample determination result (ng/mL)
Note: "-" represents that peak area is less than quantitative limit.
Claims (10)
1. flavacin B in a Chinese medicine1The method of assay, it is characterised in that described method is: middle drug solns to be measured is adjusted
Joint pH value, to 2～5, adds magnetic nanoparticle, stirring and evenly mixing, and mixed liquor, in additional the action of a magnetic field lower leaf, removes upper strata clear
Liquid, lower sediment adds dichloromethane and the acetone mixture of volume ratio 2:1, stirs desorption, divides under additional the action of a magnetic field
Layer, collects the supernatant, redissolves with absolute methanol, cross 0.22 μm filter membrane after naturally volatilizing, and filtrate uses high performance liquid chromatography to enter
Row flavacin B1Measure；Described magnetic nanoparticle is 2-amino-5-sulfydryl-1,3,4-thiadiazoles 3-trimethyl silane-1-
Ferroferric oxide magnetic nanoparticle modified by propanethiol.
2. flavacin B in Chinese medicine as claimed in claim 11The method of assay, it is characterised in that described magnetic nanoparticle
Addition is calculated as 10-20g/L with Chinese medicine liquor capacity to be measured.
3. flavacin B in Chinese medicine as claimed in claim 11The method of assay, it is characterised in that described dichloromethane and third
Ketone mixed liquor and Chinese medicine liquor capacity to be measured are than for 0.3-1.2:1.
4. flavacin B in Chinese medicine as claimed in claim 11The method of assay, it is characterised in that described stirring and evenly mixing time
Being 5～15min, the stirring desorption time is 2～10min.
5. flavacin B in Chinese medicine as claimed in claim 11The method of assay, it is characterised in that described externally-applied magnetic field refers to
Using Magnet as externally-applied magnetic field source.
6. flavacin B in Chinese medicine as claimed in claim 11The method of assay, it is characterised in that described high performance liquid chromatography
Testing conditions is: chromatographic column: C18Post, 4.6mm × 250mm ,≤5 μm；Flowing phase: acetonitrile, methanol, volumetric concentration 0.1% formic acid
Aqueous solution, volume ratio 10-25:10-25:50-80；Flow velocity: 0.8-1.2mL/min；Column temperature: 20-30 DEG C；Fluorescence detector.
7. flavacin B in Chinese medicine as claimed in claim 61The method of assay, it is characterised in that described C18Carbon used by post
Octadecylsilane chemically bonded silica particle diameter is 5 μm or 3.5 μm.
8. flavacin B in Chinese medicine as claimed in claim 61The method of assay, it is characterised in that described flowing phase: acetonitrile,
Methanol, volumetric concentration 0.1% aqueous formic acid, volume ratio 20:20:60.
9. flavacin B in Chinese medicine as claimed in claim 51The method of assay, it is characterised in that described fluorescence detector swashs
Send out wavelength: 360nm, launch wavelength: 440nm.
10. flavacin B in Chinese medicine as claimed in claim 11The method of assay, it is characterised in that described magnetic Nano
Grain is prepared as follows:
(1) by FeCl3·6H2O and FeCl2·4H2O mixes with deionized water, 85 DEG C of heating in water bath under conditions of nitrogen is protected
And stir 30min, it is slowly injected into the ammonia of mass concentration 25-28%, after addition ammonia terminates, continues reacting by heating 30min, from
The heart, takes precipitate with deionized water and washs 2 times, then wash 1 time with 0.02mol/L sodium-chloride water solution, be deposited at 40～60 DEG C
It is dried, obtains ferroferric oxide magnetic nanoparticle；Described FeCl3·6H2O and FeCl2·4H2O mass ratio is 2～3:1, institute
State deionized water volumetric usage with FeCl3·6H2O and FeCl2·4H2O gross mass is calculated as 12～13mL/g, described ammonia volume
Consumption is with FeCl3·6H2O and FeCl2·4H2O gross mass is calculated as 1.4～1.8mL/g；
(2) step (1) ferroferric oxide magnetic nanoparticle deionized water is configured to the storing solution of 40g/L, by storing solution
In additional the action of a magnetic field lower leaf, removing the supernatant, lower sediment adds mass concentration 10%3-trimethyl silane-1-rosickyite
Alcohol-water solution, stirs, and adds glycerol, heats under nitrogen protection and is stirred vigorously 2h, be cooled in 90 DEG C of water-baths
After room temperature, in additional the action of a magnetic field lower leaf, removing the supernatant, lower sediment is washed with deionized 3 times successively, without water beetle
Alcohol washs 3 times, deionized water wash 5 times, and precipitation granule is dried at 40～60 DEG C, obtains 3-trimethyl silane-1-propanethiol
Modified by magnetic nanoparticles thing；Described 3-trimethyl silane-1-propanethiol aqueous solution and storing solution volume ratio are 4～5:1, described
Glycerol and storing solution volume ratio are 3～4:1；
(3) step (2) 3-trimethyl silane-1-propanethiol modified by magnetic nanoparticles thing deionized water is configured to 40g/L
Storing solution, washs 2 times with methanol, adds mass concentration 1%2-amino-5-sulfydryl-1, and 3,4-thiadiazoles aqueous solutions, at ultrasound wave
Power is ultrasonic 2h under conditions of 600W, obtains granule precipitate with deionized water and washs 3 times, then washs 2 times with absolute methanol, in
Being dried 2h at 40～60 DEG C, i.e. obtain 2-amino-5-sulfydryl-1,3,4-thiadiazoles 3-trimethyl silane-1-propanethiol magnetic are received
Rice grain trim, is magnetic nanoparticle；Described 2-amino-5-sulfydryl-1,3,4-thiadiazoles aqueous solution and reserve liquid
Long-pending ratio is 5～8:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610528482.7A CN106053662A (en) | 2016-06-30 | 2016-06-30 | Method for measuring aflatoxin B1 content in traditional Chinese medicine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610528482.7A CN106053662A (en) | 2016-06-30 | 2016-06-30 | Method for measuring aflatoxin B1 content in traditional Chinese medicine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106053662A true CN106053662A (en) | 2016-10-26 |
Family
ID=57202072
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610528482.7A Pending CN106053662A (en) | 2016-06-30 | 2016-06-30 | Method for measuring aflatoxin B1 content in traditional Chinese medicine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106053662A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106770806A (en) * | 2017-03-03 | 2017-05-31 | 浙江工业大学 | It is a kind of to determine aflatoxin B1, the method for B2 contents in Chinese patent drug simultaneously |
| CN106885853A (en) * | 2017-02-14 | 2017-06-23 | 湖北省农业科学院农业质量标准与检测技术研究所 | The quick pre-treating method for determining AVM pesticide residue in edible oil and quantitative analysis method |
| CN107764614A (en) * | 2017-09-21 | 2018-03-06 | 四川省丹丹郫县豆瓣集团股份有限公司 | A kind of pre-treating method of bean paste aflatoxin B1 detection |
| CN109331783A (en) * | 2018-10-23 | 2019-02-15 | 南京师范大学常州创新发展研究院 | Mechanochemistry Magnetic solid phases extracting process, magnetic Nano material and preparation method thereof |
| CN110108821A (en) * | 2019-04-22 | 2019-08-09 | 广西壮族自治区疾病预防控制中心 | A kind of dispersive solid-phase extraction material and the preparation method and application thereof |
| CN112540132A (en) * | 2020-10-26 | 2021-03-23 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Method for separating and enriching aflatoxin in vegetable oil |
| CN116124925A (en) * | 2022-12-20 | 2023-05-16 | 河南工业大学 | Aflatoxin B 1 Determination method of double early warning molecules |
| CN116338054A (en) * | 2023-04-19 | 2023-06-27 | 广东维安检测科技有限公司 | Detection method of aflatoxin B1 in traditional Chinese medicine based on liquid chromatography |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102680673A (en) * | 2012-05-29 | 2012-09-19 | 西安金磁纳米生物技术有限公司 | Immune magnetic particle for purifying aflatoxin samples and preparing method and application method thereof |
| CN104475011A (en) * | 2014-12-15 | 2015-04-01 | 江南大学 | Preparation method of magnetic mesoporous silicon dioxide adsorbent for removing aflatoxin in edible oil |
-
2016
- 2016-06-30 CN CN201610528482.7A patent/CN106053662A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102680673A (en) * | 2012-05-29 | 2012-09-19 | 西安金磁纳米生物技术有限公司 | Immune magnetic particle for purifying aflatoxin samples and preparing method and application method thereof |
| CN104475011A (en) * | 2014-12-15 | 2015-04-01 | 江南大学 | Preparation method of magnetic mesoporous silicon dioxide adsorbent for removing aflatoxin in edible oil |
Non-Patent Citations (4)
| Title |
|---|
| ALDAHIR ALBERTO HERNÁNDEZ-HERNÁNDEZ ET AL.: "Food Analysis by Microextraction Methods Based on the Use of Magnetic Nanoparticles as Supports: Recent Advances", 《FOOD ANAL. METHODS》 * |
| MAHDI HASHEMI ET AL.: "Application of magnetic solid phase extraction for separation and determination of aflatoxins B1 and B2 in cereal products by high performance liquid chromatography-fluorescence detection", 《JOURNAL OF CHROMATOGRAPHY B》 * |
| MAHDI HASHEMI ET AL.: "Enhanced spectrofluorimetric determination of aflatoxin M1 in liquid milk after magnetic solid phase extraction", 《SPECTROCHIMICA ACTA PART A: MOLECULAR AND BIOMOLECULAR SPECTROSCOPY》 * |
| 刘伟伟 等: "超顺磁性免疫磁珠体系用于植物油中黄曲霉毒素B1的检测研究", 《分析测试学报》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106885853A (en) * | 2017-02-14 | 2017-06-23 | 湖北省农业科学院农业质量标准与检测技术研究所 | The quick pre-treating method for determining AVM pesticide residue in edible oil and quantitative analysis method |
| CN106770806A (en) * | 2017-03-03 | 2017-05-31 | 浙江工业大学 | It is a kind of to determine aflatoxin B1, the method for B2 contents in Chinese patent drug simultaneously |
| CN106770806B (en) * | 2017-03-03 | 2019-10-29 | 浙江工业大学 | It is a kind of to measure aflatoxins B in Chinese patent drug simultaneously1, B2The method of content |
| CN107764614A (en) * | 2017-09-21 | 2018-03-06 | 四川省丹丹郫县豆瓣集团股份有限公司 | A kind of pre-treating method of bean paste aflatoxin B1 detection |
| CN109331783A (en) * | 2018-10-23 | 2019-02-15 | 南京师范大学常州创新发展研究院 | Mechanochemistry Magnetic solid phases extracting process, magnetic Nano material and preparation method thereof |
| CN109331783B (en) * | 2018-10-23 | 2021-08-24 | 南京师范大学常州创新发展研究院 | Mechanochemical magnetic solid phase extraction method, magnetic nano material and preparation method thereof |
| CN110108821A (en) * | 2019-04-22 | 2019-08-09 | 广西壮族自治区疾病预防控制中心 | A kind of dispersive solid-phase extraction material and the preparation method and application thereof |
| CN112540132A (en) * | 2020-10-26 | 2021-03-23 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Method for separating and enriching aflatoxin in vegetable oil |
| CN116124925A (en) * | 2022-12-20 | 2023-05-16 | 河南工业大学 | Aflatoxin B 1 Determination method of double early warning molecules |
| CN116124925B (en) * | 2022-12-20 | 2023-11-21 | 河南工业大学 | Aflatoxin B 1 Determination method of double early warning molecules |
| CN116338054A (en) * | 2023-04-19 | 2023-06-27 | 广东维安检测科技有限公司 | Detection method of aflatoxin B1 in traditional Chinese medicine based on liquid chromatography |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106053662A (en) | Method for measuring aflatoxin B1 content in traditional Chinese medicine | |
| CN105424820B (en) | A method of 11 kinds of Ginsenosides are carried out at the same time qualitative and quantitative | |
| CN104730172B (en) | The aptamer affinity column new purification method of ochratoxin A in a kind of Chinese medicine | |
| CN107091892A (en) | The method that the content of synthetic coloring matter in food is tested using high performance liquid chromatography | |
| CN104587014B (en) | Method for extracting flavonoid active ingredient from traditional Chinese medicine fructus aurantii | |
| CN103884785A (en) | Selenium detection method | |
| CN106770806B (en) | It is a kind of to measure aflatoxins B in Chinese patent drug simultaneously1, B2The method of content | |
| CN104090036B (en) | A kind of enrichment and the method detecting low concentration Anthraquinones effective constituent | |
| CN107199012B (en) | A kind of magnetism fullerene nanomaterial and its application in Solid Phase Extraction | |
| CN104730158B (en) | A kind of content assaying method of JIAWEI HUOXIANG ZHENGQI RUANJIAONANG | |
| CN107389821A (en) | A kind of method of active ingredient in measure ageratum oral liquid | |
| CN109085285B (en) | Quality control method of changyanning granules | |
| CN103930118A (en) | Andrographis paniculata and testing method for preparation thereof | |
| Zhang et al. | Metal–organic framework assisted matrix solid‐phase dispersion microextraction of saponins using response surface methodology | |
| CN105954404A (en) | Method for determining content of salivary acid in serum by using UIO-66-NH2 material | |
| CN108593792A (en) | Magnetic solid phase extraction-HPLC- the ultraviolet detection methods of environment incretion interferent in water sample | |
| Xiang et al. | Two‐dimensional chromatography in screening of bioactive components from natural products | |
| CN101703583B (en) | Method for detecting quality of Xinning capsule | |
| CN110487951A (en) | A kind of quality determining method of Hedan tablet | |
| CN116265941A (en) | Method for detecting triptolide content in compound preparation containing tripterygium medicinal material | |
| CN101317935A (en) | Rujietai formulation and quality detection method | |
| CN105823830B (en) | One surveys the methods for commenting tanshin polyphenolic acid B and schizandrin content in method measurement Yixinfumai particle more | |
| CN106610406A (en) | Micro-extraction method of honeysuckle | |
| Raynie | Practical Understanding of Partition Coefficients | |
| Guo et al. | Simultaneous preconcentration and quantification of ultra-trace tin and lead species in seawater by online SPE coupled with HPLC-ICP-MS |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20161026 |